cyanobacterial quinomics studies of quinones in cyanobacteria

The Pennsylvania State University

The Graduate School

Eberly College of Science

CYANOBACTERIAL QUINOMICS

STUDIES OF QUINONES IN CYANOBACTERIA

A Thesis in

Biochemistry, Microbiology, and Molecular Biology

by

Yumiko Sakuragi

Submitted in Partial Fulfillment of the Requirements

for the Degree of

Doctor of Philosophy

August 2004

The thesis of Yumiko Sakuragi has been reviewed and approved* by the following:

Date of Signature

Donald A. Bryant Ernest C. Pollard Professor in Biotechnology Professor of Biochemistry and Molecular Biology Thesis Advisor Co-Chair of Committee

John H. Golbeck Professor of Biochemistry and Biophysics Professor of Chemistry Co-Chair of Committee

J. Martin Bollinger Jr. Associate Professor of Biochemistry and Molecular Biology

A. Daniel Jones Senior Scientist

B. Tracy Nixon Associate Professor of Biochemistry and Molecular Biology Special Signatory

Robert A. Schlegel Professor of Biochemistry and Molecular Biology Head of the Department of Biochemistry and Molecular Biology

*Signatures are on file in the Graduate School.

iii

ABSTRACT

Roles and functions of isoprenoid quinones (phylloquinone, plastoquinone-9) and

α-tocopherol were investigated in cyanobacteria. Comparative genome analyses of 14

cyanobacteria suggested that phylloquinone (PhyQ) biosynthesis in most but not all

cyanobacteria occurs similarly to menaquinone biosynthesis in Escherichia coli. This was

further supported by the discovery that two cyanobacteria, Synechococcus sp. PCC 7002

and Gloeobacter violaceus PCC 7421, synthesize menaquinone-4 (MQ-4). Targeted

inactivation of the menB, menF, and menG genes resulted in the incorporation of

plastoquinone-9 (PQ-9) and demethyl-MQ or demethyl-PhyQ into Photosystem I (PS I)

complexes. In the PS I complexes containing demethyl-PhyQ, the rate of electron transfer

from A1 to the iron-sulfur clusters slowed by a factor of two, while the kinetics of the

P700+ [FA/FB]- backreaction increased by a factor of 3 to 4. These results were explained

by a lowering of the equilibrium constant between Q-/Q and FX-/FX in the demethyl-PhyQ

containing PS I complexes by a factor of ~10.

Populations of α-tocopherol mutants of the cyanobacterium Synechocystis sp.

PCC 6803, previously isolated in the presence of glucose, were found to be

phenotypically and genotypically heterogeneous. Newly isolated, “authentic” tocopherol

mutants were unable to grow in the presence of glucose at pH 7.0; this was suggested to

be due to a significant reduction of the amounts of sigA and rbcL transcripts in cells

under these conditions. The slr2031 product, which has been previously shown to be

involved in sulfur, nitrogen, and carbon metabolism, and genes encoding inorganic

carbon uptake mechanisms, were found to be constitutively down-regulated in the

iv

“authentic” tocopherol mutants. The results indicate that α-tocopherol is involved in the

transcriptional regulation of these metabolic genes and plays an important role in the

coordination of nitrogen, sulfur, and carbon metabolism in Synechocystis sp. PCC 6803.

The PQ-9 biosynthesis pathway was predicted to be similar to that for ubiquinone

biosynthesis based on comparative genome analyses of 14 cyanobacteria. However,

targeted inactivation mutagenesis of eight genes encoding putative methyltransferase

genes similar to UbiE/MenG in E. coli did not affect PQ-9 biosynthesis in Synechocystis

sp. PCC 6803. Based on the results obtained, a possible PQ-9 biosynthesis pathway is

proposed.

v

TABLE OF CONTENTS

Page List of Figures vii List of Tables xiii Acknowledgements xv Chapter 1 General Introduction 1

Cyanobacterial oxygenic photosynthesis 3 Phylloquinone and Photosystem I 6 Plastoquinone and Photosystem II 9 Role of α-tocopherol 12 Cyanobacterial quinomics 13 References 16

Chapter 2 Comparative genome analysis and identification of menaquinone-4 biosynthetic pathways in cyanobacteria

32

Abstract 33 Introduction 35 Results 38 Discussion 45 Summary 49 Materials and Methods 50 References 54

Chapter 3 Recruitment of a foreign quinone into the A1 site of Photosystem I. Interruption of menG in the phylloquinone biosynthetic pathway of Synechocystis sp. PCC 6803 results in the incorporation of 2-phytyl-1,4-naphthoquinone and alteration of the equilibrium constant for electron transfer between A1 and FX.

72


vi

Page Chapter 4 Physiological characterization of tocopherol biosynthesis mutants in the cyanobacterium Synechocystis sp. PCC 6803: demonstration of a conditionally lethal phenotype in the presence of glucose at pH 7.0

129


Chapter 5 Transcriptional regulation of the metabolic and sigma factor genes in the vitamin E-deficient mutant of Synechocystis sp. PCC 6803

194


Chapter 6 Comparative genome analysis and genetic study of plastoquinone biosynthetic pathway in cyanobacteria

242


Appendix 291

vii

LIST OF FIGURES

Page

Figure 1.1 Structure of phylloquinone, plastoquinone-9, and α-tocopherol 27

Figure 1.2 Scheme of oxygenic photosynthesis in cyanobacteria 28

Figure 1.3 The spatial arrangement and the redox potentials of the electron

transfer cofactors in Photosystem I

29

Figure 1.4 The electron transfer cofactors in Photosystem II 30

Figure 1.5 Biosynthetic pathway of PQ-9 and α-tocopherol in higher plants 31

Figure 2.1 Menaquinone biosynthetic pathway in E. coli 62

Figure 2.2 The men gene arrangements 63

Figure 2.3 Phylogenetic trees based on the amino acid sequences of MenA,

MenB, and MenD

64

Figure 2.4 HPLC profiles of solvent extracts from PS I complexes and whole

cells

65

Figure 2.5 Absorption spectra of solvent-extracted components analyzed by

HPLC

66

Figure 2.6 LC-mass spectrometric analysis of the solvent extracts from

Synechococcus sp. PCC 7002

67

Figure 2.7 Restriction maps of the menB, menF, and menG coding regions in

Synechococcus sp. PCC 7002

68

Figure 2.8 PCR analyses on genomic DNAs isolated from the menB rubA, menF,

and menG mutants of Synechococcus sp. PCC 7002

69

viii

Figure 2.9 Reverse-phase HPLC analyses of the solvent extracts from the PS I

complexes

70

Figure 2.10 Absorption spectra of eluting components analyzed for PS I

complexes isolated from Synechococcus sp. PCC 7002

71

Figure 3.1 Relationship between the molecular structure of 2-phytyl-1,4-NQ and

the axes of the electronic g-tensor

117

Figure 3.2 Construction and verification of the menG mutant strain of

Synechocystis sp. PCC 6803

118

Figure 3.3 Mass spectra recorded for pigments and quinone extracts from the PS I

complexes isolated from the wild-type and menG mutant strains of Synechocystis

sp. PCC 6803

119

Figure 3.4 77 K fluorescence emission spectra from whole cells of Synechocystis

sp. PCC 6803

120

Figure 3.5 SDS-PAGE analysis of PS I complexes isolated from the wild-type

and menG mutant strains of Synechocystis sp. PCC 6803

121

Figure 3.6 Photoaccumulated Q-band EPR spectra and simulations of PS I

complexes from Synechocystis sp. PCC 6803

122

Figure 3.7 Transient spin-polarized EPR spectra of the charge separated P700+ Q-

state in PS I complexes.

123

Figure 3.8 Simulations of the Q-band spectrum of the menG mutant carried out

for various angles between the largest principal A11 and the gxx axis.

124

Figure 3.9 Spin-polarized EPR transient of wild type and menG mutant 125

ix

Figure 3.10 Decay-associated transient EPR spectra at ambient temperature 126

Figure 3.11 Flash-induced absorbance changes at 812 nm in PS I complexes

isolated from Synechocystis sp. PCC 6803

127

Figure 3.12 Steady-state rates of flavodoxin reduction in PS I complexes isolated

from the wild-type and menG mutant strains.

128

Figure 4.1 Biosynthetic pathway of α-tocopherol in Synechocystis sp. PCC 6803 176

Figure 4.2 Colony morphologies of three tocopherol mutants previously isolated

in the presence of glucose.

177

Figure 4.3 Growth curves of wild type and cell lines derived from small and large

colonies of tocopherol mutants selected in the presence of glucose

178

Figure 4.4 PCR analysis of the genomic DNA extracted from the newly isolated

tocopherol mutants selected in the presence of glucose

179

Figure 4.5 Growth curves of wild type and the “authentic” tocopherol mutants in

the presence and in the absence of glucose

180

Figure 4.6 Growth curves of wild type and the “authentic” slr1736 mutant in the

presence of glucose

181

Figure 4.7 Growth analyses of wild type and the “authentic” slr1736 mutant of

Synechocystis sp. PCC 6803 in the presence of various carbon sources

182

Figure 4.8 Chlorophyll a and quinone contents in wild type and the “authentic”

tocopherol mutants grown in the absence and in the presence of glucose

183

Figure 4.9 Oxygen-evolution activities of the whole-chain and the Photosystem

II-dependent electron transport chains in wild type and the “authentic” tocopherol

184

x

mutants of Synechocystis sp. PCC 6803

Figure 4.10 Immunoblotting analysis for the D1 and PsbO proteins in whole cells

of wild type and three “authentic” tocopherol mutants

185

Figure 4.11 Time-dependent RT-PCR analysis of the sodB and nblA transcripts in

wild type and the “authentic” slr1736 mutant of Synechocystis sp. PCC 6803

186

Figure 4.12 Relative phycobiliprotein contents in wild type and the “authentic”

tocopherol mutants of Synechocystis sp. PCC 6803

187

Figure 4.13 Growth curves of wild type and the “authentic” slr1736 mutant the

presence of glucose at 3% (v/v) CO2 and air

188

Figure 4.14 Cultures of wild type and the “authentic” tocopherol mutants of

Synechocystis sp. PCC 6803 grown at various pH values

189

Figure 4.15 Growth curves of wild type and the “authentic” slr1736 mutant of

Synechocystis sp. PCC 6803 at various pH values in the presence of glucose in air

and 1% (v/v) CO2

190

Figure 4.16 Cultures of wild type and the “authentic” slr1736 mutant of

Synechocystis sp. PCC 6803 grown under various conditions for 48 h at pH 7.0

191

Figure 4.17 PS II-dependent O2-evolution activities of wild type and the

“authentic” slr1736 mutant in the absence and in the presence of glucose

192

Figure 4.18 Phycobiliprotein contents in wild type and the tocopherol mutants of

Synechocystis sp. PCC 6803 grown at 1% (v/v) CO2.

193

Figure 5.1 Time-dependent RT-PCR analysis on the cpcA, nblA, and sbtA

transcripts in wild type and slr1736 mutant grown at pH 7.0 and 8.0

233

xi

Figure 5.2 Time-dependent RT-PCR analysis of the alternative sigma factor

transcripts in wild-type and slr1736 mutant grown at pH 7.0 and 8.0

234

Figure 5.3 Time-dependent RT-PCR analysis of the sigA, sigG, and Ci genes in

wild-type and slr1736 mutant grown at pH 7.0 and 8.0

235

Figure 5.4 Thin-section micrographs of the wild-type cells grown in the absence

and in the presence of glucose for 24 h

236

Figure 5.5 Thin-section micrographs of the slr1736 mutant grown in the absence

and in the presence of glucose for 24 h

237

Figure 5.6 Time-dependent RT-PCR analysis of the slr2031 and CCM gene

transcripts in wild type and slr1736 mutant grown at pH 7.0 and 8.0

238

Figure 5.7 PCR analysis of the genomic DNAs extracted from the slr2031::aadA

and pmgA::aacC1 mutants

239

Figure 5.8 Cultures of wild type, the slr1736, slr2031, and pmgA mutants of

Synechocystis sp. PCC 6803 in the presence of glucose at pH 7.0 and 8.0

240

Figure 5.9 Summary and a model of the non-antioxidant role of α-tocopherol in

Synechocystis sp. PCC 6803

241

Figure 6.1 UQ biosynthesis pathway in Escherichia coli 280

Figure 6.2 Hypothetical PQ-9 biosynthesis pathway in cyanobacteria 281

Figure 6.3 An alignment of the Ycf21/UbiC homologs in cyanobacteria and

eukaryotic phototrophs

282

Figure 6.4 Phylogenetic analyses based on nucleotide sequences of 16S rRNA

and amino acid sequences of Ubi proteins in cyanobacteria and proteobacteria

283

xii

Figure 6.5 Phylogenetic trees based on amino acid sequences of UbiE homologs

in cyanobacteria

285

Figure 6.6 HPLC analysis of the sll1653 mutant of Synechocystis sp. PCC 6803

and the 2-phytyl-1,4-benzoquinone (MPBQ) standard.

286

Figure 6.7 PQ-content in whole cells of the wild-type and methyltransferase

mutant strains of Synechocystis sp. PCC 6803

287

Figure 6.8 Restriction maps of coding regions and their flanking sequences for the

UbiE homologs in Synechocystis sp. PCC 6803

288

Figure 6.9 PCR analyses of genomic DNA isolated from the single, double, and

triple methyltransferase (UbiE homolog) mutants of Synechocystis sp. PCC 6803

289

Figure 6.10 Aromatic amino acid biosynthesis and an alternative proposal for 4-

hydroxybenzoate synthesis in cyanobacteria

290

xiii

LIST OF TABLES

Page

Table 2.1 Whole genome analyses for the MQ/PhyQ biosynthesis enzymes in 14

cyanobacteria, Escherichia coli, Bacillus subtilis, Chlorobium tepidum and

Halobacterium sp. NRC

60

Table 3.1 Doubling times of Synechocystis sp. PCC 6803 wild type and the

menG mutant strains 114

Table 3.2 Chlorophyll content in cells grown photoautotrophically at 32 °C

under various illumination conditions. 115

Table 3.3 Magnetic parameters of PhyQ (A1-) and 2-phytyl-1,4-NQ in the A1

binding site 116

Table 6.1 Amino acid sequence similarities between UbiA homologs in 14

cyanobacteria and E. coli 272

Table 6.2 Amino acid sequence similarities between UbiH homologs in 14


Table 6.3 Amino acid sequence similarities between UbiD homologs in 14


Table 6.4 Amino acid sequence similarities between UbiX homologs in 14


Table 6.5 List of the UbiE homologs in 14 cyanobacteria 276

Table 6.6 Ycf21/UbiC homologs in cyanobacteria and eukaryotic phototrophs 277

Table 6.7 Sequences of primers used for PCR analyses 278

xiv

Table 6.8 Construction of methyltransferase mutants in Synechocystis sp. PCC

6803

279

xv

ACKNOWLEDGEMENTS

I would like to thank my advisor Dr. Donald A. Bryant and my co-advisor Dr.

John H. Golbeck for their guidance and support. I would also like to acknowledge my

collaborators who have contributed to the work on phylloquinone: Dr. Gaozhong Shen,

Dr. Boris Zybailov, Dr. Art van der Est, Dr. Robert Bittl, Dr. Stephan Zech, and Dr.

Dietmar Stehlik; and my collaborators who have contributed to the work on

plastoquinone and α-tocopherol: Dr. Dean DellaPenna, Hiroshi Maeda, and Dr. Zigang

Cheng.

My special thanks are also due to the current and former members of both the

Bryant and Golbeck laboratories. Lastly, I would like to thank my husband, Dr. Niels-

Ulrik Frigaard, for his support and advice throughout my studies.

1

Chapter 1

General Introduction

Cyanobacteria are a phylogenetically coherent group of organisms, yet

morphologically, physiologically, and genetically they are very diverse (Bryant, 1994).

They are found in a variety of terrestrial and aqueous environments such as open oceans,

coastal waters, fresh water lakes, rivers, municipal reservoirs, ponds, and even kitchen

sinks in domestic households. Some perform N2 fixation, and some are capable of

chemotaxis or phototaxis or both. While the majority are obligate photoautotrophs, a few

are able to grow chemoheterotrophically. Genome sizes vary from 2 Mbp to ~15 Mbp.

Despite these differences, there is one thing that is common among all cyanobacteria: the

unique ability to perform oxygenic photosynthesis. All cyanobacteria perform oxygenic

photosynthesis, which uses CO2 as the source of carbon, water as the source electrons,

and light as the source of energy, in order to produce biomass. As a result, molecular

oxygen is produced as a byproduct (Barber and Anderson, 2002). Through the activity of

oxygenic photosynthesis, cyanobacteria have affected, and are continuing to affect, our

environment since their first appearance more than 3 billion years ago. This activity of

cyanobacteria has transformed the atmosphere of our planet from an extremely reducing

to a rather oxidizing environment, and this change has supported the evolution of diverse

forms of life on Earth. In an era of global warming, these organisms attract more

attention given that about half of the global CO2 fixation known to occur on Earth is

carried out by cyanobacteria (Bryant, 2003).

2

Much has been learned about the molecular mechanisms of oxygenic

photosynthesis owing to decades of study, which have dissected and largely solved the

mystery of water oxidation and the detailed functions of the photosynthetic apparatus

primarily from the viewpoint of protein functions. Such a perspective, however, has

somewhat overlooked the biological importance of non-proteineous molecules that are

also involved in the process. A group of such molecules are the quinones. In

cyanobacteria these are phylloquinone (PhyQ, 2-methyl-3-phytyl-1,4-naphthoquinone,

also known as vitamin K1), plastoquinone (PQ-9, 2,3-dimethyl-5-solanyl-1,4-

benzoquinone). These two compounds are isoprenoid quinones, which are composed of a

quinonoid nucleus with a polyprenyl substituent (Fig 1.1). There are two types of quinone

substructures: 1,4-naphthoquinone (1,4-NQ), and 1,4-benzoquinone (1,4-BQ). PhyQ is a

methyl- and phytyl-substituted 1,4-NQ, while PQ-9 is a methyl- and solanyl-substituted

1,4-BQ. The phytyl substituent is a partially saturated tetra-isoprene unit (equivalent to

20 carbon atoms), while the solanyl substituent is a nona-isoprene unit (equivalent to 45

carbon atoms). α-Tocopherol (2,5,7,8-tetramethyl-6-chromanol, also known vitamin E)

is a tetramethyl-substituted chromanol, another type of quinone nucleus that is derived

from a phytyl-substituted 1,4-BQ after ring closure (Fig 1.1).

These quinones are synthesized only in oxygenic phototrophs including

cyanobacteria, algae and higher plants (Collins and Jones, 1981; Threlfall and Whistance,

1971), and their functions are tightly connected to oxygenic photosynthesis. PhyQ and

PQ-9 are bound cofactors of the Photosystem I (PS I) and Photosystem II (PS II)

complexes, respectively, and in this context they mediate electron transfer as one-electron

carriers (see below). PQ-9 also serves as a membrane-associated two-electron and two-

3

proton carrier, both in the photosynthetic electron transport and the respiratory electron

transport chains. α-Tocopherol, on the other hand, is thought to provide protection

against oxidative stress in animals and plants, although its role(s) in cyanobacteria has not

yet been demonstrated (see below).

Cyanobacterial oxygenic photosynthesis

Fig 1.2 illustrates the process of oxygenic photosynthesis in cyanobacteria. In

general, photosynthesis is described in the context of two processes known as the light

reactions and dark reactions. The light reactions are the processes in which the energy of

photons is transformed into an electrochemical membrane potential that drives ATP

synthesis and into a strong reducing power that ultimately generates NADPH. The dark

reactions are the processes in which CO2 is reduced or “fixed” into biomass at the

expense of NADPH and ATP as the reducing power and energy source, respectively.

The light reactions are catalyzed by the photosynthetic electron transport chain

that resides in the thylakoid membranes (Bryant, 1994; Hervás et al., 2003). This electron

transport chain involves 3 membrane integral protein complexes (PS I, PS II, cytochrome

b6f complex) and 2 water-soluble electron carriers (cytochrome c6 or plastocyanin, and

ferredoxin or flavodoxin), as well as the lipid-soluble electron carrier PQ-9 (Fig 1.2). X-

ray crystallographic structures of PS I, PS II, and cytochrome b6f complexes are now

available at resolutions of 2.5 Å (Jordan et al., 2001), 3.5 Å (Ferreira et al., 2004), and 3.0

Å (Kurisu et al., 2001), respectively. Upon illumination, photons absorbed by the

peripheral antenna complexes known as phycobilisomes are transferred predominantly to

the PS II complexes, in which they trigger photochemistry that drives the light-dependent

4

oxidation of water and the reduction of plastoquinone (Barry et al., 1994; Bricker and

Ghanotakis, 1996; Britt, 1996; Diner and Babcock, 1996). Water oxidation occurs in the

thylakoid lumen and results in the release of molecular oxygen and protons (2H2O O2

+ 4H+ + 4e-), while the reduction and protonation of PQ-9 occurs on the stromal side of

the membrane and releases doubly reduced plastoquinone, or plastoquinol (PQH2), into

the membrane matrix (2 PQ-9 + 4 H+ + 2 e- 2 PQH2). PQH2 shuttles electrons to the

quinol oxidation site (o) of the cytochrome b6 f complex in a diffusion-dependent fashion,

where it is reoxidized and becomes available for a new round of photochemical reduction

by the PS II complexes. At this step, the electron transfer bifurcates into two separate

pathways within the cytochrome b6f complex; one electron is transferred to the Reiske

iron-sulfur protein and subsequently to a soluble electron carrier such as cytochrome c6 or

plastocyanin in the lumen with concomitant release of two protons into the lumen, while

the other electron is transferred to heme bL and heme bH and reduces PQ-9 at the quinone

reduction site (r) near the stromal surface of the thylakoid membranes (Kallas, 1994;

Hauska et al., 1996). After two rounds of PQH2 oxidation, one PQ-9 at the quinone

reduction site becomes doubly reduced and is protonated by concomitant uptake of two

protons from the stroma (cytoplasm). The resulting PQH2 is released into the membrane

matrix where it joins the pool of PQH2 and participates in electron donation to the

cytochrome b6f complex (2PQH2 (O) + PQ (r) + 2H+Lumen 2PQ(O) + PQH2(r) + 4H+

Stroma

+ 2e-). This process is known as the Q cycle, and the result is an amplification of the

proton gradient across the membranes, which is coupled to ATP synthesis by ATP

synthase (Michell, 1976). The final steps of the electron transport chain involve the PS I

complex. Essentially, this light-dependent cytochrome c6/plastocyanin-

5

ferredoxin/flavodoxin oxidoreductase shuttles an electron from cytochrome c6 or

plastocyanin in the lumen to ferredoxin or flavodoxin in the stroma (Golbeck, 1994;

Brettel and Leibl, 2001). The resulting strong reductant donates an electron to ferredoxin-

NADPH+ oxidoreductase, which catalyzes the reduction of NADP+ to NADPH (Morand

et al., 1994).

It is noteworthy that the cyanobacterial photosynthetic electron transport chain

shares the membrane-associated PQ-9 pool and cytochrome b6f complexes with the

respiratory electron transport chain (Fig 1.2). In this case, electrons enter the electron

transport chain through NADH dehydrogenase in the form of NAD(P)H that is generated

through glycolysis and the TCA cycle, and the oxidative pentose phosphate pathway. The

terminal electron acceptor is molecular oxygen, which is reduced and protonated by

cytochrome c oxidase resulting in generation of water (Schmetterer, 1994).

NADPH and ATP generated through the photosynthetic electron transport chain

are used to fuel the dark reactions. A CO2 molecule is first incorporated into the C5

backbone of ribulose-1,5-bisphosphate, which is then split into two molecules of 3-

phosphoglycerate (Tabita, 1987, 1994). This reaction is catalyzed by ribulose-1,5-

bisphosphate carboxylase/oxygenase (RuBisCO), which is probably the most abundant

protein on Earth (Garrett and Grisham, 1999). In cyanobacteria, RuBisCO is housed

within protein-encapsulated structures known as carboxysomes (Badger and Price, 2003),

which play a role in locally increasing the CO2 concentration. 3-Phosphoglycerate is

reduced to 3-phosphoglyceraldehyde, which is processed through the reductive pentose

phosphate pathway to regenerate ribulose-1,5-bisphosphate. The processes are known as

the Calvin cycle and cause the net production of organic molecules that constitute the

6

metabolic precursors for the biosynthesis of proteins, nucleic acids, sugars, pigments, and

other metabolites. The net production of biomass in the Calvin cycle can be expressed as

the following equation:

6 CO2 + 12 NADPH + 18 ATP C6H12O6 + 12 NADP+ + 18 ADP + 18 Pi

PhyQ and Photosystem I

The PS I complex consists of twelve protein subunits, 96 Chl a molecules, 22 β-

carotene molecules, two PhyQ molecules, three iron-sulfur clusters, and some structural

lipids and water molecules (Jordan et al., 2001). Tightly bound electron transfer cofactors

are found within the core of the complex formed by PsaA and PsaB proteins. These

included a chlorophyll a (Chl a) dimer (P700), two Chl a monomers (A0), two PhyQ

molecules (A1), and three [4Fe-4S] clusters (denoted as FX, FA, FB). These cofactors are

arranged in two branches related by C2 symmetry. The spatial arrangement of these

cofactors and their estimated in situ redox potentials are shown in Fig. 1.3. Upon

photoexcitation, the primary donor P700 reduces the primary acceptor, A0, resulting in

the initial charge separated state [P700+ A0-]. Subsequent electron transfer to the

secondary acceptor A1 stabilizes the charge-separated state [P700+ A0 A1-] and minimizes

the rapid charge recombination that occurs between P700+ and A0-. The electron is

subsequently transferred to FX [P700+ A0 A1 FX-], and then to the terminal acceptors FA

[P700+ A0 A1 FX FA-] and FB [P700+ A0 A1 FX FA FB

-], which are located in the peripheral

PsaC subunit. The reduction of ferredoxin or flavodoxin takes place upon direct protein-

protein interaction between these acceptors and the stromal proteins: PsaC, PsaD, and

PsaE (Zhao et al., 1991, 1993; Li et al., 1991; Golbeck, 1994). On the lumenal side, the

7

photooxidized P700+ accepts an electron from reduced cytochrome c6 or plastocyanin

(Durán et al., 2004).

Since the first discovery of PhyQ in oxygenic phototrophs by Dam in 1941 (cited

in Hauska, 1988), the role of PhyQ in PS I as the secondary acceptor A1 was obscure for

more than two decades. After its initial discovery by Thornber and coworkers in the

1970’s, in the 1980s it was found to be exclusively associated with PS I (Thornber et al.,

1976; Takahashi et al., 1985; Shoeder and Locau, 1986) and to participate in electron

transfer as shown by spin-polarized EPR analysis (Petersen et al., 1987) and optical

kinetic analysis (Brettel et al., 1987; see also reviews by Golbeck, 1994, 2003 and

references therein). Further supporting evidence was derived from solvent extraction

studies, in which extraction of PhyQ from PS I with hexane or ether resulted in a rapid,

nano-second-scale charge recombination between P700+ and A0-. Stable charge

separation was restored by the addition of exogenous PhyQ (Biggins and Mathis, 1988;

Itoh and Iwaki, 1989).

This in vitro reconstitution method was subsequently used to study the structural

and thermodynamic requirements for A1 by introducing diverse quinones of abiotic origin

into the A1 site, including benzoquinones, naphthoquinones, and anthraquinones with

various ring substitutions (Iwaki and Itoh, 1989, 1991; Itoh and Iwaki, 1991; see also

review by Itoh 2001). These authors concluded that the successful restoration of stable

charge separation occurs only when quinones with redox potentials between those of A0

and FX are used. They additionally found that neither the structure of the head group nor

the presence of an alkyl substituent is important. This conclusion contradicted earlier

studies, in which the presence of an alkyl substituent was shown to be essential when

8

hexane/hexane-methanol-extracted PS I complexes were studied (Biggins and Mathis,

1998). These contradictory interpretations imply that different solvent-extraction methods

affect the architecture of PS I differently; hence, the inconsistency in conclusions may

reflect artifacts of the methods employed. Such artifacts can be avoided if “biologically”

or “physiologically” modified materials are used. Materials that have not gone through

harsh extraction processes, which result in co-extraction of other important PS I

constituents such as water, Chl a, carotenoid, and lipid molecules (Biggins and Mathis,

1988; Itoh and Iwaki, 1989), should produce more consistent and interpretable results.

Some of these molecules are located in the vicinity of the quinone-binding site; in

particular, as revealed by 2.5Å-resolution crystallographic structure (Jordan et al., 2002),

β-carotenes and chlorophylls form hydrophobic contacts with the phytyl side chain of

PhyQ and seem to provide structural stability for this important electron carrier.

Genetic manipulation of the PhyQ biosynthetic pathway was first introduced by

Chitnis and colleagues (Johnson et al., 2000) and provided an alternative and very

powerful experimental system, by which the selective elimination of PhyQ from PS I

could be achieved in vivo. Based on the menaquinone biosynthetic pathway in

Escherichia coli (Meganathan, 2001), a part of the PhyQ biosynthetic pathway in

Synechocystis sp. PCC 6803 was first predicted. Targeted insertional mutagenesis of

genes encoding dihydroxynaphthoate phytyltransferase (MenA) and

dihydroxynaphthoate-CoA synthase (MenB) successfully interrupted the PhyQ

biosynthesis in the mutant strains (Johnson et al., 2000). The PS I complexes isolated

from these mutants completely lacked PhyQ and accumulated PQ-9 (Johnson et al., 2000;

Zybailov et al., 2000; Semenov et al., 2000). As confirmed by continuous-wave EPR,

9

electron spin-polarized transient EPR, and electron spin-echo modulation experiments

(Zybailov et al., 2000), the orientation of the carbonyl bonds relative to the membrane

normal and the distance between PQ-9 and P700+ were the same as with PhyQ in the

wild-type PS I complexes. In the PS I complexes isolated from the mutants, PQ-9 was

shown to participate in electron transfer from A0 to FX (Zybailov et al., 2000; Semenov et

al., 2000), although the rate of forward electron transfer from A1 to FX slowed at least by

a factor of 100. The estimated in situ redox potential of PQ-9 differs from that of PhyQ

by ca. 130 mV (Shinkarev et al., 2002), which is consistent with the faster rate of charge

recombination between P700+ and FeS- in PS I (Semenov et al., 2000). Despite this

significant defect in the efficiency of forward electron transfer, the PS I complexes

containing PQ-9 supported nearly 85% of the wild-type activity of PS I, as measured by

the rate of cytochrome c6:flavodoxin oxidoreduction (Johnson et al., 2000). More recent

studies have shown that PS I complexes are functional when the A1 site is occupied by

PhyQ, PQ-9 and even anthraquinones with various substituents (Golbeck et al., 2001;

Zybailov, 2003). The combined results indicate that PS I has an innate capacity to

accommodate and use a wide range of redox-active quinones with various structures and

substituents.

PQ-9 and Photosystem II

Cyanobacterial Photosystem II is a light-driven, water:plastoquinone

oxidoreductase and is composed of at least 19 protein subunits, seven carotenoid

molecules, two pheophytin molecules, two PQ-9 molecules, two bicarbonate molecules,

one none-heme Fe, one heme b, and one heme c (Zouni et al., 2001; Ferreira et al.,

10

2004). Four manganese atoms and one calcium atom are bound to the oxygen-evolving

complex that is attached to the lumenal surface of the PS II complex and is responsible

for water oxidation. As shown in Fig 1.4, the electron transfer cofactors are arranged in

two branches that are related by a pseudo-C2 symmetry axis within the core of the PS II

complex that is formed by the D1 and D2 proteins. Aside from the differences in the

specific types of the cofactors and their arrangement, the principle of the photochemistry

is largely the same in PS II as in PS I. Upon photoexcitation of a Chl a monomer (PD1,

the primary donor P680), an electron is transferred to a pheophytin molecule (PheoD1)

(Danielius et al., 1987). This initial charge separation [P680+ Pheo-] is further stabilized

by electron transfer to a tightly bound PQ-9 molecule (QA) [P680+ Pheo QA-], which is

then followed by transfer to a second PQ-9 molecule (QB) [P680+ Pheo QA QB-] (Renger,

1992; Barry et al., 1994). The second electron transfer doubly reduces QB, which, after

protonation, dissociates from the complex into the thylakoid membrane matrix (Bouges-

Bocquet, 1973; Velthuys and Amesz, 1974; Govindjee and van Rensen, 1993). The

oxidized P680+ is reduced by the redox-active tyrosine Z (TyrZ, Y161 in the D1 protein)

(Gerken et al., 1988; Diner and Babcock, 1996), which is then reduced by a tetra-

manganese cluster that catalyzes the abstraction of electrons from water molecules

(Bricker and Ghanotakis, 1996). With the exception of QA, all redox-active cofactors that

are involved in electron transfer are in principle bound by subunit D1 (PsbA) (Ferreira et

al., 2004).

Prolonged exposure to high light intensity is known to cause irreversible damage

to PS II, and this phenomenon is generally known as photoinhibition (see review by Aro

et al., 1993, and references therein). Under such conditions, QA becomes doubly reduced

11

and leaves the binding pocket (Vass et al., 1992). This leads to rapid charge

recombination between P680+ and Pheo- and to formation of the triplet P680T. P680T can

readily react with molecular oxygen in its vicinity and generate singlet oxygen (1O2).

Singlet oxygen, and other reactive oxygen species generated from it, are responsible for

the photodamage. Photoinhibition can also occur under normal light intensity, when the

donation of electrons to P680 occurs slower than their removal by the acceptor side

(Chen et al., 1995; Aro et al, 1993; Anderson and Chow, 2002). In such cases, highly

oxidizing Tyr Z+ and P680+ cation radicals are formed, which can extract electrons from

the surrounding environment and also cause irreversible damage to PS II. In either case,

the D1 protein is rapidly degraded (Gong and Ohad, 1991; Philbrick et al., 1991; Aro et

al., 1993).

Genetic manipulation of the quinone species present in the PS II quinone-binding

sites has not been demonstrated. The PQ-9 biosynthesis pathway has been intensively

studied, and the complete pathway is known in higher plants (Threlfall and Whistance,

1971). The synthesis occurs from the precursor homogentisate after two enzymatic steps

involving homogentisate solanyltransferase and 2-methyl-6-solanyl-1,4-benzoquinone

(MSBQ) methyltransferase (Soll et al., 1980, 1985; Collakova and DellaPenna, 2001;

Cheng et al., 2003; also see review by Threlfall and Whistance, 1971) (Fig. 1.5). This

pathway in plants overlaps with the α-tocopherol biosynthesis pathway, since both utilize

homogentisate as a precursor, and both pathways share the step that introduces the second

methyl group, a reaction which is catalyzed by MPBQ/MSBQ methyltransferase (Cheng

et al. 2003) (Fig 1.5). Recent studies have shown that the cyanobacterial PQ-9

biosynthetic pathway involves neither homogentisate (Dänhardt et al., 2002) nor MPBQ

12

activity (Cheng et al., 2003). Therefore, PQ-9 seems to be synthesized by a completely

different pathway in cyanobacteria.

Role of α-Tocopherol

α-Tocopherol was first discovered by Evans and Bishop (1922) as a factor

essential for reproduction in rats. Forty years later, its antioxidant activity was recognized

by Epstein and colleagues (Epstein et al., 1966). Because α-tocopherol is an essential

component of our diet, much work since then has been done on its significance in animal

systems. Studies in animals, animal cell cultures, and artificial membranes have shown

that tocopherols scavenge or quench various reactive oxygen species and lipid oxidation

by-products that would otherwise propagate lipid peroxidation chain reactions in

membranes (Kamal-Eldin and Appelqvist, 1996). Upon interaction with a prooxidant, α-

tocopherol can undergo one-electron transfer and form a relatively stable tocopheroxyl

cation radical, which is then recycled back to α-tocopherol by an antioxidant network

consisting of ascorbate, glutathione, and NADPH/NADH (see review by Packer et al.,

2001). α-Tocopherol can also undergo two-electron transfer and form α-

tocopherylquinone. In higher plants, this antioxidant function of α-tocopherol has been

discussed in connection with protection against oxidative stress caused by various

environmental factors (Munné-Bosch and Leonor, 2000). In the eukaryotic green algae

Chlamydomonas reinhardtii, the herbicide-mediated interruption of the α-tocopherol

biosynthetic pathway rendered PS II more susceptible to oxidative stress induced by an

13

extreme high light illumination (Trebst et al., 2002). Under these conditions, the D1

protein was rapidly degraded.

In addition to these antioxidant functions, other “non-antioxidant” functions

related to modulation of signaling and transcriptional regulation in mammals have also

been reported (Chan et al., 2001; Azzi et al., 2002; Ricciarelli et al., 2002). For example,

α-tocopherol has been shown to bind to phospholipase A2 specifically at the substrate-

binding pocket and to act as a competitive inhibitor, which thereby decreased the release

of arachidonic acid for eicosanoid synthesis (Chandra et al., 2002). α-Tocopherol has

also been suggested to modulate the phosphorylation state of protein kinase Cα in rat

smooth-muscle cells, possibly via phosphorylation of protein phosphatase 2A (Ricciarelli

et al., 1998). α-Tocopherol is also directly involved in transcriptional regulation in

animals, including the expression of the genes encoding liver collagen αI, α-tocopherol

transfer protein, and α-tropomyosin collagenase (Yamaguchi et al., 2001; Azzi et al.,

2002). Whether α-tocopherol in cyanobacteria performs one or both of these roles has not

yet been demonstrated.

Cyanobacterial Quinomics

The goal of this study was to develop experimental systems in which the type and

amounts of the quinone species found in cyanobacteria could be manipulated in vivo.

Such systems would allow the functions of the complexes in which they occur to be

studied. This has been accomplished by using comparative genome analyses, combined

with a reverse genetic approach, to predict and verify the biosynthetic pathways of PhyQ,

14

α-tocopherol, and PQ-9 (Chapter 2, 3, 6). Consequences of altering the nature of these

quinone species are studied in the context of PS I function (Chapter 3), stress responses

(Chapter 4), and gene regulation (Chapter 5). To express the relatively wide scope of and

inclusiveness of these studies, I introduce the term “quinomics” to describe my

investigations of the cyanobacterial “quinome”.

15

ABBREVIATIONS

BQ benzoquinone

Chl a chlorophyll a

DHNA 1,4-dihydroxy-2-naphthoate

MPBQ 2-methyl-6-phytyl-1,4-benzoquinone

MSBQ 2-methyl-6-solanyl-1,4-benzoquinone

NQ naphthoquinone

PhyQ phylloquinone

PQ-9 plastoquinone-9

PQH2 plastoquinol

PS I Photosystem I

PS II Photosystem II

RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase

16

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27

Fig 1.1: Structures of phylloquinone (PhyQ), plastoquinone-9 (PQ-9), and

α-tocopherol.

28

Fig 1.2: Scheme for oxygenic photosynthesis in cyanobacteria.

ATPase, ATP synthase; COX, cytochrome c oxidase; Cyt b6f, the cytochrome b6 f complex; Cyt c6, cytochrome c6; Fd, ferredoxin; Fl, flavodoxin; FNR, ferredoxin:NADP+ oxidoreductase; NDH, NADH dehydrogenase; PBS, phycobilisome; PC, plastocyanin; PS I, Photosystem I; PS II, Photosystem II.

29

Fig 1.3: The spatial arrangement and redox potentials of the electron transfer

cofactors in Photosystem I.

The soluble electron donor cytochrome c6 (Cytc6) and electron acceptor ferredoxin (Fd) are also shown.

30

Fig 1.4: The electron transfer cofactors in Photosystem II.

(A) Illustration of electron transfer between the cofactors, (B) the spatial arrangement of the cofactors obtained from the 3.5-Å resolution X-ray crystal structure, adapted from Ferreira KN, Iverson TM, Maghlaoui K, Barber J, Iwata S (2004) Science 303: 1831-1838.

A

B

31

Fig1.5: Biosynthetic pathway of PQ-9 and α-tocopherol in higher plants.

BQ, benzoquinone; HDDP, 4-hydroxyphenylpyruvate dioxygenase; HTP, homogentisate phytyltransferase; MSBQ/MPBQ MT, 2-methyl-6-solanyl-1,4-benzoquinone/2-methyl-6-phytyl-1,4-benzoquinone methyltransferase; SAM, S-adenosyl-L-methionine; TC, tocopherol cyclase; γ-TMT, γ-tocopherol methyltransferase.

32

Chapter 2

Comparative Genome Analysis and Identification of Menaquinone-4 Biosynthetic

Pathways in Cyanobacteria

Publications:

Yumiko Sakuragi, Boris Zybailov, Gaozhong Shen, Ramakrishnan Balasubramanian,

Bruce A. Diner, Irina Karygina, Yulia Pushkar, Dietmar Stehlik, Donald A. Bryant and

John H. Golbeck, Recruitment of a foreign quinone into the A1 site of Photosystem I.

Spectroscopic characterization of a menB rubA double deletion mutant in Synechococcus

sp. PCC 7002 containing plastoquinone-9 but devoid of FX, FB, FA, in preparation

Yumiko Sakuragi and Donald A. Bryant, a review article ‘Genetic Manipulation of the

Quinone Pathway in Photosystem I’ in Photosystem I: the Plastocyanin:Ferredoxin

Oxidoreductase in Photosynthesis’, Golbeck JH (ed), in preparation

33

ABSTRACT

Phylloquinone (PhyQ, 2-methyl-3-phytyl-1,4-naphthoquinone) is an electron transfer

cofactor at the A1 site of Photosystem I and is synthesized only in oxygenic phototrophs

such as cyanobacteria, algae, and higher plants. The biosynthetic pathway of PhyQ was

investigated by means of comparative genomics using the menaquinone (MQ)

biosynthesis genes of Escherichia coli as queries. The homologs of menA, menB, menC,

menD, menE, menF, menG and menH were found to be conserved in most of the

cyanobacteria. In some of these organisms, these genes formed clusters similar to those

found in Escherichia coli, Bacillus subtilis, and Chlorobium tepidum, suggesting that the

PhyQ biosynthetic pathway has evolved from that for MQ. In support of this hypothesis it

was demonstrated that Gloeobacter violaceus and Synechococcus sp. PCC 7002 wild-

type strains synthesize MQ-4 (2-methyl-3-geranylgeranyl-1,4-naphthoquinone) that

functions at the A1 site in Photosystem I complexes. Targeted insertional mutagenesis of

menB, menF, and menG in Synechococcus sp. PCC 7002 results in the complete

interruption of MQ-4 biosynthesis and demonstrates that the predicted pathway indeed

functions in this cyanobacterium. Phylogenetic analysis of MenA (dihydroxynaphthoate

phytyltransferase) showed that the MenA sequences of the plant Arabidopsis thaliana

and Oryza sativa group with cyanobacterial sequences, whereas MenB

(dihydroxynaphthoate-CoA synthase) and MenD (2-succinyl-6-hydroxy-2,4-

cyclohexadiene-1-carboxylate synthase) do not. This leads to the hypothesis that PhyQ

biosynthesis is a result of mosaic evolution derived from a non-cyanobacterial MQ

34

pathway together with a cyanobacterial-type MenA, possibly derived from

endosymbiosis.

ABBREVIATIONS

Chl a chlorophyll a

DHNA dihydroxynaphthoate

HPLC high-performance liquid chromatography

MQ-4 menaquinone-4

PCR polymerase chain reaction

PhyQ phylloquinone

PQ plastoquinone-9

PS I Photosystem I

35

INTRODUCTION

Phylloquinone (PhyQ, 2-methyl-3-phytyl-1,4-naphthoquinone), also known as

vitamin K1, is synthesized only in oxygenic phototrophs such as cyanobacteria, algae, and

higher plants. PhyQ plays a crucial role in photosynthesis as a cofactor in Photosystem I

by mediating electron transfer between the primary acceptor A0 (Chl a) and the terminal

acceptor Fx (4Fe-4S cluster) after photoexcitation of the primary donor P700 (Golbeck,

1994, 2003; Brettel and Leibl, 2001). Two molecules of PhyQ are found within the

protein environments of the PS I complex, which is composed of 12 protein subunits, 96

Chl a molecules, 22 β-carotenes, 3 [4Fe-4S] clusters, and 4-5 structural lipids (Jordan et

al., 2001). PS I is a light-driven cytochrome c6/plastocyanin:ferredoxin/flavodoxin

oxidoreductase and is responsible for providing electrons that are ultimately used for the

generation of NADPH, the reducing power for cellular anabolism. The quantum

efficiency of this enzyme is largely dependent on the stability of a charge-separated state

between P700+ and FX-, and the presence of PhyQ ensures an optimal rate of transfer

between these cofactors.

Biosynthesis of PhyQ has been studied by isotope tracer and direct enzymatic

activity assays in higher plants (see a review by Pennock and Threlfall, 1981). The results

have indicated that PhyQ biosynthesis resembles menaquinone (MQ) biosynthesis in E.

coli (see reviews by Meganathan 1996, 2001). MQ biosynthesis occurs through eight

enzymatic steps as summarized in Fig 2.1. The first committed step is the isomerization

of chorismate to isochorismate by isochorismate synthase (MenF), followed by the

36

condensation of isochorismate and thiamine-pyrophosphate-succinic semialdehyde by 2-

succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase (MenD).

Dehydration of SHCHC is catalyzed by o-succinylbenzoate (OSB) synthase (MenC) and

the subsequent CoA thioesterification by OSB-CoA synthetase (MenE); the ring

cyclization reaction is catalyzed by 1,4-hydroxynaphthoyl-CoA synthase (MenB), and

deesterification by 1,4-hydroxynaphthoyl-CoA thioesterase (MenH) results in 1,4-

dihydroxy-2-naphthoate (DHNA). DHNA polyprenyltransferase (MenA) catalyzes the

condensation of DHNA and polyprenyl diphosphate, yielding 2-polyprenyl-1,4-

naphthoquinone (demethylmenaquinone). The final methylation at the C2 position is

carried out by 2-polyprenyl-1,4-naphthoquinone methyltransferase (MenG) with S-

adenosyl-methinone as a methyl donor, resulting in the production of MQ. PhyQ and MQ

have the same general molecular structure except that PhyQ has a partially saturated, C20

phytyl substituent and MQ found in other bacteria has an unsaturated polyprenyl

substituent typically consisting of 30 to 50 carbon units (Collins and Jones, 1981).

The targeted inactivation of the menA, menB, menD, and menE homologs in

Synechocystis sp. PCC 6803 was shown to result in the complete interruption of PhyQ

biosynthesis. This demonstrated that a MQ-like pathway is responsible for PhyQ

biosynthesis in this organism (Johnson et al., 2000; Johnson et al., 2002). The following

questions remained to be answered: is this MQ-like PhyQ biosynthesis pathway

ubiquitous among cyanobacteria and higher plants; what is its evolutionary origin; how

has it evolved among the various oxygenic phototrophs? In this study, MQ biosynthesis

was investigated in 14 cyanobacteria whose genomes have been or are currently being

37

sequenced. The results indicate that the MQ-like pathway is conserved among most,

although not all, of the cyanobacteria and higher plants. A detailed analysis of the gene

arrangements and phylogenetic reconstruction lead to the hypothesis that the PhyQ

biosynthetic pathway has evolved from the MQ pathway in both cyanobacteria and

higher plants.

38

RESULTS

As of April 26, 2004, complete genomic sequences of 8 cyanobacteria are

available: Synechocystis sp. PCC 6803 (Kaneko et al., 1996), Nostoc PCC 7120 (Kaneko

et al., 2001), Thermosynechococcus elongatus BP-1 (Nakamura et al., 2002ab),

Gloeobacter violaceus (Nakamura Y et al., 2003), Prochlorococcus marinus MED4 and

Prochlorococcus marinus MIT9313 (Rocap et al., 2003), Prochlorococcus marinus

SS120 (Dufresne et al., 2003), Synechococcus sp. WH8102 (Palenik et al., 2003); and

incomplete sequences exist for Synechococcus sp. PCC 7002 (Jürgen Marquardt, Tao Li,

Jindong Zhao, and Donald A. Bryant, unpublished), Nostoc punctiforme (Gene Bank

accession number: NZ_AAAY00000000), Trychodesmium erythraeum (Gene Bank

accession number: NZ_AABK00000000), Synechococcus elongatus sp. PCC 7942 (Gene

Bank accession number: NZ_AADZ01000001), Anabaena variabilis ATCC 29413 (Gene

Bank accession number: NZ_AAEA01000001), and Crocosphaera watsonii WH 8501

(Gene Bank accession number: NZ_AADV01000004). Similarity searches with the

MenA through MenH protein sequences of E. coli as queries were performed against

each genome, and the results are summarized in Table 2.1. Of 14 cyanobacteria

examined, 11 were shown to possess the complete set of 8 Men protein homologs.

Similarities based on amino acid sequences were typically in the range of 30-70%,

although the MenB sequences were very highly conserved among a wide range of

organisms including cyanobacteria, the γ-proteobacterium E. coli, the green sulfur

bacterium Chlorobium tepidum, the Gram-positive bacterium Bacillus subtilis, and the

archeon Halobacterium sp. NRC-1 (similarity >70%). In the genome of G. violaceus,

39

homologs of only MenA and MenG were detected. When the Synechocystis sp. PCC

6803 MenB sequence was used to search the G. violaceus genome database, the best hit

was Gll2549 with only 38 % similarity. Further database searching has revealed that

Gll2549 is highly similar to 6-oxo camphor hydrolase in Rhodococcus sp. NCIMB 9784

(69%). When Synechocystis sp. PCC 6803 MenC, MenD, MenE, and MenF sequences

were used to search the G. violaceus database, the best hits were Gll3099 (35%), Gll2804

(44%), Glr1146 (45%), and Gll0757 (43%), respectively. These ORFs also showed

higher similarities to other hypothetical proteins: Gll3099 is similar to chloromuconate

cycloisomerase in Clostridium acetobutylicum (57%); Gll2804 is similar to acetolactate

synthase in Synechocystis sp. PCC 6803 (Sll1981, 86%); Glr1146 is similar to a long-

chain fatty-acid CoA ligase in Bacillus cereus (54%); and Gll0757 is similar to

anthranilate synthase component I in Synechocystis sp. PCC 6803 (Slr1979, 66%). No

homologs of MenD (A. variabilis) or MenC and MenF (C. watsonii) have been identified

to date, but these genomes are not yet completely sequenced.

Some of the men genes involved in PhyQ biosynthesis are found to form clusters

in several cyanobacterial genomes. A cluster composed of menF, menA, menC, and menE

is conserved in T. erythraeum, N. punctiforme, Nostoc sp. PCC 7120, Synechococcus sp.

WH 8102, and all three Prochlorococcus species (Fig 2.2). In a separate region of the

genome, menD and menB are also found to form a cluster. The observed gene

arrangements in these cyanobacteria are similar to parts of the gene clusters found in E.

coli, C. tepidum, B. subtilis and Halobacterium sp. NRC-1 (Fig 2.2). Combined with the

conservation in the amino acid sequences among Men proteins, the conserved

40

arrangements of the men genes are strong indications that PhyQ biosynthesis in

cyanobacteria evolved from the MQ pathway of other bacteria.

Given that oxygenic photosynthesis is believed to have originated in

cyanobacteria and that PhyQ is associated exclusively with PS I, one may expect that the

common ancestor of higher plants and algae acquired the entire PhyQ biosynthetic

pathway from cyanobacteria upon endosymbiosis. Interestingly, the men gene

arrangement in Arabidopsis thaliana does not resemble that in cyanobacteria (Fig 2.2).

Lack of conserved gene arrangements does not necessarily indicate the absence of an

evolutionary relationship; however, it is sufficient to cast doubt about it. Thus the

evolutionary relatedness between cyanobacterial and higher plant PhyQ biosynthetic

genes was evaluated further. As shown in Table 2.1, A. thaliana contains the whole set of

men genes in its nuclear chromosomes, which indicates that PhyQ biosynthesis in A.

thaliana is similar to that in cyanobacteria and to MQ synthesis in other prokaryotes. In a

phylogenetic tree based on the MenA sequences, the two higher plants, A. thaliana and

Oryza sativa, form a clade within a domain composed entirely of cyanobacteria, which

suggests that MenA in higher plants is evolutionarily more related to MenA in

cyanobacteria than to MenA of other eubacteria (Fig 2.3A). On the contrary, in trees

based on the MenB and MenD sequences, these two higher plants form a clade outside

the cyanobacterial domain. This suggests that MenB and MenD in higher plants are more

closely related to their homologs in other eubacteria than to those in cyanobacteria (Fig

2.3B and C). Trees based on the MenC, MenE, and MenF sequences showed the topology

similar to those observed for the MenB and MenD sequences; however, the detailed

branching orders within the cyanobacterial domain were variable probably due to low

sequence conservation of these proteins (< ~50%). The observed variation in the

41

phylogenetic relationships therefore suggests that not all men genes in higher plants

originated from cyanobacteria.

By comparative genome analyses and phylogenetic reconstructions, it was so far

hypothesized that PhyQ biosynthesis in cyanobacteria and higher plants evolved from

that of MQ, which suggests that early cyanobacteria synthesized and utilized MQ for the

PS I complexes. This hypothesis was supported by the discovery that G. violaceus and

Synechococcus sp. PCC 7002 synthesize MQ-4 instead of PhyQ. Fig 2.4A shows the

HPLC profile of the solvent extract obtained from the PS I complexes isolated from

Synechocystis sp. PCC 6803 wild type. PhyQ, which typically elutes at 22.4 min, was

shown to be present. In the HPLC profiles of the solvent extracts obtained from the PS I

complexes isolated from Synechococcus sp. PCC 7002 wild type and from the G.

violaceus whole cells, no such peak was present as judged by the absence of UV-

absorbing compounds at this retention time (Fig 2.4B and C). Further examination of the

chromatograms led to the discovery of a new peak at ca. 14 min that had an intense UV-

absorption with maxima at 248, 263, 270, and 332 nm (Fig 2.5B and C), which coincide

well with the absorption spectrum of phylloquinone obtained from Synechocystis sp. PCC

6803 (Fig 2.5A). This spectrum is characteristic of a 1,4-naphthoquinoid compound with

two alkyl substitutions at the C2 and C3 positions (Dunphy and Brodie, 1971). The

absence of PhyQ in G. violaceus was further confirmed by the absence of the PhyQ

absorption in the eluate at 22.5 min (Fig 2.5C). The same results were obtained for

Synechococcus sp. PCC 7002. Mass spectrometric analysis of the solvent extracts of PS I

complex isolated from Synechococcus sp. PCC 7002 showed that the compound eluting

at ca. 14 min has a m/z of 444, as opposed to a m/z of 450 for PhyQ (Figure 2.6). This

difference is best explained by the presence of a geranylgeranyl substituent with four

unsaturated isoprenoid units. Thus, Synechococcus sp. PCC 7002 synthesizes MQ-4

42

instead of PhyQ. The absence of PhyQ was confirmed by the absence of any component

with a m/z of 450 at 22.5 min (Fig 2.6). The solvent extracts from the PS I complexes

isolated from Synechococcus sp. PCC 7002 revealed the presence of 2.3 MQ-4 molecules

per 100 Chl a molecules, demonstrating that MQ-4 plays a role as the A1 cofactor in the

PS I complexes in this cyanobacterium. Similar quantitative analysis for G. violaceus has

not yet performed.

Targeted insertional inactivation of menB, menF, and menG in Synechococcus sp.

PCC 7002 was performed to verify the involvement of these gene products in the MQ-4

biosynthesis (Fig 2.7). The menF and menG mutations were introduced into the wild

type, whereas the menB mutation was introduced into the rubA mutant for the purpose of

generating PS I complexes that contain plastoquinone-9 but lack iron-sulfur clusters for

future studies concerning electron transfer kinetics and thermodynamics (Y. Sakuragi, B.

Zybailov, G. Shen, R. Balasubramanian, B. A. Diner, I. Karygina, Y. Pushkar, D. Stehlik,

D. A. Bryant and J. H. Golbeck, manuscript in preparation). The full segregation of the

mutated alleles from the respective wild-type alleles was analyzed by PCR as shown in

Fig 2.8. In the rubA mutant, PCR using the designed primers that are targeted to the

menB region resulted in a product of 1.0 kb, which is expected based on the restriction

map, as shown in Fig 2.7. Likewise, in the wild type, PCR using the designed primers

that are targeted to the menF and menG regions resulted in products of 1.7 kb, and 1.8 kb,

respectively. No products with corresponding sizes were detected for the mutants; instead

products with larger sizes were detected. In the menB rubA and the menF mutants, the

size of the products were ca. 2.1 kb and 2.8 kb, respectively. The difference between the

PCR products from the wild type and the mutants corresponds to the size of the 1.1-kb

gentamicin-resistance cartridge derived from pMS266. In the menG mutant a product of

2.2 kb was observed. The difference between the PCR products from the wild type and

43

the menG mutant is 0.3 kb, which corresponds to the difference between the 1.1-kb

gentamicin-resistance cartridge derived from pMS266 and the 0.8-kb deletion in the N-

terminal region (Fig 2.7C). These results show that the menB rubA double mutant and

menF and menG single mutants are free of the respective wild-type alleles and therefore

homozygous in the menB::GmR, menF::GmR , and menGB::GmR alleles, respectively.

When the pigment extracts from the PS I complexes isolated from the menB rubA

and the menG mutants were analyzed, no peak with UV-absorption was detected at ca. 14

min, demonstrating that the menB and menG homologs are indeed required for MQ-4

synthesis in Synechococcus sp. PCC 7002 (Fig 2.9). In extracts of PS I complexes from

the menF mutant, a small peak was detected at this retention time; however, the

absorption properties of the component eluted at this retention time were significantly

different from those of MQ-4 with an absorption maximum at 292 nm (Fig 2.10B). This

component is virtually absent in the wild type and its chemical nature has not yet been

determined. These results confirm that the interruption of the menF gene also results in

the complete loss of MQ-4 and that MenF is required for MQ-4 synthesis in

Synechococcus sp. PCC 7002. In the menF and the menB rubA mutants, a peak at 35.1

min was detected, which is absent in the extracts of PS I complexes in the wild type (Fig

2.9). The component eluted at this retention time showed absorption properties with a

single peak at 256 nm (Fig 2.10C), which is characteristic of PQ-9 (Crane and Dilley,

1963). Mass spectrometric analysis of the component has shown a m/z of 748, which is

characteristic of PQ-9. These results are in good agreement with a previous study in

which the interruption of PhQ biosynthesis in Synechocystis sp. PCC 6803 resulted in the

incorporation of PQ-9 in PS I (Johnson et al., 2000). Likewise, no MQ was detectable in

the chromatogram of PS I complexes isolated from the menG mutant (Fig 2.9D). A new

peak was detected at 11.9 min, which showed a strong UV-absorption with a maximum at

44

248 nm and a shoulder at 265 nm. Because the loss of a methyl group is expected to

lower the hydrophobicity and size of a molecule, it was expected that

demethylphylloquinone would elute earlier than MQ-4 in the reverse phase HPLC

analysis. Therefore, it is highly plausible that the component eluting at 11.9 min is due to

demethylmenaquinone. Combined with the sequence similarities to E. coli counterparts,

these results confirm that menB, menG, and menF encode dihydroxynaphthoic acid-CoA

synthase, 2-geranylgeranyl-1,4-naphthoquinone methyltransferase, and isochorismate

synthase, respectively, in Synechococcus sp. PCC 7002. Together with the previous

identification of menA, menB, menD, menE, and menG in Synechocystis sp. PCC 6803, it

is concluded that PhyQ/MQ-4 biosynthesis in cyanobacteria requires MenF, MenD,

MenE, MenB, MenA, and MenG.

45

DISCUSSION

Comparative genome analyses were conducted on 14 cyanobacteria, E. coli, B.

subtilis, Halobacterium sp. NRC-1 and A. thaliana. Based on the results, it was

concluded that PhyQ biosynthesis in cyanobacteria is similar to the MQ biosynthesis

pathway in E. coli. Supporting this hypothesis is the discovery that G. violaceus and

Synechococcus sp. PCC 7002 synthesize MQ-4 instead of PhyQ and that Synechococcus

sp. PCC 7002 utilizes it as the A1 cofactor in PS I complexes. G. violaceus is thought to

be one of the earliest branching lineages among the cyanobacteria (Nelissen et al., 1995).

The fact that this organism synthesizes MQ-4 is a strong indication that PhyQ

biosynthesis probably evolved from MQ biosynthesis pathway in other bacteria. It is

therefore highly plausible that the early cyanobacteria synthesized MQ-4, and PhyQ

biosynthesis evolved from the MQ-4 biosynthesis in cyanobacteria. This is consistent

with the fact that the occurrence of PhyQ biosynthesis is restricted to cyanobacteria and

to algae and higher plants, which are evolutionarily related to cyanobacteria. A recent

study has shown that the red alga Cyanidium caldarium also synthesizes MQ-4 and

utilizes it in its PS I complexes (Yoshida et al., 2004). Therefore, MQ-4 synthesis may be

a widely occurring phenomenon among oxygenic phototrophs.

Based on phylogenetic reconstructions, it was concluded that not all men genes in

higher plants are of cyanobacterial origin. It is believed that plastids, and hence the ability

to perform oxygenic photosynthesis, in eukaryotic phototrophs were acquired from a

cyanobacterium upon endosymbiosis (Margulis, 1970). Therefore, the presence of a gene

46

with strong similarity to the cyanobacterial menA in the plant nuclear chromosome is

probably a result of gene transfer from the plastids. This gene is presumably derived from

the endosymbiotic cyanobacterium and would have been transferred to a host nuclear

chromosome during endosymbiotic interaction (Douglas, 1998; Delwiche and Palmer,

1997; Martin et al., 1998). On the other hand, the presence of the non-cyanobacterial-type

menB and menD in the plant nuclear chromosome suggests they originated in other

organisms. One possibility is that menB and menD were already present in the eukaryotic

host cells before the endosymbiotic cyanobacterium was incorporated into them. It is

believed that mitochondria in eukaryotic cells originated from a proteobacterium,

possibly in the α-subdivision (Olsen et al., 1994; Andersson et al., 1998; Martin and

Müller, 1998; Emelyanov 2001; Horner et al., 2001; Emelyanov, 2003), through an

endosymbiosis that preceded the acquisition of plastids. Given that some, although not

all, proteobacteria synthesize both ubiquinone and MQ (Collins and Jones, 1981), it is

possible that the endosymbiotic proteobacterium brought the ability to synthesize MQ

into the eukaryotic host cells. Another possibility is that menB and menD genes were

acquired via horizontal gene transfer from bacteria to plant cells. Interestingly, the men

genes in the plastid genomes of the red algae Cyanidium caldarium and Cyanidioschyzon

merolae form a cluster (menD-menF-menB-menA-menC-menE), which is very similar to

the cluster found in a variety of prokaryotes (Fig 2.2). Phylogenetic analyses show that

neither the MenA nor the MenB sequences of the red algae Cyanidioschzon merolae

forms a cluster with the cyanobacterial sequences (Fig 2.3). These observations therefore

support the occurrence of horizontal gene transfer from bacteria other than cyanobacteria

to these eukaryotic phototrophs.

47

Targeted mutagenesis of the menB, menF, and menG homologs in Synechococcus

sp. PCC 7002, encoding for DHNA synthase, isochorismate synthase, and 2-methyl-1.4-

naphthoquinone methyltransferase, respectively, resulted in the expected interruption of

PhyQ biosynthesis. This confirms that the pathway responsible for MQ-4 synthesis in

Synechococcus sp. PCC 7002 is similar to that in other bacteria.

PhyQ mediates electron transfer between A0 and FX of PS I during the charge

separation process upon photoexcitation of P700. The optimal rate of electron transfer

from A0 to FX is ensured by the thermodynamically downhill arrangement of these

cofactors. The molecular structures of PhyQ and MQ-4 are identical except for the level

of saturation of the C3 polyprenyl side chain, and thus the redox potentials of the two

quinones are also expected to be identical. Therefore, it is not surprising that MQ-4 can

function at the A1 site in the PS I complexes. The prenylation reaction of DHNA in

Synechococcus sp. PCC 7002, G. violaceus, and C. caldarium is likely to be different

from that in PhyQ-synthesizing species either by i) higher substrate specificity of the

MenA protein for geranylgeranyl diphosphate compared to phytyl diphosphate; and/or ii)

substantially higher intracellular concentration of geranylgeranyl diphosphate than phytyl

diphosphate in these cells.

The whole-genome analysis of G. violaceus seems to suggest that menB, menC,

menD, menE, and menF homologs are absent in this organism. ORFs that show similarity

to the proteins encoded by these men genes are more related to other proteins that do not

play roles in MQ-4 biosynthesis. One example is the MenB-like protein Gll2549, which

shows high similarity to 6-oxo camphor hydrolase. This enzyme is a member of the

48

crotonase superfamily that catalyzes the cleavage of carbon-carbon bonds. Therefore, it is

possible that Gll2549 participates in the PhyQ pathway and catalyzes the ring cyclization

reaction of o-succinylbenzoate. However, MenB is a very highly conserved enzyme

among a wide variety of organisms (Table 2.1), and the absence of its homolog together

with four other Men proteins in G. violaceus suggests the presence of an alternative

pathway that can lead to the synthesis of DHNA. Alternatively, it was also possible that

G. violaceus does not synthesize PhyQ, MQ-4 or related compounds at all. Previous

studies have shown that PhyQ-less mutants recruit PQ into the A1 site of PS I, and,

despite a slightly lower catalytic efficiency, as measured by the flavodoxin reduction rate,

the cells are able to grow photoautotrophically (Johnson et al., 2000). However, HPLC

analyses performed in this study demonstrated that MQ-4 is present in the whole cell

extracts of G. violaceus (see above). This result largely rules out the possibility of PQ-9

dependent PS I function in this organism. It also suggests that a part of the MQ-4

biosynthetic pathway in this organism is very different from those in the rest of the

oxygenic phototrophs analyzed in this study.

In this study, a comparative genomic approach was shown to be useful in

predicting the entire biosynthetic pathway for a secondary metabolite. Combined with

reverse genetics, one can now genetically modify the quinone species in PS I in

cyanobacteria. The advantage of this approach is that one can study the biochemical and

biophysical properties of PS I, as well as the physiology of the cells, by using a

biologically intact system. Further examples are provided in the following chapters.

49

SUMMARY

Synechococcus sp. PCC 7002 and G. violaceus were found to synthesize MQ-4

instead of PhyQ. Targeted inactivation of the menF, menB, and menG genes in

Synechococcus sp. PCC 7002 demonstrated that these genes are required for MQ-4

biosynthesis. The PS I complexes isolated from the menB rubA and menF mutants

contained PQ-9, while the PS I complexes isolated from the menG mutant contained an

UV-absorbing compound, which may be demethyl-MQ-4. Comparative genome analyses

of 14 cyanobacteria and four eukaryotic phototrophs suggested that PhyQ biosynthesis in

cyanobacteria evolved from MQ biosynthesis in bacteria and that this pathway in higher

plants may have developed as a result of mosaic evolution.

50

MATERIALS AND METHODS

Comparative Genome Analyses

The Men protein sequences of E. coli were used as queries to detect the presence

of homologs in cyanobacteria using the Blast-P search method. Retrieved sequences were

named after their locus tags in the genomes. Sequence similarities between the homologs

were obtained based on pairwise alignment by BLOSUM30 using ClustalW. Multiple

amino acid sequence alignments were also generated by BLOSUM30 using ClustalW.

For the phylogenetic reconstructions based on the MenA, MenB, and MenD sequences,

unambiguous regions in the alignments were removed. The resulting “trimmed” multiple

alignments were subjected to phylogenetic reconstruction by the Parsimony method using

PAUP*4.0 beta. The bootstrap values are results of 10,000 repetitions.

Generation of Mutant and Verification of Segregation

Wild type and mutant strains of Synechococcus sp. PCC 7002 were grown as

described previously (Frigaard et al., 2004). A DNA fragment containing the menB

coding region was amplified from the isolated genomic DNA by PCR using the primer

B1 (5’-CGTCGGGACACATCTTTCAC-3’) and the primer B2 (5’-

CGGTATCTACTCTACCAAATTGGG-3’). The resulting fragment was digested with

HindIII and BamHI, and inserted into the HindIII-BamHI sites of pUC19 (pUC19menB).

A fragment containing the menF coding region was amplified by PCR using the primer

F1 (5’-TTTGGGCAGCATCCCGGGTAAGCTGG-3’) that contains the engineered KpnI

cleavage site and the primer F2 (5’-GGAACCTATGAAGTTAGTCTGCGTTTCG-3’).

The resulting fragment was digested with KpnI and HindIII, and inserted into the KpnI-

51

HindIII fragment of the pUC19 cloning vector (pUC19menF). A fragment containing the

menG coding region was amplified by PCR using the primer G3 (5’-

GATGGTATTGTCGTCAACACCGC-3’) and the primer G4 (5’-

CACCGCCGCTATGGTACCAGGCG-3’). The resulting fragment was digested

(pUC19menG). The accC1 gene, derived from the plasmid pMS266 and conferring

gentamycin resistance, was inserted into the unique PstI site and SmaI cleavage sites in

the plasmids pUC19menB and pUC19menF, respectively. The accC1 gene was inserted

into the EcoRV sites of pUC19menG, resulting in a deletion of 740 bp encompassing the

N-terminal and the upstream region of the menG gene. The menB::accC1 construct was

used to transform the rubA mutant of Synechococcus sp. PCC 7002 as previously

described (Shen et al., 2002). The menF::accC1 construct was used to transform a psaB-

strain. After achieving complete segregation of the menF::accC1 locus, the wild type

psaB gene was introduced into the menF::accC1 psaB mutant to generate the

menF::accC1 single mutant by homologous recombination at the psaB locus. The

menG::accC1 construct was introduced into the wild-type strain. The full segregation of

each allele was tested by PCR using the primers B1 and B2 for the menB::accC1

transformants, the primers F1 and F2 for the menF::accC1 transformant, and the primers

G1 (5’- CGTTTCAATTTCCCAGGCAAAGTC-3’) and G2 (5’-

CCGAGGAAGTAAACCGCATATTC-3) for the menG::accC1 transformant.

52

Isolation of Thylakoid Membranes and PS I Particles

Thylakoid membranes were isolated from cells in the late-exponential growth

phase as described previously (Shen et al., 1993). Cells were broken at 4° C by three

passages through a French pressure cell at 124 MPa. The thylakoid membranes were

resuspended and solubilized for 2 h at 4° C in the presence of 1% (w/v) n-dodecyl-β-D-

maltoside. PS I complexes were separated from other membrane components by

centrifugation on 5-20% (w/v) sucrose gradients with 0.05% n-dodecyl-β-D-maltoside in

the buffer. Further purification was achieved by a second centrifugation on sucrose

gradients in the buffer in the absence of n-dodecyl-β-D-maltoside. Trimeric PS I

complexes were used in these studies (Golbeck, 1995).

Quinone and Chlorophyll Analysis

Isolated PS I was either lyophilized or dried under N2 gas before extraction with

acetone:methanol (7:2 v/v). Pigments and quinones were extracted with cold

acetone:methanol (7:2 v/v) after brief ultrasonication. Alternatively, 20 µL of the PS I

preparations were extracted with 400 µL of acetone:methanol. After centrifugation and

filtration through a PTFE filter membrane with a 0.2-µm pore size (Whatman

International Ltd., Maldstone, UK), the extract in the organic phase was directly injected

into an Agilent Technology 1100 series HPLC system (Agilent Technology, Palo Alto,

CA, USA) equipped with a SUPELCO (Sigma-Aldrich Corp., St. Louis, MO, USA)

Discovery® C18 column (25 cm x 4.6 mm, 5 µm). Analyses were carried out using the

following protocol: 80%A/20%B from 0 to10 min, a linear change to 20%A/80%B in 40

min, and 20%A/80%B for 5 min, where solvent A and solvent B are 100% methanol and

100% isopropanol, respectively. The flow rate was 0.75 ml min-1. Detection of eluates

was performed with a diode array detector (Agilent 1100 series). The chlorophyll a,

53

phylloquinone, and plastoquinone contents of each sample were determined based on

integrated peak areas and their molar absorption coefficients, which are 17.4 mM-1 cm-1

at 618 nm, 18.9 mM-1 cm-1 at 270 nm (Dunphy and Brodie, 1971), and 15.2 mM-1 cm-1 at

254 nm (Crane and Dilley, 1963), respectively. The absorption coefficient of chlorophyll

a at 618 nm was calculated from the ratio of the absorption peaks at 618 nm and 666 nm

in methanol-isopropanol (6:4, v/v) and from its absorption coefficient at 666 nm in

methanol (MacKinney, 1941). The peak assignments were confirmed by mass

spectrometry using atmospheric-pressure chemical ionization with a Quattro II time-of-

flight mass spectrometer (Micromass, Beverly, MA, USA) operated in the negative-ion

mode.

54

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1005

60

Tab

le 2

.1: W

hole

gen

ome

anal

yses

for

the

MQ

/Phy

Q b

iosy

nthe

sis e

nzym

es in

14

cyan

obac

teri

a, E

. col

i, B.

subt

ilis,

C.

tepi

dum

, and

Hal

obac

teriu

m sp

. NR

C-1

. Sim

ilarit

y to

Syn

echo

cyst

is sp

. PC

C 6

803

prot

eins

are

show

n in

par

enth

eses

(%).

The

sim

ilarit

y is

bas

ed p

airw

ise

alig

nmen

t of t

he e

ntire

sequ

ence

s con

stru

cted

by

BLO

SUM

30. (

Tabl

e co

ntin

ues o

n ne

xt p

age)

S.

680

3 S.

700

2 N

osto

c T.

ery

N

. pun

A.

var

C

. wat

S.

elo

T.

elo

G

. vio

Men

A

Slr1

518

b

(67%

) A

lr003

3 (7

0%)

Tery

1070

(7

3%)

Npu

n060

4 (7

0%)

Ava

r026

348

(68

%)

a Cw

at14

3901

(3

4%)

Selo

0206

47

(66%

) Tl

r219

6 (7

0%)

Gll1

578

(53%

)

Men

B

Sll1

127

b (8

9%)

All2

347

(91%

) Te

ry10

85

(83%

) N

pun2

498

(91%

) A

var4

1360

1 (9

1%)

Cw

at15

9701

(9

1%)

Selo

1873

01

(89%

) Tl

l245

8 (8

8%)

-

Men

C

Sll0

409

b (5

2%)

Alr0

034

(54%

) Te

ry10

67

(54%

) N

pun0

605

(52%

) A

var0

2638

2 (5

4%)

- Se

lo02

0304

(5

0%)

Tlr1

174

(55%

) -

Men

D

Sll0

603

b (6

2%)

Alr0

312

(61%

) Te

ry10

75

(61%

) N

pun2

496

(59%

) -

Cw

at13

1201

(6

1%)

Selo

0271

8 (6

1%)

Tll0

130

(56%

) -

Men

E Sl

rl049

2 b

(44%

) A

lr003

5 (5

6%)

Tery

1066

(5

6%)

Npu

n060

6 (5

4%)

Ava

r026

381

(52%

) C

wat

0260

26

(52%

) Se

lo02

305

(45%

) Tl

l122

1 (5

0%)

-

Men

F Sl

r081

7 b

(54%

) A

ll003

2 (5

8%)

Tery

al71

(5

7%)

Npu

n060

3 (6

0%)

Ava

r026

437

(58%

) -

Selo

0214

74

(53%

) Tl

l121

3 (5

4%)

-

Men

G

Slr1

653

b (7

0%)

Alr5

252

(72%

) Te

ry33

21

(63%

) N

pun3

758

(65%

) a A

var2

8710

1 (4

8%)

Cw

at17

9201

(6

8%)

Selo

0203

83

(66%

) Tl

l237

3 (6

3%)

Gll0

127

(53%

)

Men

H

Sll1

916

b (4

3%)

All0

111

(68%

) Te

ry19

86

(46%

) N

pun0

061

(55%

) A

var5

1370

1 (6

5%)

Cw

at02

4832

(4

5%)

Selo

0200

62

(52%

) Tl

r206

6 (4

3%)

-

S. 6

803,

Syn

echo

cyst

is sp

. PC

C 6

803;

S.7

002,

Syn

echo

cocc

us sp

. PC

C 7

002;

Nos

toc,

Nos

toc

sp. P

CC

712

0; N

. pun

, Nos

toc

punc

tifor

me;

T.e

lo, T

. elo

ngat

us

BP-

1; T

. ery

, Tri

chod

esm

ium

ery

thra

eum

; G.v

io, G

. vio

lace

us P

CC

742

1; A

.var

, Ana

baen

a va

riab

ilis A

TCC

294

13; S

. elo

, Syn

echo

cocc

us e

long

atus

sp.

7942

; C.w

at, C

roco

spha

era

wat

soni

i WH

8501

. a N-te

rmin

al se

quen

ce w

as tr

unca

ted.

b No

locu

s tag

s are

ava

ilabl

e.

61

Tabl

e 2.

1 co

ntin

ued

S. 8

102,

Syn

echo

cocc

us sp

. WH

8102

; P.M

ED4,

Pro

chlo

roco

ccus

mar

inus

MED

4; P

.MIT

, Pro

chlo

roco

ccus

mar

inus

MIT

9313

; P.S

S120

, Pro

chlo

roco

ccus

mar

inus

SS1

20; A

rabi

dops

is, A

rabi

dops

is t

halia

na; E

. col

i, Es

cher

ichi

a co

li; C

. tep

, Chl

orob

ium

tepi

dum

; B. s

ub, B

acill

us su

btili

s. a N

-term

inal

sequ

ence

was

trun

cate

d.

S.

810

2 P.

MED

4 P.

MIT

P.

SS12

0 Ar

abid

opsi

s E.

col

i C

. tep

B.

sub

Men

A

Synw

2307

(5

6%)

Pmm

0176

(5

8%)

Pmt2

059

(55%

) Pr

o020

1 (5

5%)

At1

g606

00

(55%

) B

3930

(4

7%)

CT1

1511

(4

4%)

Bsu

3849

0 (4

1%)

Men

B

Synw

0998

(8

0%)

Pmm

0608

(7

7%)

Pmt0

405

(79%

) Pr

o105

3 (7

9%)

At1

g605

50

(78%

) B

2262

(7

8%)

CT1

846

(77%

) B

su30

75

(81%

)

Men

C

Synw

2306

(3

9%)

Pmm

0175

(4

1%)

Pmt2

058

(43%

) Pr

o020

0 43

%)

At1

g689

00

(46%

) B

2261

(3

8%)

CT1

847

(35%

) B

su30

780

(35%

)

Men

D

Synw

0997

(4

6%)

Pmm

0607

(4

6%)

Pmt0

406

(47%

) Pr

o105

4 (4

6%)

At1

g688

90

(45%

) B

2264

(4

3%)

CT1

839

(44%

) B

su30

77

(49%

)

Men

E Sy

nw23

05

(35%

) Pm

m01

74

(34%

) Po

mt2

057

(36%

) Pr

o019

9 (3

5%)

At3

g489

90

(43%

) B

2260

(4

6%)

CT1

848

(39%

) B

su30

74

(39%

)

Men

F Sy

nw23

08

(42%

) Pm

m01

77

(48%

) Pm

t206

0 (4

7%)

Pro0

202

(47%

) A

t1g7

4710

(5

1%)

B22

65

(44%

) C

T183

8 (4

4%)

Bsu

3078

(4

7%)

Men

G

Synw

1674

(5

6%)

Pmm

0431

(5

1%)

Pmt0

276

(37%

) Pr

o042

7 (5

7%)

At1

g233

60

(50%

) B

3833

(4

7%)

CT0

462

(52%

) B

su22

750

(45%

)

Men

H

Synw

0681

(3

8%)

Pmm

1628

(3

5%)

Pmt0

128

(38%

) Pr

o179

0 (4

0%)

At5

g385

20

(43%

) B

2263

(3

5$)

CT1

845

(31%

) B

su30

76

(34%

)

62

Fig 2.1: Menaquinone biosynthetic pathway in E. coli.

DHNA, dihydroxynaphthoate; DMQ, demethylmenaquinone; OSB, o-succinylbenzoate; SHCHC, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate; TPP-SS, thiamine-pyrophosphate-succinic semialdehyde.

63

Fig 2.2: The men gene arrangements.

Nostoc, Nostoc sp. PCC 7120; Bsu, Bacillus subtilis; Ctep, Chlorobium tepidum; Eco, Escherichia coli; Hal, Halobacterium sp. NRC-1; Arabidopsis, A. thaliana; Cyme, Cyanidiochyzon merolae; Cyca, Cyanidium caldarium; Npun, Nostoc punctiforme; Pmm, Prochlorococcus marinus MED4; Pmt, P.marinus MIT9313; Synw, Synechococcus sp. WH8102; Tery, Trichodesmium erythraeum. P. marinus SS220 shows the same arrangement as Pmm.

64

Fig 2.3:Phylogenetic trees based on the amino acid sequences of (A) MenA, (B) MenB, and (C) MenD.

The multiple alignments were generated by BLOSUM30 using Clustal W, and the trees were constructed by the Parsimony method using PAUP*4.0 beta. Cyanobacteria, red algae and higher plants are indicated with black circles, black diamonds, and stars, respectively. The locus tags used to indicate organisms are summarized in Table 2.1, except for the following: Cymecp095, Cymecp092, Cymecp094 for MenA, MenB, and MenD in C. merolae, respectively; Oj1217B09.18 and P0671D01.18 for MenA and MenB in Oryza sativa, respectively. AF2036 and AF0435 are hypothetical proteins in the archeon Archeoglobus fulgidis DSM4304. Ml2270 is a MenD homolog in Mycobacterium leprae. Bootstrap values were obtained after 10,000 repetitions and those larger than 50 are shown.

65

Fig2.4: HPLC profiles of solvent extracts from the PS I complexes and whole cells. Chromatograms of reverse-phase HPLC analysis of solvent extracts obtained from (A) PS I complexes isolated from the wild-type Synechocystis sp. PCC 6803 (B) PS I complexes isolated from wild-type Synechococcus sp. PCC 7002, and (C) whole cells of the wild-type G. violaceus. Chromatograms were recorded at 270 nm. Chl a, chlorophyll a; β-car, β-carotene; MQ-4, menaquinone-4.

66

Fig 2.5: Absorption spectra of solvent-extracted components analyzed by HPLC.

Components eluting (A) at 22.5 min from the PS I complex of the wild-type Synechocystis sp. PCC 6803, (B) at 14.5 min from the PS I complex of the wild-type Synechococcus sp. PCC 7002, and (C) at 14.5 min from the whole cell of the wild-type G. violaceus (solid line) (see Fig 2.4). A component eluting at 22.5 min in G. violaceus is also shown by dotted line and does not appear to be a quinone.

67

Fig 2.6: LC-mass spectrometric analysis of the solvent extracts from Synechococcus sp. PCC 7002. (A) Chromatograms at the m/z of 444.3 (top) and 450.3 (bottom), (B) mass spectra at the retention time at 30 min (top) and 12.4 min (bottom). PQ-9 elutes at 30 min.

68

Fig 2.7: Restriction maps of the menB (A), menF (B), and menG (C) coding regions in Synechococcus sp. PCC 7002. Short black arrows show the primers used for verification of segregation by PCR.

69

Fig 2.8: PCR analyses on genomic DNAs isolated from the menB rubA, menF, and menG mutants of Synechococcus sp. PCC 7002. Primers used for the analyses are shown by the black arrows in Fig 2.7. Control amplifications using template DNA from the rubA and wild-type (WT) strains are also shown.

70

Fig 2.9: Reverse-phase HPLC analyses of the solvent extracts from PS I complexes. (A) wild type, (B) the menB rubA mutant, (C) the menF mutant, and (D) the menG mutant of Synechococcus sp. PCC 7002. The retention time for MQ-4 is indicated by the black arrows.

71

Fig 2.10: Absorption spectra of eluting components analyzed for PS I complexes isolated from Synechococcus sp. PCC 7002 using the reverse-phase HPLC analysis (see Fig 2.9). (A) MQ-4 at 13.5 min in wild type, (B) unidentified component eluting at 13.5 min in the menF mutant, (C) PQ-9 at 35.1 min in the menB rubA mutant, (D) unidentified component (possibly demethyl-MQ-4) eluting at 11.9 min in the menG mutant.

72

Chapter 3

Recruitment of a Foreign Quinone into the A1 Site of Photosystem I.

Interruption of menG in the Phylloquinone Biosynthetic Pathway of Synechocystis sp.

PCC 6803 Results in the Incorporation of 2-Phytyl-1,4-Naphthoquinone and

Alteration of the Equilibrium Constant between A1 and FX

Publication:

Yumiko Sakuragi, Boris Zybailov, Gaozhong Shen, Parag R. Chitnis, Art van der Est,

Robert Bittl, Stephan Zech, Dietmar Stehlik, John H. Golbeck and Donald A. Bryant.

(2002) Biochemistry 41: 394-405

73

ABSTRACT

A gene (menG), encoding a methyltransferase was identified in Synechocystis sp. PCC

6803 as responsible for transferring the methyl group to 2-phytyl-1,4-naphthoquinone

(demethylphylloquinone) in the biosynthetic pathway of phylloquinone, the secondary

electron acceptor in Photosystem I (PS I). Mass spectrometric measurements showed that

targeted inactivation of the menG gene prevents the synthesis of phylloquinone and leads

to the accumulation of 2-phytyl-1,4-naphthoquinone in PS I complexes. Growth rates of

the wild-type and the menG mutant strains under photoautotrophic and photomixotrophic

conditions were virtually identical. The chlorophyll a content in whole cells of the menG

mutant was similar to that in the wild type when the cells were grown at a light intensity of

50 µE m-2 s-1 but slightly lower when grown at 150 µE m-2 s-1. Low temperature chlorophyll

fluorescence emission measurements showed a larger increase in the ratio of PS II to PS I

in the menG mutant strain relative to the wild type as the light intensity was elevated from

50 µE m-2 s-1 to 300 µE m-2 s-1. CW EPR studies at 34 GHz and transient EPR studies at

multiple frequencies showed that the quinone radical in the menG mutant has the same

overall linewidth as in the wild type, but the pattern of hyperfine splittings showed two

lines in the low-field region consistent with the presence of an aromatic proton at ring

position 3. The spin polarization pattern indicated that 2-phytyl-1,4-naphthoquinone is in

the same orientation as phylloquinone, and out-of-phase, spin-echo modulation

spectroscopy showed the same P700+ to Q- center-to-center distance in the two samples.

Transient EPR studies indicated that forward electron transfer from Q- to FX is slowed from

74 290 ns in the wild-type to 600 ns in the menG mutant. The redox potential of

2-phytyl-1,4-naphthoquinone was estimated to be 50 to 60 mV more oxidizing than

phylloquinone in the A1 site, which translates to a lowering of the equilibrium constant

between Q-/Q and FX-/FX by a factor of ca. 10. The kinetics of the P700+ [FA/FB]-

backreaction increased from 80 msec in the wild-type to 20 msec in the menG mutant

strain, thus supporting a thermally-activated uphill electron transfer through the quinone

rather than a direct route for charge recombination between [FA/FB]- and P700+.

75

ABBREVIATIONS

bp basepair(s)

Chl chlorophyll

CW continuous wave

ENDOR electron nuclear double resonance

EPR electron paramagnetic resonance

HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid

HPLC high performance liquid chromatography

kb kilobase

MS mass spectrometry

NQ naphthoquinone

ORF open reading frame


PhyQ phylloquinone


PS photosystem

Tricine N- [2-hydroxy-1,1-bis(hydroxymethylethyl)]glycine

76

INTRODUCTION

Photosystem I (PS I) in cyanobacteria is a membrane-integral pigment-protein

complex consisting of 12 protein subunits (PsaA through PsaF, PsaI through PsaM, and

PsaX) and a variety of cofactors, including 96 molecules of chlorophyll a (Chl a), two

molecules of 2-phytyl-3-methyl-1,4-naphthoquinone (phylloquinone, PhyQ), three

[4Fe-4S] clusters, and about 22 molecules of β-carotene (Jordan et al., 2001). After the

absorption of a photon by one of the antenna molecules, charge separation takes place

between a special pair of Chl a molecules (P700) and a Chl a monomer (A0). The

charge-separated state is stabilized on the donor side by electron transfer from

plastocyanin or cytochrome c6 to P700+, and on the acceptor side by electron transfer to

PhyQ in the A1 site, through the iron-sulfur clusters at FX, FA and FB, and ultimately to

ferredoxin or flavodoxin. This light-driven enzyme therefore functions as cytochrome

c6/plastocyanin:ferredoxin/flavodoxin oxidoreductase that ultimately provides reducing

power to the cell in the form of NADPH.

Two molecules of PhyQ are present per P700 in PS I complexes of Synechocystis

sp. PCC 6803 and Synechococcus sp. PCC 7942 (Biggins and Mathis, 1988; Malkin, 1986;

Schoeder and Lockau, 1986). Both quinones have been located on the 2.5 Å-resolution

electron density map of Synechococcus elongatus PS I complexes (Jordan et al., 2001), and

there is a good agreement with EPR studies that provide distance (Bittl et al., 1997) and

orientation (Kamlowski et al., 1998; Zech et al., 2000) information for the EPR-active

quinone. For example, the same orientation of the carbonyl oxygen bonds of PhyQ, tilted

77 ca. 30 degrees from the membrane plane, is deduced from the orientation of the magnetic

gxx [(Zech et al., 2000) and earlier references therein] and the electron density map (Jordan

et al., 2001). PhyQ is structurally related to menaquinone-9 (2-

nonaprenyl-3-methyl-1,4-naphthoquinone). Both have 3-methyl-1,4-naphthoquinone core

structures with a poly-isoprenoid chain at the C2 ring position: menaquinone has an

unsaturated C45 isoprenoid chain, while PhyQ has a partially saturated C20 phytyl chain.

Because of their similarities in structure, it is likely that the PhyQ biosynthetic pathway is

similar to the menaquinone biosynthetic pathway in Escherichia coli (Johnson et al., 2000,

see also Chapter 2). The biosynthetic pathway of PhyQ is interesting for two reasons.

Firstly, the genes and enzymes of this pathway have not yet been completely characterized

in cyanobacteria. Secondly, knowledge of this pathway should enable one to devise

biological and chemical strategies to modify the quinone present in the A1 site.

A pathway of PhyQ biosynthesis in Synechocystis sp. PCC6803 was recently

proposed and included the genes menA, menB, menC, menD, menE, menF, and menG

(Johnson et al., 2000) (see Fig 2.1). Inactivation of the menA and menB genes, predicted to

encode 1,4-dihydroxy-2-naphthoic acid phytyltransferase and 1,4-dihydroxy-2-naphthoate

synthase, respectively, prevented the synthesis of PhyQ and led to the incorporation of

plastoquinone-9 (PQ-9) into the A1 site of PS I (Johnson et al., 2000). PQ-9 is the

secondary quinone acceptor in Photosystem II (PS II) and additionally functions as the

molecule that shuttles electrons to the cytochrome b6f complex. It was reported that PQ-9 is

present in the A1 site of PS I with the same orientation and asymmetric spin-density

distribution as PhyQ in wild type PS I complexes (Zybailov et al., 2000). PQ-9 is a

78 benzoquinone with a C45 alkyl tail and is structurally and functionally only distantly

related to PhyQ. The fact that PQ-9 could be inserted into the A1 site and could function in

lieu of PhyQ indicates that the binding site is not highly specific for PhyQ. Rather, the

redox properties of the quinone appear to be conferred largely by the protein environment

of the A1 site rather than by the identity of the quinone (Semenov et al., 2000).

The object of this study is the protein encoded by ORF sll1653, which was

originally annotated as gerC2, a spore germination protein in Bacillus subtilis. This ORF is

homologous to menG, which codes for a methyltransferase enzyme in E. coli and other

bacteria. In Synechocystis sp. PCC 6803, this gene is anticipated to encode a

2-phytyl-1,4-naphthoquinone (2-phytyl-1,4-NQ) methyltransferase enzyme (Johnson et

al., 2000). 2-phytyl-1,4-NQ differs from PhyQ only by the absence of the methyl group at

ring position C3 and by a higher redox potential (Depew and Wan, 1988). The relationship

between the molecular structure of this quinone and the axes of the molecular g-tensor is

depicted in Fig 3.1.

This study has two aims. The first is to confirm that sll1653 codes for the

methyltransferase responsible for synthesizing 2-phytyl-3-methyl-1,4-NQ from

2-phytyl-1,4-NQ. Indeed, it was found that inactivation of ORF sll1653 resulted in the

synthesis of PS I complexes containing 2-phytyl-1,4-NQ in the A1 site. The second is to

study the roles of the methyl group in quinone binding and electron transfer in PS I. Here, it

was shown that 2-phytyl-1,4-NQ is present in the A1 site with the same orientation and

asymmetric spin-density distribution as PhyQ in native PS I. It was also reported that

79 2-phytyl-1,4-NQ functions in forward electron transfer with some attendant alteration of

the PS I activity and electron-transfer kinetics.

80

RESULTS

Analysis of the Genotype of the menG Mutant Strain

As shown in Fig 3.2A, the menG gene (ORF sll1653) was inactivated by insertion

of a 1.3-kb DNA cassette, encoding the aphII gene and conferring resistance to kanamycin,

in the unique KpnI site within the coding sequence. Full segregation of the menG and

menG::aphII alleles was confirmed by PCR amplification and by Southern

blot-hybridization analysis of the genomic DNA. For PCR analysis, the primers flanking

the menG coding sequence (Fig 3.2A small arrows) were used to amplify the DNA

fragments from the wild-type and the menG mutant genomic DNAs. As expected, a 1.2-kb

fragment was amplified by PCR for wild type (Fig 3.2B). For the menG::aphII mutant

strain a 2.5-kb fragment was amplified by PCR; no DNA fragment with a size equal to that

expected from the wild-type menG allele was detected. This result indicates that the menG

mutant is homozygous for the menG::aphII gene interruption. To verify the menG

mutation further, the genomic DNA isolated from the wild-type and the menG mutant

strains were digested with restriction enzymes BglII and HindIII (see in Fig 3.2A) for the

Southern-blot hybridization analysis. Hybridization was performed using the PCR product

from the wild type genome as a probe. A single band with the size of 2.3 kb was observed

for the wild type strain (Fig 3.2C). For the menG mutant, two bands with the sizes of 2.7 kb

and 1.5 kb were observed due to the presence of the BglII site in the Kmr cartridge. All of

these results confirmed that the menG::aphII allele has fully segregated from the wild-type

gene.

81 High Performance Liquid Chromatography/Mass Spectrometry Analysis

To determine whether the menG gene is involved in PhyQ biosynthesis,

solvent-extracted quinone fractions from PS I complexes isolated from the wild-type and

the menG mutant strains were subject to orthogonal acceleration/time-of-flight mass

spectrometry. Pigments and quinones extracted from the isolated PS I trimers were

separated by reverse-phase HPLC, and the molecular mass of each eluate was determined

by mass spectrometry. In the wild-type complexes, PhyQ was detected by its characteristic

retention time, its UV absorption spectrum, and its m/z of 450 (Fig 3.3A). In PS I from the

menG mutant strain, no compound absorbing at 270 nm eluted at this retention time, nor

was a m/z of 450 detected. Rather, a UV-absorbing component eluted near the retention

time of Chl a that had a m/z of 436, which corresponds to the mass of 2-phytyl-1,4-NQ (Fig

3.3B).

Growth Rate under Photoautotrophic and Photomixotrophic Conditions

As indicated by HPLC and mass spectrometry, mutation of the menG gene

resulted in the replacement of PhyQ by 2-phytyl-1,4-NQ in PS I complexes. To measure

the effectiveness of 2-phytyl-1,4-NQ in supporting photosynthesis, growth rates of the

wild-type and menG mutant strains were compared. The results are summarized in Table

3.1. Growth of the wild-type and menG mutant strains were virtually the same under low

(50 µE m-2 s-1) and moderate (150 µE m-2 s-1) illumination conditions, in either the presence

or absence of glucose (Table 3.1). When cells were grown photoautotrophically, doubling

times of both the wild-type and the menG mutant strains were ca. 10 h under low light

intensity conditions and ca. 8 h under moderate light intensity conditions. When cells were

82 grown photomixotrophically, doubling times of the wild-type and the menG mutant strains

were both ca. 8 h under low light intensity conditions, and ca. 6 h under the moderate light

intensity conditions. The observed differences in the growth rates between the wild-type

and the menG mutant strains were all within the range of error. These results suggest that

under these illumination regimes, replacement of PhyQ with 2-phytyl-1,4-NQ has no

significant effect on growth.

PS II/PS I Ratio Measured by 77 K Fluorescence Emission Spectra

The relative ratios of PS II to PS I were compared by measuring 77 K fluorescence

emission spectra of wild-type and the menG mutant cells grown at various light intensities.

The results are summarized in Fig 3.4. When cells were grown under low-light conditions,

which is typical for Synechocystis sp. PCC 6803 (Fig 3.4A), the emission amplitude from

PS I at 721 nm was much greater than that from PS II at 695 nm. When cells were grown at

higher light intensities, an obvious decrease at 721 nm was found in the emission spectra

of the menG mutant cells when compared to wild-type cells (Fig 3.4B and 3.4C). In

wild-type cells the emission peak at 721 nm due to Chl a associated with PS I decreased

approximately 10% in cells grown at a light intensity of 300 µE m-2 s-1 relative to cells

grown at a light intensity of 50 µE m-2 s-1, while emission peaks at 685 and 695 nm due to

Chl a associated with PS II remained nearly constant. However, the peak amplitude at 721

nm decreased substantially in the menG mutant cells grown at higher light intensities. The

amplitude of this peak in cells grown at a light intensity of 300 µE m-2 s-1 was less than

70% that for cells grown at 50 µE m-2 s-1. The PS II/PS I ratio is slightly higher in the

menG mutant than in wild type when grown at 50 µE m-2 s-1 and significantly higher when

83

grown at 300 µE m-2 s-1. These results suggest that there may be either a defect in the

assembly of PS I complexes or an accelerated degradation of PS I complexes in the menG

mutant cells grown at high light intensities.

Chlorophyll Content of Whole Cells Grown at Various Light Intensities

The chlorophyll contents of wild-type and the menG mutant cells grown under

various light intensities were also determined (Table 3.2). When cells were grown under

low or moderate light intensities (50 or 150 µE m-2 s-1), the chlorophyll content of the

menG mutant cells was similar to that of the wild-type cells. However, when the light

intensity was increased to 300 µE m-2 s-1, the chlorophyll content in the wild-type cells

decreased to approximately 70% of the initial value (Table 3.2). A significantly lower

value, approximately 58% of the value obtained at lower light intensities, was observed for

menG mutant cells grown at a light intensity of 300 µE m-2 s-1. This value is only 80% that

of the wild-type strain under the same conditions, and this decrease can be correlated with

the loss of fluorescence emission from PS I observable in the decreased emission at 721 nm

in Fig 3.4. In combination, the results from Table 3.2 and Fig 3.4 suggest that content of PS

I complexes is lower in the menG mutant strain when cells were grown at high light

intensity. This difference could be due to a defect either in biogenesis or in stability of the

PS I complexes in the mutant strain.

Polypeptide Composition of Isolated PS I Trimers

To analyze whether the interruption of the menG gene has any effect on the PS I

subunit composition, PS I trimers were analyzed by SDS-polyacrylamide gel

electrophoresis. Polypeptide bands were visualized by silver staining. All of the eleven

84 polypeptides detected in the wild-type, PsaA though F and PsaI, J, L and M, were found to

be present in the menG mutant strain (Fig 3.5). No additional polypeptides were observed

in PS I complexes isolated from the menG mutant strain. These results indicate that the

replacement of PhyQ by 2-phytyl-1,4-NQ does not affect the subunit composition of the

PS I complexes in the menG mutant strain.

EPR spectroscopy of Q- and P700+ Q-

Fig 3.6A shows continuous wave EPR spectra measured at 34 GHz (Q-band) of

the photoaccumulated semiquinone radical (Q-) in PS I complexes isolated from the

wild-type (solid line, top) and the menG mutant strains (solid line, bottom). The PhyQ

anion radical is characterized (Fig 3.6A, solid line) by principal g-tensor values (Zybailov

et al., 2000) and the principal hyperfine A-tensor for the three protons in the methyl group

at the C2 position (Rigby et al., 1996) as summarized in Table 3.3. The hyperfine splitting

due to the remaining aromatic hydrogen atoms and the methylene group of the phytyl chain

at the C3 position remain unresolved and are included in the inhomogeneous line

broadening using linewidths of 3.0, 6.0, and 4.0 G for each of the three g-tensor

components (Fig 3.6B, dotted line). The deviations of the experimentally obtained

spectrum (Fig 3.6B, solid line) from the simulated spectrum (Fig 3.6A, dotted line) seen in

the mid- and high field regions are due to contamination by A0-.

The Q-band spectrum of the photoaccumulated semiquinone radical in the PS I

complexes isolated from menG mutant strain is shown in Fig 3.6B (solid line). The

spectrum has about the same overall width as that of wild type, which indicates that the

g-tensor of the semiquinone radical is about the same as that of the PhyQ anion radical. The

85 pattern of hyperfine splitting, however, is different in the menG mutant sample. The usual

four hyperfine lines are missing and instead, only two lines are present in the low-field

region. Such properties are expected if the semiquinone radical in the A1 site is replaced

with a 2-phytyl-1,4-NQ that lacks the methyl group at ring position 3 (see below). Indeed,

a satisfactory simulation of the spectrum of the menG mutant strain was obtained by

modifying the wild-type simulation, with the A-tensor of the methyl group replaced by that

of an aromatic C-H fragment with the same tensor components evaluated from the spin

polarized spectra described in the next section (Fig 3.6B, dotted line).

Transient EPR Spectroscopy

Fig 3.7 compares transient, spin-polarized EPR spectra (X-, Q-, W-band) of the

transient charge separated P700+ Q- state in the PS I complexes isolated from the wild-type

(dashed line) and the menG mutant strains (solid line). The two sets of spectra coincide

quite well in the high-field region, which is dominated by the P700+ contribution, but are

quite different in the low-field region. The W-band spectrum indicates a slightly larger

g-anisotropy for the menG mutant than that of the wild type PS I complex. The best fit

yields gxx = 2.00633 (menG) versus 2.00622 (wild type) while gyy = 2.00507 and gzz =

2.00218 remain unchanged from the wild type (Zech et al., 2000) as summarized in Table

3.3.

Closer inspection reveals that the main difference in the low field region concerns

the partially resolved hyperfine pattern. It is sufficiently resolved to permit an initial,

qualitative analysis directly from the spectra. For wild-type, the characteristic hyperfine

pattern due to the 1:3:3:1 quartet associated with a methyl group in ring position 2 is

86 particularly obvious in the Q-band spectrum. The fact that it is partially resolved results

from the increased spin density at the carbon ring position to which the methyl group is

attached (specific to the semiquinone radical ion in the A1 site). The components of the

axially symmetric hyperfine tensor have been determined by ENDOR to be A11 = 12.8

MHz and A⊥ = 9.0 MHz (see Rigby et al., 1996, for summary and further references; see

also Kamlowski et al., 1998; Zech et al., 2000). In the menG mutant, the CH3 group is

expected to be replaced by an aromatic C-H fragment. Indeed, the quartet hyperfine pattern

due to the CH3 group is missing in the menG mutant spectrum. Instead it is replaced by a

doublet (menG), the center position of which is shifted down field with respect to the

center of the quartet (wild type). Such a shift is expected with an altered orientation of the

dominant hyperfine principal axis with respect to the molecular axis frame. As concluded

from the observed shift direction, the dominant hyperfine axis will point more in the

direction of the gxx(Q) tensor axis in the menG mutant while it points more in the direction

of the gyy tensor axis in the wild type. Since the dominant hyperfine axis A11 of the CH3

hyperfine tensor is usually almost parallel to the C-CH3 bond axis, its direction is closer to

(about 30 degrees off) the gyy tensor axis. In contrast, the direction of the dominant

hyperfine axis of the aromatic C-H fragment (which is in-plane and perpendicular to the

C-H bond, see Carrington and MacLachlan, 1967) is closer to (about 30 degrees off) the

gxx(Q) tensor axis. The associated downfield shift of the center of the hyperfine doublet is

therefore in agreement with the experimental result.

To prepare for a quantitative simulation of the spectra, the following findings

were taken into account. The spin polarization pattern shows that 2-phytyl-1,4-NQ has the

87 same orientation in the A1 site as native PhyQ (Fig 3.7). The same P700 to A1

center-to-center distance has been verified by out-of-phase, spin-echo modulation

spectroscopy (data not shown). An independent determination of the hyperfine tensor

components of the C-H fragment has been obtained from pulsed ENDOR spectroscopy of

the same P700+ Q- state in the menG mutant (data not shown). The principal hyperfine

tensor components are given in Table 3.3. While the two large components, A11 and A22 are

clearly resolved, the third component is part of the line manifold in the center part of the

ENDOR spectrum and cannot be evaluated. With the reasonable assumption that the spin

density of the relevant carbon ring position does not change with respect to wild type

(corresponding to Aiso = -10.3 MHz with the appropriate McConnell relation) one can

estimate the third hyperfine principal component to be A33 = -3.6 MHz.

Although the axis associated with the largest hyperfine component is expected to

be in-plane and perpendicular to the C-H bond, this direction can be influenced by

significant spin densities on the neighboring molecular atoms, such as the spin density that

is known to reside on the carbonyl groups of the semiquinone radical. Therefore,

simulations of the Q-band spectrum (Fig 3.8) were carried out for various angles between

the largest principal axis A11 and the gxx (Q) axis (in-plane along the carbonyl bond

direction). The A11 axis is perpendicular to the C-H bond direction when this angle is 30°.

Indeed, of the simulations shown, the one for this value (Fig 3.8, bottom, long dashed

curve) comes closest to the experimental spectrum (Fig 3.8, top). A slightly smaller angle

than 30 degrees improves the simulation result to some degree. This indicates a weak

influence from spin density on neighboring atoms. The only significant spin density is

88 expected for the oxygen atom of the carbonyl group in ring position 1, as concluded from

the alternating spin density distribution due to asymmetric H-bonding to the opposite

carbonyl group in position 4 (see Kamlowski et al., 1998; Zech et al., 2000, and references

therein). However, the oxygen atom is already quite distant from the proton spin of the C-H

fragment, and thus the deviation of the C-H fragment hyperfine tensor from the (3:2:1)

ratio of the undisturbed C-H fragment is small, as indeed is observed here.

Kinetics of Forward Electron Transfer from A1- to FX

Forward electron transfer kinetics from the quinone to the iron sulfur clusters in

PS I complexes isolated from the wild-type and the menG mutant are compared in Fig 3.9

and Fig 3.10, which show spin-polarized EPR transients and decay-associated spectra,

respectively. The transients in Fig 3.9 labeled a, b and c were taken at the corresponding

field positions indicated with arrows in Fig 3.10. In wild-type PS I complexes, two

consecutive spectra are observed as electron transfer from A1- to FX occurs. At early times

(< ~1 µs) an emission/absorption/emission (EAE) pattern due to P700+ A1- is observed;

this pattern changes to an emissive spectrum due to P700+ in the state P700+ FeS- at later

times (> ~1 µs). The identity of the iron-sulfur cluster partner to P700+ in the state giving

the late spectrum requires some discussion. It has been shown that electron transfer

proceeds via FX (van der Est et al., 1994), which suggests the spectrum is due to P700+ FX-.

However, there is evidence (Leibl et al., 1995) that electron transfer from FX- to FA/FB is

faster than the transfer from A1 to FX. In this case, the late spectrum would be due to P700+

FA- and/or P700+ FB

-. Calculations (Kandrashkin et al., 1998) show the polarization

patterns corresponding to P700+ FX-, P700+ FA

-, and P700+ FB- show only minor

89 differences. Thus, the state giving the late spectrum P700+ FeS- is labeled P700+ FeS- and

the identity of the iron sulfur cluster(s) is left open. In the kinetic traces (Fig 3.9), the

electron transfer is seen most clearly at field position b. The positive (absorptive) signal at

early times (< ~50 ns) is due to P700+ Q-, while the negative (emissive) signal at late times

(< ~1 µs) is due to P700+ FeS-. The electron transfer dominates the decay of the early signal

while relaxation of the spin polarization causes the decay of the late signal. At positions a

and c the intensity of the late signal is weak and thus the transients decay primarily with the

electron transfer rate. As can be seen at all three field positions shown in Fig 3.10, the rate

of electron transfer is considerably slower in the mutant than in the wild type sample. For

the mutant PS I complexes, a fit of the entire data set as described in (van der Est, 1994)

yields an average electron transfer lifetime of 600 ± 100 ns. This compares with a value of

290 ± 70 ns for the wild-type complexes.

Kinetics of Charge Recombination Measured by Optical Spectroscopy

The reduction of P700+ was monitored at 811 nm after a brief laser flash in PS I

trimers isolated from the wild-type and the menG mutant strains (Fig 3.11). Because no

terminal electron acceptors such as ferredoxin or flavodoxin were present, the reduction

rate of P700+ represents the rate of charge recombination between [FA/FB]- to P700+. The

kinetics of P700+ reduction in the wild-type PS I complexes were fitted by one stretched

exponent with (1/e) lifetimes of 0.70, 30.7, and 94.7 ms (Fig 3.11A). The latter two values

are similar to the two lifetime components found previously for the P700-FA/FB complexes

from Synechocystis sp. PCC 6803 and are attributed to charge recombination between

[FA/FB]- and P700+ (Vassiliev et al., 1997). The first value is probably due to charge

90 recombination between P700+ and FX

- in damaged PS I complexes that lack an intact PsaC

subunit. The kinetics of P700+ reduction in PS I complexes from the menG mutant (Fig

3.11B) were also fitted by two exponentials with (1/e) lifetimes of 10.3 and 29.3 ms, These

values are attributed to charge recombination between [FA/FB]- and P700+ and are ca. 3

times faster than the wild-type rates. Even though the reason for the heterogeneity in P700+

reduction is not understood, it is noteworthy that the two kinetic phases attributed to charge

recombination between [FA/FB]- and P700+ are equally slowed in the menG mutant

samples.

Steady-State Rates of Flavodoxin Reduction

Steady-state rates of flavodoxin reduction as a function of light intensity were

measured for PS I complexes isolated form the wild-type and menG mutant strains to

assess the relative efficiencies of forward electron transfer. The rates at saturating light

intensity were determined by treating light as a substrate in a Michaelis-Menten kinetic

analysis (Fig 3.12). The maximal rate of flavodoxin reduction was found to be 1527 ± 247

µmol (mg Chl a)-1 h-1 for the wild-type PS I complexes and 1845 ± 399 µmol (mg Chl a)-1

h-1 for the menG mutant PS I complexes. Assuming that 100 Chl a molecules are present

per P700 in all PS I complexes, these maximal rates of electron transfer correspond to 37.9

± 6.2 e- PS I-1 s-1 in the wild-type PS I complexes and 45.8 ± 8.4 e- PS I-1 s-1 in the menG

mutant PS I complexes. Although the value obtained for the PS I complexes from the

menG mutant was slightly higher than that for the wild-type, this difference is within the

margin of error and therefore is not statistically significant (Fig 3.12). Hence, the

steady-state rate of electron transfer for the PS I complexes of the menG mutant, which

91 contain 2-phytyl-1,4-NQ, was virtually identical to the steady-state rate of electron transfer

for the PhyQ-containing PS I complexes of the wild-type.

92

DISCUSSION

The menG gene, encoding the 2-phytyl-1,4-NQ (demethylphylloquinone)

methyltransferase, was identified by targeted inactivation of ORF sll1653 in Synechocystis

sp. PCC 6803. This open reading frame was originally assigned as gerC2, which encodes

the spore germination protein C2 in Bacillus species and other spore-former bacteria

(Kaneko et al., 1996). In fact, the deduced amino acid sequence for ORF sll1653 shows

only 27% sequence identity to the menG gene product of E. coli. Targeted inactivation of

ORF sll1653 was accomplished by insertion of a kanamycin resistance cartridge in the

coding region, which was confirmed by PCR amplification of the region containing the

ORF and by Southern-blot hybridization analysis. The presence of 2-phytyl-1,4-NQ in PS I

complexes was confirmed by high performance liquid chromatography and mass

spectrometry, and its function in electron transfer was demonstrated by CW and transient

EPR spectroscopy. The orientation and distance of 2-phytyl-1,4-NQ relative to P700 was

confirmed to be the same as for PhyQ in wild-type PS I complexes by transient EPR,

out-of-phase, spin-echo modulation, and pulsed ENDOR spectroscopy. It can be safely

concluded that ORF sll1653 in Synechocystis sp. PCC 6803 is menG; that it encodes

2-phytyl-1,4-NQ methyltransferase, the enzyme required for the last step of the PhyQ

biosynthetic pathway; and that 2-phytyl-1,4-NQ functionally replaces PhyQ at the A1 site

in PS I complexes.

Despite the absence of PhyQ and the presence of 2-phytyl-1,4-NQ in PS I

complexes, the growth rates of the wild-type and menG mutant cells were identical. As

observed for many other cyanobacteria (Fujita, 1997), the ratio of PS II to PS I is known to

93 change in Synechocystis sp. PCC 6803 as a function of the light intensity during cell

growth (G. Shen, J. H. Golbeck, and D. A. Bryant, unpublished observation). An

interesting observation was that the change in the ratio of PS II to PS I was more

pronounced in the menG mutant strain than in the wild type as the light intensity was

increased from 50 µE m-2 s-1 to 300 µE m-2 s-1. In wild-type cells, the PS II to PS I ratio

increased by a factor of 1.1, while in the menG mutant cells the PS II to PS I ratio increased

by a factor of 2 as estimated from the amplitudes of the fluorescence emission spectra at 77

K. It is noteworthy that the overall chlorophyll content in the wild-type and menG mutant

cells were similar at light intensities of 50 and 150 µE m-2 s-1 and differed only at 300 µE

m-2 s-1. Therefore, the difference in the PS II to PS I ratio between wild type and the menG

mutant strain can be primarily attributed to a change in the content of PS I, i.e. the number

of PS I complexes declines in the menG mutants to a greater extent than in the wild-type

cells as the light intensity is raised. By way of comparison, the menA and menB mutant

strains, which contain plastoquinone-9 in the A1 site, have been observed to grow

significantly more slowly and can be grown only under very low light intensity conditions

(< 20 µE m-2 s-1). The ratio of PS II to PS I is already greater than 2:1 at low light intensity,

and might be expected to be even greater as the light intensity is increased (the cells,

however, do not survive). Thus, it appears that Synechocystis sp. PCC 6803 must exceed

ca. 2:1 ratio of PS II to PS I before light becomes toxic to the growing cells.

To understand further the behavior of 2-phytyl-1,4-NQ in electron transfer, one

must consider the redox potential of this quinone in the A1 site. In aqueous solution, the

E(Q/Q-)7 of 2-methyl-1,4-NQ is –203 mV vs. NHE (normal hydrogen electrode) and the

94 E(Q/Q-)7 of 2,3-dimethyl-1,4-NQ is –240 mV vs. NHE (Swallow, 1982). The E(Q/Q-)7 of

2-phytyl-3-methyl-1,4-NQ (PhyQ) in aqueous solution (containing 5 M 2-propanol and

2M acetone) is reported to be –170 mV. The presence of the second alkyl group, ortho to

the first, therefore lowers the redox potential of the quinone by ca. 37 mV. However, the A1

site is highly hydrophobic (Iwaki and Itoh, 1991, 1994), and reduction potentials of

quinones in organic solvent are probably more relevant to their properties in PS I

complexes. In dimethylformamide (DMF), the E1/2 value for 2-methyl-1,4-NQ has been

reported as –650 mV vs. SCE and the E1/2 value for 2,3-dimethyl-1,4-NQ has been reported

to be –746 mV (Prince et al., 1983) vs. SCE (standard calomel electrode). The lower redox

potential for the dimethyl quinone is logical chemically; a methyl group is an electron

donor and should therefore destabilize the semiquinone radical in either aqueous solution

or in organic solvent. A prenyl substituent on the quinone ring is not as effective as a

second alkyl group at electron donation and lowers the E1/2 value by only 50 to 60 mV

(Prince et al., 1983). For example the E1/2 of menaquinone-2

(2-all-trans-polyprenyl-3-methyl-1,4-NQ) is –746 mV vs. SCE (to the best of our

knowledge, the reduction potential of 2-phytyl-1,4-NQ in DMF is not available). Using the

redox potentials for 2-methyl-1,4-NQ and 2,3-dimethyl-1,4-NQ as models for

2-phytyl-1,4-NQ and PhyQ (both differ by the addition of one methyl group), the addition

of a second methyl group ortho to the first should lower the redox potential by ca. 96 mV.

If one further employs the concept of an ‘acceptor number’ that was utilized for PQ-9 in the

menA and menB mutants (Semenov et al., 2000), the redox potential of 2-phytyl-1,4-NQ in

the A1 site would be 61 mV more oxidizing than PhyQ. An acceptor number corrects the

95 E1/2 value for the degree of the nucleophilic (donor) or electrophilic (acceptor) properties of

the solvent (Gutmann, 1976) and was employed initially by Itoh (Iwaki and Itoh., 1994) to

estimate the redox potential of PhyQ in the A1 site of PS I. It is a dimensionless number that

expresses the acceptor properties of a solvent relative to SbCl5.

The estimated redox potential of 2-phytyl-1,4-NQ in the A1 site can now be

compared to estimates based upon the experimentally measured rate of electron transfer.

Forward electron transfer is ca. 3-times slower when 2-phytyl-1,4-NQ rather than PhyQ

occupies the A1 site. According to the Moser-Dutton formulation (Moser et al., 1992), the

rate of electron transfer in proteins depends on the Gibbs free energy between the

donor-acceptor pair, the edge-to-edge distance between the donor-acceptor pair, and the

reorganization energy. The distance between the 2-phytyl-1,4-NQ anion radical and P700+

was experimentally determined here to be the same as the center-to-center distance

between the PhyQ anion radical and P700+. The orientation of 2-phytyl-1,4-NQ relative to

the membrane plane was also shown to be identical to that of PhyQ. Hence, the

edge-to-edge distance between 2-phytyl-1,4-NQ and FX is likely to be the same as the

distance between PhyQ and FX. It seems reasonable to assume similar reorganization

energies when 2-phytyl-1,4-NQ and PhyQ occupy the A1 site, given that the only

difference between the two quinones is the presence or absence of a single methyl group.

Using the values of 11.3 Å for the edge-to-edge distance between the quinone and FX

(Klukas et al., 1999), and a value of 0.7 eV for the reorganization energy, a (1/e) lifetime of

ca. 290 ns for Q- in the wild-type translates to an 81 mV difference in Gibbs free energy

between Q-/Q and FX-/FX. Retaining the same values for the distance and reorganization

96 energy, a (1/e) lifetime of 600 ns for Q- in the menG mutant translates to a 28 mV

difference in the Gibbs free energy between Q-/Q and FX-/FX. According to this analysis,

the addition of methyl group to 2-phytyl-1,4-NQ lowers the redox potential of the quinone

in the A1 site by 53 mV. This value is only a rough estimate, especially given the

uncertainties in the edge-to-edge distance between the cofactors and given the assumed

(unchanged) reorganization energy of the site. Nevertheless, this value agrees quite well

with the difference in redox potentials of 2-methyl-1,4-NQ and 2,3-dimethyl-1,4-NQ in

organic solvents, especially after correction for the site’s “acceptor number”. The

replacement of PhyQ by 2-phytyl-1,4-NQ would therefore lower the equilibrium constant

between Q-/Q and FX-/FX from 27 to 3, a factor of ca. 10. Estimating the absolute redox

potential of 2-phytyl-1,4-NQ in the A1 site is somewhat more difficult because the redox

potential of A1 is not known with certainty. Three values have been published for the redox

potentials of the Q-/Q couple [-810 mV vs. NHE (Vos and Gorkom, 1990), -800 mV vs.

NHE (Chamrovoskii and Cammack, 1983) and -754 mV vs. NHE (Iwaki and Itoh, 1994)].

If one uses the lower redox potentials, the replacement of PhyQ by 2-phytyl-1,4-NQ would

therefore raise the redox potential of the Q-/Q couple to ca. –750 mV, a value that is

probably still lower than the redox potential of the FX-/FX couple.

The kinetics of reduction of P700+ after a single flash represents dissipative

charge recombination between P700+ and terminal electron acceptors [FA/FB]- in the

absence of an acceptor such as ferredoxin or flavodoxin. It is not yet clear whether electron

transfer occurs directly from [FA/FB]- to P700+, or whether the electron travels backward

97 by a thermally activated, uphill electron transfer through the quinone. The latter

presupposes that each redox pair (Q-/Q ↔ FX-/FX; FX

-/FX ↔ FB-/FB; FB

-/FB ↔ FA-/FA) is

described by an equilibrium constant that can be determined from the midpoint potentials

of the acceptors. The large distance between P700 and the terminal iron-sulfur clusters [46

Å center-to-center distance from P700 to FA (Schubert et al., 1998)] argues against the

direct recombination mechanism, although experimental data to support thermally

activated uphill electron transfer have been lacking. If the charge recombination between

[FA/FB]- and P700+ were to occur through backward electron transfer, then a change in the

equilibrium constant between Q-/Q and FX-/FX should logically affect the rate. The

equilibrium constant between Q-/Q and FX-/FX would be correspondingly higher as the

redox potential of Q-/Q were driven more negative, thereby resulting in a depopulation of

reduced quinone. Conversely, the equilibrium constant between Q-/Q and FX-/FX would be

correspondingly lower as the redox potential of Q-/Q were driven more positive, thereby

resulting in a repopulation of reduced quinone. Consequently, the back-reaction kinetics in

the mutant would be faster than in wild-type PS I complexes.

The latter description is precisely the behavior observed. The forward

electron-transfer kinetics from Q- to FX becomes correspondingly slower as PhyQ [1/e

lifetime of 200 ns (van der Est et al., 1994)], 2-phytyl-1,4-NQ (1/e lifetime of 600 ns), and

PQ-9 [1/e lifetime of 15 µs (Semenov et al., 2000)] occupy the A1 site. Conversely, the

charge recombination kinetics between [FA/FB]- and P700+ become more rapid as PhyQ

[1/e lifetime of 80ms (Vassiliev et al., 1997)], 2-phytyl-1,4-NQ (1/e lifetime of 20 ms) and

PQ-9 [1/e lifetime of 3 ms (Semenov et al., 2000)] occupy the A1 site. The trend among the

98 wild type, menA or menB, and menG mutants therefore strongly supports the backward,

through-carrier route of electron transfer for charge recombination. Since the redox

potential of the quinone in the A1 site influences the rate of charge recombination from the

terminal electron acceptors to P700+ as well as the forward electron transfer between the

quinone and the terminal iron-sulfur clusters, this suggests that charge recombination

occurs by thermally activated, uphill electron transfer through the secondary quinone

acceptor in PS I.

Finally, although the kinetics of electron transfer within PS I are altered when

PhyQ is replaced by 2-phytyl-1,4-NQ, these changes do not affect the growth rate of the

organism. An explanation for this apparent discrepancy is that the slower rate of forward

electron transfer from A1- to FX is still not the rate-limiting step in the overall electron

transfer reaction to flavodoxin (or probably to ferredoxin). Indeed, the maximal rate of

flavodoxin reduction of the PS I complexes isolated from the menG mutant was virtually

identical to that observed for wild-type PS I complexes. Analysis of the rate of electron

donation from PS I to flavodoxin is complicated by the two-electron reduction using this

acceptor. Since the potentials at pH 7 for the oxidized flavodoxin/flavodoxin semiquinone

and the flavodoxin semiquinone/hydroquinone couple are –212 and –436 mV (Schubert et

al., 1998), respectively, it is likely that only the latter couple is physiologically relevant.

Studies show that the reduction of flavodoxin semiquinone is biphasic; the fast, first-order

phase corresponds to electron transfer within a preformed complex, but the slower phase is

concentration-dependent, with a second-order rate constant of 1.7 x 108 M-1 s-1. Given the

nearly wild-type electron-transfer rates from cytochrome c6 to flavodoxin in the menG

99 mutant, it is clear that forward electron transfer continues to out-compete the faster back

reaction rate in the mutant. Hence, PS I complexes are remarkably robust and are tolerant

to changes caused by replacement of wild-type components with “suboptimal” ones. This

robustness still allows for a remarkable level of functionality of the bound electron-transfer

cofactors under suboptimal conditions.

100

SUMMARY

The menG gene, encoding the 2-phytyl-1,4-NQ (demethylphylloquinone)

methyltransferase, was identified in Synechocystis sp. PCC 6803. The PS I complexes

isolated from the menG mutant accumulated 2-phytyl-1,4-NQ, which functioned as the A1

cofactor in the electron transfer. The rate of forward electron transfer from A1 to the

iron-sulfur clusters was slowed by a factor of two, while the rates of the P700+ [FA/FB]-

backreaction increased by a factor of 3 to 4. These results were explained by the lowering

of the equilibrium constant between Q-/Q and FX-/FX by a factor of ~10. Despite the defect

in the forward electron transfer, the PS I complexes containing 2-phytyl-1,4-NQ showed

catalytic activity similar to those of PS I complexes containing phylloquinone, which

demonstrates that PS I complexes are remarkably robust and can function with presumably

“suboptimal” electron transfer cofactors.

101


Generation of the menG Mutant Strain of Synechocystis sp. PCC 6803

To generate the construct for inactivation of the menG gene, a 1.2-kb DNA

fragment containing the sll1653 ORF was amplified through PCR from the genome of the

Synechocystis sp. PCC 6803. For cloning convenience, a new EcoRI site was created in the

3'-end primer with one nucleotide change. The PCR-amplified DNA fragment was cloned

into pUC19 vector and confirmed by sequencing. To inactivate the menG gene, a 1.3-kb

kanamycin-resistance cartridge encoded by the aphII gene was inserted into the KpnI site

in the middle of the menG gene. The resulting plasmid was linearized by digestion with

EcoRI and was used to transform the Synechocystis sp. PCC 6803 wild-type cells. The

transformation and the transformant screening were carried out as described (Shen et al.,

1993). After several rounds of restreaking to single colonies, full segregation of the

menG::aphII from the menG alleles was verified by PCR and Southern blot-hybridization

analyses.

Growth of Synechocystis sp. PCC 6803

Wild-type Synechocystis sp. PCC 6803 was grown in medium B-HEPES. This

medium is prepared by supplementing BG-11 medium (Stainer et al., 1971) with 4.6 mM

of HEPES [4-(2-hydroxyethyl)piperazine-N’-(2-ethnesulfonic acid)]-KOH and 18 mg L-1

ferric ammonium citrate. The menG mutant cells were grown in medium B-HEPES

supplemented with 50 µg mL-1 kanamycin. The growth temperature was maintained at 32o

C and the light intensity was adjusted to 50, 150, and 300 µE m-2 s-1 by adding fluorescent

102 lamps or by shielding with paper as required. For photomixotrophic growth, the medium

was supplemented with 5 mM glucose. Growth of the cells was monitored by measuring

the absorbance at 730 nm using a Cary-14 spectrophotometer that had been modified for

computerized data acquisition by On-Line Instruments, Inc (Bogart, GA). Cells from a

liquid starter culture in the exponential phase (OD730nm < 0.7) were adjusted to the same

initial cell density (OD730nm = 0.05) for growth-curve measurements. The liquid cultures

were bubbled with 1% (v/v) CO2 balanced with air.

DNA Isolation, PCR, and Southern Blotting

Chromosomal DNA from Synechocystis sp. PCC 6803 wild-type and mutant

strains was isolated as previously described (Shen, et al., 1993). PCR primers used to

amplify the menG DNA fragment for evaluation of the menG alleles were positioned as

shown in the Fig 3.2. The sizes of the PCR products from the wild type and menG mutant

strains were determined by agarose gel electrophoresis. For Southern blot-hybridization

analysis, the chromosomal DNAs were subjected to restriction enzyme digestion, agarose

gel electrophoresis, and capillary transfer to nitrocellulose membranes. Hybridization

probes were generated by labeling the wild-type menG PCR fragment with the

random-primed DNA labeling kit (Boehringer-Mannheim, Indianapolis, IN).

Hybridization conditions and detection were as previously described (Shen and Bryant

1995)

103 Chlorophyll Extraction and Analysis

Chlorophyll was extracted from whole cells, thylakoid membranes and PS I

particles with 100% methanol, and its concentration was determined

spectrophotometrically as described (MacKinney, 1941).

Isolation of Thylakoid Membranes and PS I Particles

Thylakoid membranes were isolated from cells in the late exponential growth

phase as described previously (Shen et al., 1993). Cells were broken at 4°C by three

passages through a French pressure cell at 124 MPa. The thylakoid membranes were

resuspended and solubilized for 2 h at 4°C in the presence of 1% (w/v)

n-dodecyl-β-D-maltoside. PS I complexes were separated from other membrane

components by centrifugation on 5-20% (w/v) sucrose gradients with 0.05%

n-dodecyl-β-D-maltoside in the buffer. Further purification was achieved by a second

centrifugation on sucrose gradients in the buffer in the absence of

n-dodecyl-β-D-maltoside. Trimeric PS I complexes were used in these studies (Golbeck,

1995).

SDS-Polyacrylamide Gel Electrophoresis Analysis

Methods used for SDS-polyacrylamide gel electrophoresis were identical to those

previously described (Shen and Bryant, 1995). A Tricine/Tris discontinuous buffer system

was used to resolve the polypeptide composition of the PS I complexes prepared from the

wild type and the menG mutant strains of Synechocystis sp. PCC 6803 (Schägger and van

104 Jagow, 1987) The separating gel contained 16% acrylamide and 6 M urea, and the resolved

proteins were visualized by silver staining (Blum et al., 1987)

Analysis of Quinones by High Performance Liquid Chromatography and Mass Spectrometry

For quinone extraction, PS I particles isolated from the wild-type and the menG

mutant strains were exchanged into distilled water by dialysis for 4 h and lyophilized. The

pigments were extracted from the lyophilized samples with acetone:methanol, 1:1 (v/v) at

4 °C, vacuum dried at room temperature in the dark, and resuspended in 100% methanol.

The extracts were separated by reverse-phase high performance liquid chromatography

(HPLC) using a C18 column as previously described (Johnson et al., 2000). Eluates were

analyzed using atmospheric-pressure chemical ionization with a Micromass Quattro II

mass spectrometer operated in the negative-ion mode.

Flavodoxin Photoreduction

Steady-state rates of flavodoxin reduction were measured for the PS I trimers

isolated from the wild-type and the menG mutant strains as previously described (Jung et

al., 1995). Cyanobacterial flavodoxin was overexpressed in E. coli and purified as

described (Fillat et al., 1991). PS I trimers were resuspended to a final concentration of 5

µg Chl mL-1 in 25 mM Tris-HCl buffer, pH 8.3, containing 50 mM MgCl2, 15 µM

cytochrome c6, 15 µM flavodoxin, 6.0 mM sodium ascorbate, and 0.05 %

n-dodecyl-β-D-maltoside. Measurements were made by monitoring the rate of change in

the absorption at 467 nm using a modified Cary 219 spectrophotometer fitted with a 465

nm interference filter on the surface of the photomultiplier. The 4-sided clear cuvette was

105 illuminated from two sides using high intensity, red light-emitting diodes (LS1, Hansatech

Ltd., Norfolk, UK). Initial rates of the reaction were recorded under various light

intensities and plotted as a function of the light intensity to assess the relative efficiency of

forward electron transfer to flavodoxin.

77 K Fluorescence Emission Spectra

Fluorescence emission spectra were measured at 77 K using an SLM 8000C

spectrofluorometer as previously described (Shen and Bryant, 1995). Cells from the

exponential phase of growth were harvested and resuspended in 25 mM HEPES/NaOH,

pH 7.0, containing 60% glycerol. Samples were adjusted to equal cell density (OD730nm =

1.0) prior to freezing in liquid nitrogen. The excitation wavelength was 440 nm, the

excitation slit width was 4 nm, and the emission slit width was 2 nm. Each spectrum shown

is the average of four spectra.

Q-band EPR of Photoaccumulated PS I Complexes

Photoaccumulation experiments were performed using a Bruker ER300E

spectrometer and an ER 5106-QT resonator equipped with an opening for in-cavity

illumination similar to that described in (Yang et al., 1998). Low temperatures were

maintained with an ER4118CV liquid nitrogen cryostat and an ER4121 temperature

controller. The microwave frequency was measured with a Hewlett-Packard 5352B

frequency counter and the magnetic field was measured with a Bruker ER035M NMR

gaussmeter. The pH of the sample was adjusted to 10.0 with 1.0 M glycine buffer, and

sodium dithionite was added to a final concentration of 50 µM. After incubation for 10 min

in the dark, the sample was placed into the resonator and the temperature was adjusted to

106 205 K. The sample was illuminated for 40 min with a 20 mW He-Ne laser operating at 630

nm. A dark background spectrum was subtracted from the photoaccumulated spectrum.

EPR spectral simulations were carried out on a 466 MHz Power Macintosh G3 computer

using a Windows 3.1 emulator (SoftWindows 3.0, Insignia Solutions, UK) and SimFonia

software (Bruker Analytik GMBH, Germany).

Time-Resolved EPR Spectroscopy at Multiple Frequencies

Transient EPR spectra as well as pulsed EPR/ENDOR data were obtained using

the same instrumentation described earlier (Zybailov et al., 2000; Bittl et al., 1997).

Flash-Induced Absorbance Changes at 811 nm

The transient absorbance changes were measured with a home-built, double-beam

spectrophotometer described in (Vassiliev et al., 1997). The sample was prepared at a

chlorophyll concentration of 7 µM in 50 mM Tris buffer, pH 8.3. Measurements were

made at pH 8.3 in 50 mM Tris buffer in the presence of 2 mM sodium ascorbate and 5 µM

2,6-dichlorophenolindophenol. The samples were measured in a quartz cuvette with a

1-cm path for the measuring light. The excitation beam was provided by a

frequency-doubled Nd-YAG laser (532 nm) with the flash energy attenuated to ca. 20 mJ

using the Q-switch delay and neutral density filters. The measuring beam was provided by

a 5 mW semiconductor laser operating at 811 nm. Kinetic analysis was performed using a

non-linear regression algorithm in Igor Pro (WaveMetrics Inc., Lake Oswego, OR)

(Vassiliev et al., 1997). All kinetic constants are reported as 1/e lifetimes (τ).

107 X-band Transient EPR Spectroscopy at Room Temperature

Room-temperature, transient EPR experiments were carried out using a modified

Bruker ESP 200 equipped with a home-built broad-band amplifier (bandwidth >500 MHz)

for direct detection experiments. Excitation was provided by a Continuum YAG/OPO laser

system operating at 680 nm and 1 Hz. The EPR signals were digitized using a LeCroy

LT322 500 MHz digital oscilloscope and transferred to a PC for storage and analysis. The

samples were measured using a flat cell and a Bruker rectangular resonator fitted with a

piece of rough-surfaced glass in front of the window to provide optimal illumination. The

response time of the system in direct detection mode is limited by the bandwidth of the

resonator and is estimated as ~50 ns, and the decay of the spin polarization limits the

accessible time range to times shorter than a few microseconds. The same set-up can also

be used with field modulation and lock-in detection. In this mode the response time is ~50

µs but the sensitivity is much higher, and the charge recombination can be monitored in the

millisecond time range. In both modes of operation, full time/field data sets are collected

and analyzed to determine the lifetimes of the species and their decay-associated spectra as

described in detail (van der Est et al., 1994; Semenov et al., 2000).

108

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resonance spectroscopy. J Biol Chem 275: 8531-8539

114

Table 3.1: Doubling times of Synechocystis sp. PCC 6803 wild type and the menG

mutant strains (hours).

Strains Growth conditions

Wild type menG

Photoautotrophic, 50 µE m-2 s-1 (n = 4) 9.8 ± 0.0.85 9.8 ± 0.95

Photoautotrophic, 150 µE m-2 s-1 (n = 3) 8.1 ± 0.12 8.1 ± 0.25

Photomixotrophic, 50 µE m-2 s-1 (n = 4) 7.9 ± 0.90 7.5 ± 0.47

Photomixotrophic, 150 µE m-2 s-1 (n = 4) 5.6 ± 0.73 6.3 ± 0.56

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Table 3.2: Chlorophyll content in cells grown photoautotrophically at 32° C under

various illumination conditions.

Chlorophylls were extracted from whole cells with methanol. The absorbance coefficient

of 82 (µg/ml) –1 at 666 nm was used to calculate the chlorophyll content in 1 ml of the

cultures with an optical density of 1.0 at 730 nm.

µg Chlorophyll / OD730nm (n = 3) Light intensity

(µE m-2 s-1) Wild type menG

50 3.55 ± 0.24 3.40 ± 0.14

150 3.65 ± 0.21 3.40 ± 0.29

300 2.48 ± 0.34 1.98 ± 0.15

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Table 3.3: Magnetic parameters of PhyQ (A1-) and 2-phytyl-1,4-NQ (Q-) in the A1

binding site.

g-tensors a

gxx gyy gzz

A1- b 2.00622 2.00507 2.00218

Q- 2.00633 2.00507 2.00218

Hyperfine coupling principal values (MHz)

A11 A22 A33

A1- b 12.8 9.0 9.0

Q- -15.5 ± 0.5 -11.8 ± 0.5 <5

a the principal axes of the g-tensors are shown in Fig 3.1. b Zech et al., (2000) J. Phys. Chem.

B 104, 9728-9739 and references therein.

117

Fig 3.1: Relationship between the molecular structure of 2-phytyl-1,4-NQ and the axes of the electronic g-tensor. The axes of the electronic g-tensor of 2-phtyl-1,4-NQ (indicated by arrows) are parallel to the molecular axes system.

118

Fig 3.2: Construction and verification of the menG mutant strain of Synechocystis sp. PCC 6803. (A) Restriction map of the genomic region surrounding the menG gene in the wild type (top) and the menG mutant strain (bottom). The small arrows indicate the position of the PCR primers used to amplified the menG coding region. (B) Electrophoretic analysis of the DNA fragments amplified from the genomic DNA of wild type and the menG mutant strain through PCR analysis. (C) Southern-blot analysis of the genomic DNA isolated from wild type and menG mutant strain. Chromosomal DNA was digested using restriction enzymes BglII and HindIII. DNA fragment obtained by PCR from the wild type genome was used as probe, as shown in top panel.

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Fig 3.3: Mass spectra recorded for pigments and quinone extracts from the PS I complexes isolated from the wild-type and menG mutant strains of Synechocystis sp. PCC 6803. (A) Wild type, and (B) the menG mutant.

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Fig 3.4: 77 K Fluorescence emission spectra from whole cells of Synechocystis sp. PCC 6803. Chlorophyll fluorescence emission spectra were measured for the whole cells of wild type (solid lines) and the menG mutant strain (dotted lines) grown under three different light intensities, (A) 50 µE m-2 s-1, (B) 150 µE m-2 s-1, (C) 300 µE m-2 s-1. Each spectrum was recorded at the same cell density and presented as the average of four measurements.

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Fig 3.5: SDS-PAGE analysis of PS I complexes isolated from the wild-type and menG mutant strains of Synechocystis sp PCC 6803. The equal amount of Chl a (5 µg) is loaded for each lane. Polypeptides were visualized after silver staining.

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Fig 3.6: Photoaccumulated Q-band EPR spectra and simulations of PS I complexes from Synechocystis sp. PCC 6803.

Wild type (solid line), and the simulated spectrum (dotted line). B, menG mutant strain (solid line), and the simulated spectrum (dotted line). See text for details of the parameters of the simulation.

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Fig 3.7: Transient spin-polarized EPR spectra (X-, Q- and W-) of the charge separated P700+ Q- state in PS I trimers. Wild-type spectra (dashed lines) and the menG mutant (solid lines). The frequencies are from bottom to top: 9, 35, and 95 GHz.

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Fig 3.8: Simulations of the Q-band spectrum of the menG mutant carried out for various angles between the largest principal axis A11 and the gxx axis. Top: experimental spectrum as in Fig 3.4 (middle). Bottom: simulations as described and with the parameters specified in the text.

125

0 2 40 2 4 6

a

b

c

MenG Wild Type

time/µstime/µs

Fig 3.9: Spin-polarized EPR transients of wild type and menG mutant.

The traces a, b, c were collected using direct detection at the field positions indicated by the similarly labeled arrows in Fig 3.10. The traces on the left are for PS I complexes isolated from the menG mutant, and those on the right are those from the wild type.

126

3460 3470 3480 3490 3500B0 /Gauss

a b c

P+Q

-

P+(FeS)

-

Wild Type

MenG

Fig 3.10: Decay-associated transient EPR spectra at ambient temperature. Spectra of PS I complexes the wild-type and menG mutant strains extracted from the full time/field datasets as described in detail in Van der Est et al., (1994) Biochemistry 33, 11789-11797. The solid curves correspond to the state P700+ Q- while the dashed curves are the spectra of P700+ FeS-. The arrows labeled a, b, c indicate the field positions corresponding to the transients shown in Fig 3.9.

127

Fig 3.11: Flash-induced absorbance changes at 812 nm in PS I complexes isolated from Synechocystis sp. PCC 6803. The experimental data are depicted as dots, and the computer-generated exponential fits are shown as solid lines, with the lifetimes of the phase indicated. Top: P700+ reduction kinetics in wild-type PS I complexes. Bottom: P700+ reduction kinetics in the menG mutant PS I complexes.

128

Fig 3.12: Steady-state rates of flavodoxin reduction in PS I complexes isolated from the wild-type and menG mutant strains. Seven independent measurements were performed for PS I complexes from each strain at each light intensity. The open and filled diamonds indicate the average values obtained for the complexes for the wild-type and menG mutant strains, respectively. The solid and dashed lines indicate fitted curves derived from the Michaelis-Menten equation for the rates obtained for PS I complexes from the wild-type and menG mutant strains, respectively. Bars on the data points indicate the standard deviation.

129

Chapter 4

Physiological Characterization of Tocopherol Biosynthesis Mutants in the

Cyanobacterium Synechocystis sp. PCC 6803: Demonstration of a Conditionally

Lethal Phenotype in the Presence of Glucose at pH 7.0

Publications:

Zigang Cheng, Scott Sattler, Hiroshi Maeda, Yumiko Sakuragi, Donald A. Bryant, Dean

DellaPenna (2003) Highly divergent methyltransferases catalyze a conserved reaction in

tocopherol and plastoquinone synthesis in cyanobacteria and photosynthetic eukaryotes.

Plant Cell 15: 2343-2356

Yumiko Sakuragi, Hiroshi Maeda, Dean DellaPenna and Donald A. Bryant, manuscript

in preparation.

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ABSTRACT

The phenotypes of three tocopherol mutant populations, slr1736, sll0418 and slr0089,

previously selected in the presence of glucose in the cyanobacterium Synechocystis sp.

PCC 6803, have been reexamined. When cultured on solid media containing glucose, the

mutant populations showed extremely heterogeneous colony morphologies, which

suggested that the strains were genotypically inhomogeneous. Newly isolated tocopherol

mutant strains were unable to grow in the presence of glucose at pH values below 7.2.

Therefore, it was concluded that “authentic” slr1736, sll0418, and slr0089 mutants are

glucose-sensitive and are not able to grow in the presence of glucose. Further

characterization of the “authentic” mutants revealed that the chlorophyll a,

phylloquinone, and plastoquinone contents decreased when cells were grown in the

presence of glucose, and Photosystem II (PS II) activity was completely lost after 24 h.

However, levels of the D1 and PsbO proteins remained nearly constant, and the levels of

the sodB transcripts (encoding the superoxide dismutase) were virtually identical between

the wild-type and mutant strains before and after transfer to medium containing glucose.

These results indicate that the loss of the D1 and PsbO proteins is not responsible for the

loss of PS II activity and strongly support the conclusion that the glucose-induced

phenotype is not directly associated with oxidative stress. The authentic mutants

exhibited chlorosis when grown in the presence of glucose. The relative phycobiliprotein

contents decreased to nearly one-third of the wild-type level, and the concomitant

accumulation of the nblA (encoding a protein essential for the degradation of

phycobilisomes) transcript was detected. These results indicate that the authentic mutants

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experience macronutrient starvation when grown in the presence of glucose. Further

growth analyses revealed that the glucose-induced growth defects, PS II inactivation, and

the macronutrient starvation responses occur at pH below 7.2. At pH 7.0 in the presence

of glucose, the PS II activity was completely lost, and the growth ceased after 24 h. The

results further demonstrate that α-tocopherol plays an essential role for the growth and

survival of Synechocystis sp. PCC 6803 in the presence of glucose and suggest that α-

tocopherol plays a role in the regulation of macronutrient metabolism in cyanobacteria.

132

ABBREVIATIONS

Chl a chlorophyll a

DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea

HEPES 4-(2-hydroxyethyl)-piperazine-N’-(2-ethanesulfonic acid)

HGA homogentisic acid

HPP 4-hydroxyphenylpyruvate

HPPD 4-hydroxyphenylpyruvate dioxygenase

HPT homogentisate phytyltransferase

MPBQ 2-methyl-6-phytyl-1,4-benzoquinol

MPBQ MT 2-methyl-6-phytyl-1,4-benzoquinol methyltransferase

PBP phycobiliprotein



RT-PCR reverse transcriptase-polymerase chain reaction

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INTRODUCTION

α-Tocopherol (vitamin E) is a lipid-soluble, organic molecule that is synthesized

only by oxygen-evolving phototrophs, including some cyanobacteria and all green algae

and plants (Threlfall and Whistance, 1971; Collins and Jones, 1981). The conservation of

α-tocopherol synthesis during the evolution of photosynthetic organisms suggests that the

molecule performs one or more critical functions. In plants, α-tocopherol is synthesized

and localized in the plastids (Soll et al., 1980, 1985), and it is particularly abundant in the

thylakoid membranes (Fryer, 1992), in which it is thought to afford protection against

various oxidative stresses (Munné-Bosch and Lenor, 2000). Because α-tocopherol is also

an essential component of animal diets, most of our knowledge of tocopherol function

has been obtained from studies in these systems. Studies in whole animals, animal cell

cultures and artificial membranes have shown that tocopherols scavenge and quench

various reactive oxygen species and lipid oxidation byproducts, which would otherwise

propagate lipid peroxidation chain reactions in membranes (Kamal-Eldin and Appelqvist,

1996). In addition to these antioxidant functions, other “non-antioxidant” functions

related to modulation of signaling and transcriptional regulation in mammals have also

been reported (Chan et al., 2001; Azzi et al., 2002; Ricciarelli et al., 2002). In

photosynthetic organisms, tocopherol functions have not yet been rigorously defined, but

it is believed that they are likely to include some or all of the functions reported in

animals, as well as other possible functions specific to photosynthetic organisms.

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The biosynthetic pathway of α-tocopherol is summarized in Fig 4.1. It was

elucidated in plants in the mid-1980’s and during the past five years, most of the genes

encoding the enzymes of this pathway have been identified in both plants and

cyanobacteria (Threlfall and Whistance, 1971; d’Harlingue and Camara, 1985; Norris et

al., 1998; Shintani and DellaPenna, 1998; Collakova and DellaPenna, 2001; Schledz et

al., 2001; Porfirova et al., 2002; Savidge et al., 2002; Shintani et al., 2002; Dähnhardt et

al., 2002; Collakova and DellaPenna, 2003; Koch et al., 2003; Cheng et al., 2003).

Briefly, homogentisic acid (HGA) is the aromatic head-group precursor for all

tocopherols, and HGA is produced from 4-hydroxyphenylpyruvate (HPP) by the enzyme

HPP dioxygenase (Norris et al., 1998; Dähnhardt et al., 2002). The committed step in

tocopherol synthesis is the condensation of HGA and phytyl-pyrophosphate by the

enzyme homogentisate phytyltransferase to produce 2-methyl-6-phytyl-1,4-benzoquinol

(MPBQ) (Collakova and DellaPenna, 2001; Schledz et al., 2001; Savidge et al., 2002;

Collakova and DellaPenna, 2003). The ring of MPBQ is methylated by MPBQ

methyltransferase to yield 2,3-dimethyl-5-phytyl-1,4-benzoquinol (Soll et al., 1985;

Shintani et al., 2002; Cheng et al., 2003), which is cyclized by tocopherol cyclase to yield

γ-tocopherol (Soll et al., 1985; Porfirova et al., 2002; Sattler et al., 2003). Finally, a

second ring methylation by γ-tocopherol methyltransferase yields α-tocopherol (Soll et

al., 1980; d’Harlingue and Camara, 1985; Shintani and DellaPenna, 1998, Koch et al.,

2003). Genes encoding HPP dioxygenase (slr0090), homogentisate phytyltransferase

(slr1736), MPBQ methyltransferase (sll0418) and γ-tocopherol methyltransferase

(slr0089) have been identified in Synechocystis sp. PCC 6803 (Shintani and DellaPenna,

135

1998; Shintani et al., 2002; Collakova and DellaPenna, 2001; Dähnhardt et al., 2002,

Cheng et al., 2003). The remaining enzyme, tocopherol cyclase, has recently been shown

to be the product of slr1737 in Synechocystis sp. PCC 6803 (Sattler et al., 2003).

In the cyanobacterium Synechocystis sp. PCC 6803, the tocopherol biosynthetic

pathway has been studied genetically and homozygous mutants of the slr1736, sll0418,

and slr0089 genes, encoding homogentisate phytyltransferase, MPBQ methyltransferase,

and γ-tocopherol methyltransferase, respectively, have been obtained (Shintani and

DellaPenna, 1998; Shintani et al., 2002; Cheng et al., 2003; Collakova and DellaPenna,

2001). As expected from the biosynthetic pathway (Fig 4.1), the slr0089 mutant

accumulates only γ-tocopherol (Shintani and DellaPenna, 1998), the sll0418 mutant

accumulates 30% of the wild-type level of α-tocopherol and a small amount of β-

tocopherol (Cheng et al., 2003), and the slr1736 mutant does not accumulate any

tocopherols (Collakova and DellaPenna, 2001). In light of the established antioxidant

activity of α-tocopherol in animal systems, one would expect that the loss or reduction of

α-tocopherol would lead to a detectable phenotypic difference between the wild-type and

mutant strains. Intriguingly, however, the tocopherol-deficient slr1736 mutant grew

similarly to the wild-type strain both in the absence and in the presence of glucose at a

moderate light intensity (110 µE m-2 s-1) (Collakova and DellaPenna, 2001). A limited

comparative analysis of the photosynthetic activity of the wild-type and slr1736 mutant

strains failed to reveal any differences. This led the authors to conclude that α-tocopherol

is dispensable for the survival of Synechocystis sp. PCC6803 under the conditions tested

(Collakova and DellaPenna, 2001).

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In this study, it is demonstrated that the “authentic” tocopherol mutants are

glucose-sensitive and that they cannot maintain growth in the presence of glucose at pH

7.2 or lower. Mutant populations obtained in previous studies probably contained

mixtures of secondary suppressor mutations that accumulate during cultivation in the

presence of glucose, although their exact nature has yet to be identified. The glucose-

sensitive phenotype of the “authentic” mutants was characterized, and the data indicate

that α-tocopherol is essential for the survival of Synechocystis sp. PCC 6803 in the

presence of glucose at pH 7.2 or lower.

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RESULTS

Extremely Heterogeneous Colony Morphologies in Tocopherol Mutants Previously

Isolated in the Presence of Glucose

When maintained on solid media containing 5 mM glucose, three tocopherol

mutants, slr1736::aphII, sll0418::aphII, and slr0089::aphII that had been isolated in

previous studies in the presence of glucose, showed much larger variations in colony size

and pigmentation than what is typically observed for the wild-type strain (Fig 4.2 E-H).

The larger colonies exhibited more intense blue-green pigmentation, which is

characteristic of wild-type colonies, while the smaller colonies appeared more chlorotic

and were yellow-green in color. Generally such non-homogenous colony morphology is

indicative of heterogeneous genotypes within a given population. However, PCR analysis

of genomic DNA isolated from three mutant strains showed that the targeted, mutated

loci were homozygous and that no corresponding wild-type allele could be detected in

any of the three mutant strains. These results eliminate the possibility that the observed

colony heterogeneity (Fig 4.2) was due to incomplete segregation of the mutant loci or to

contamination of the cultures with wild-type cells. When the tocopherol mutants were

maintained in the absence of glucose, more uniform colony morphologies were observed

(Fig 4.2 A-D). Although the size of colonies showed somewhat larger variation as

compared to wild type, all colonies showed intense blue-green pigmentation. Therefore it

was concluded that the heterogeneity of colony morphologies observed for the tocopherol

mutants is a specific response to growth in the presence of glucose.

138

The growth characteristics of two cell lines, one derived from a small colony and

the other derived from a large colony of the slr0089 mutant selected in the presence of

glucose, were analyzed in the absence and in the presence of glucose. In the absence of

glucose, the growth behavior of both cell lines was virtually indistinguishable from that

of wild type (Fig 4.3 A and B). Upon transfer to media containing glucose, however, the

cell line derived from the small colony ceased growth within 24 h and did not recover

during the time course of measurements (Fig 4.3 C). The cell line derived from the large

colony grew somewhat more slowly than the wild-type strain, but it was able to maintain

growth during the course of the experiment (Fig 4.3 D). The dramatic phenotypic

difference between the two cell lines strongly suggested that they were genotypically

distinct, and that the mutant populations previously obtained by selection in the presence

of glucose (Shintani and DellaPenna, 1998; Collakova and DellaPenna, 2001; Shintani et

al., 2002) were genotypically inhomogeneous.

Tocopherol Mutants Are Glucose Intolerant

“Authentic” tocopherol mutant strains were constructed by transforming an

immotile, glucose-tolerant wild-type strain of Synechocystis sp. PCC 6803 with genomic

DNAs isolated from the slr1736, sll0418, and slr0089 mutants selected in the presence of

glucose. Because the presence of glucose was found to be unfavorable for some

populations of these tocopherol mutants, kanamycin-resistant transformants were selected

in the absence of glucose. Segregation of each mutant allele from the corresponding wild-

type allele was tested by PCR analysis using specifically designed oligonucleotide

primers (Fig 4.4). Products anticipated for the wild-type slr1736, sll0418, and slr0089

139

genes were 1.3 kbp, 1.0 kbp, and 1.2 kbp, respectively. No such product was observed for

any of the mutant strains; instead, a product ~1.3 kbp larger was detected in each mutant

line. This difference in size corresponds to the aphII-containing, kanamycin-resistance

cartridge originally used for the insertional inactivation of each gene. It was therefore

concluded that each mutant strain is homozygous and that the slr1736::aphII,

sll0418::aphII, and slr0089::aphII alleles in each mutant line segregated completely in

the absence of glucose.

The newly isolated slr1736, sll0418, and slr0089 mutants produced uniform

colonies and grew similarly to the wild type in the absence of glucose under moderate

light intensity (< 50 µE m-2 s-1; Fig 4.5 A). In the presence of glucose, however, all three

mutants showed severe growth defects and were incapable of maintaining growth after 24

h (Fig 4.5 B and C). This growth phenotype of the mutant strains selected in the absence

of glucose is very similar to that observed for the cell line derived from the small colonies

of the mutants selected in the presence of glucose (see Fig 4.3 C), but sharply contrasts

with the growth behavior of the cell lines derived from the large colonies (Fig 4.3 D).

These results clearly demonstrate that “authentic” slr1736, sll0418, and slr0089 mutants

are glucose sensitive. The data also suggest that α-tocopherol plays an essential role for

the survival of Synechocystis sp. PCC 6803 in the presence of glucose.

When cells from these “authentic” mutants grown in the presence of

glucose for 72 h were transferred to fresh medium containing no glucose, no

growth was detected even after 3 weeks of incubation. These results indicated that

these “authentic” mutants lost viability during cultivation in the presence of

140

glucose for 72 h. The pH of the cultures grown in the presence of glucose

typically shifted from the original value (pH 8.0) to a value in the range of 7.0 to

7.3. Preconditioned media prepared from cultures of the “authentic” mutants

grown in the presence of glucose supported the normal growth of the wild-type

cells. These results show that the glucose toxicity of the mutants is not the result

of products excreted from the cells in the presence of glucose.

To test whether the growth light intensity was responsible for the observed failure

of the “authentic” tocopherol mutants to grow in the presence of glucose, the “authentic”

slr1736 mutant was grown in the presence of glucose at various light intensities. Under

both weak (< 5 µE m-2 s-1) and strong (300 µE m-2 s-1) illumination conditions, however,

the presence of glucose in the growth medium resulted in the death of the “authentic”

slr1736 mutant (Fig 4.6 A and B). This indicates that light intensity is unlikely to be a

contributing factor in the observed glucose toxicity of the tocopherol mutants. The effects

of inhibitors of the electron transport chain on the glucose toxicity of the slr1736 mutant

were also studied. Addition of a sublethal concentration of DCMU (3-(3,4-

dichlorophenyl)-1,1-dimethyl-urea) to the growth medium led to a small recovery of

growth for the slr1736 mutant in the presence of glucose (Fig 4.6 C). However,

increasing the DCMU concentration further did not improve the growth of the slr1736

mutant. Addition of potassium cyanide in various concentrations did not rescue the

slr1736 mutant from the glucose toxicity (Fig 4.6 D).

The effects of other organic carbon sources were analyzed on the growth of the

“authentic” slr1736 mutant. The presence of 5 mM pyruvate, succinate, and fumarate,

141

which are intermediates of the TCA cycle, did not cause growth defects in the slr1736

mutant. (Fig 4.7 A-D). The presence of 15 mM glutamate did not affect the growth of the

slr1736 mutant (Fig 4.7 E). The presence of ornithine, however, resulted in growth

defects similar to those caused by glucose (Fig 4.7 F). The results suggested that the

toxicity induced by glucose is caused by one or more metabolic intermediate that

accumulate during glucose and ornithine catabolism. Ornithine is an intermediate in the

citrulline cycle, in which as much as 20 % of CO2 fixation can take place (Tabita, 1987).

On the other hand, glucose is metabolized through the glycolytic pathway or oxidative

pentose phosphate pathway. Intermediates of these pathways are also shared with the

Calvin-Benson cycle (reductive pentose phosphate pathway), in which the major CO2

fixation occurs (Tabita, 1987). The fact that both glucose and ornithine caused lethal

effects on the tocopherol mutants implies that α-tocopherol is perhaps involved in carbon

metabolism by coordinating inorganic and organic carbon metabolic balance in the cells.

Chlorophyll a, Phylloquinone and Plastoquinone Content

The complete loss of, or a decrease in, tocopherol content led to small changes in

the pigmentation of the “authentic” tocopherol mutants grown in the absence of glucose.

The chlorophyll a (Chl a), phylloquinone, and plastoquinone contents were

approximately 10%, 10-20 %, and 3-30% lower, respectively, in these tocopherol

mutants than in the wild type (Fig 4.8). The slr1736 mutant cells contained nearly the

same amount of Chl a and quinones as wild type, while the sll0418 and slr0089 mutant

cells contained somewhat lower amounts. These differences, however, did not seem to

affect the photosynthetic electron transport activity; the rates oxygen evolution observed

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for both whole-chain electron transport and the Photosystem II (PS II)-dependent electron

transport for the three tocopherol mutants were very similar to, or perhaps somewhat

higher than, those of wild type (Fig 4.9, see below).

When grown in the presence of glucose, however, the mutants exhibited more

dramatic changes in the contents of Chl a, phylloquinone, and plastoquinone. The Chl a

and phylloquinone contents were largely unchanged by 24 h after transfer from medium

without glucose to medium containing glucose and decreased significantly after 48 to 72

h to as low as 50% of the corresponding wild-type level. Under these conditions, the Chl

a as well as phylloquinone level of wild type was largely unaffected during the course of

measurements (Fig 4.8 A and B). Because phylloquinone is associated exclusively with

the Photosystem I complexes in cells, the parallel decreases in the Chl a and

phylloquinone contents may imply a decrease in the Photosystem I content in the cells.

In contrast, following transfer from a medium without glucose to a medium

containing glucose, the plastoquinone content gradually decreased in the wild-type strain

(Fig 4.8 C); by 48 h, the plastoquinone content of wild type was only half of that found in

the absence of glucose. An even more severe decrease was observed in the mutants, in

which the plastoquinone content decreased to ~50 % of the wild-type level by 24 h and as

low as 15 % by 72 h after transfer to medium containing glucose (Fig 4.8 C). Recent

studies have shown that the redox state of the plastoquinone pool in the thylakoid

membranes modulates the expression levels of photosynthetic genes and affects the

cellular contents of components of the photosynthetic apparatus, including the

phycobilisomes and Photosystem I (Alfonso et al., 2000; Li and Sherman, 2000). This

143

raises the question as to whether the dramatic decrease in the plastoquinone content of the

“authentic” mutants could lead to a change in its redox state, which in turn could be

responsible for the decrease in the phycobiliprotein content and PS II activity (see

below), and ultimately cell death. The redox state of the plastoquinone pool can be

modified by various growth conditions including the intensity of illumination applied

during cell growth. Low light intensity (< 5 µE m-2 s-1) typically causes reduction of the

plastoquinone pool while high light intensity (300 µE m-2 s-1) causes its oxidation

(Cooley and Vermaas, 2001). However, incubation under either of these light conditions

did not lead to recovery of growth in the “authentic” tocopherol mutants in the presence

of glucose (Fig 4.6 A and B). Similarly, addition of various concentrations of potassium

cyanide, which promotes reduction of the plastoquinone pool, did not alleviate the

glucose sensitivity of these mutants (Fig 4.6 D), and addition of DCMU, which causes

oxidation of the plastoquinone pool, did so only to a small extent (Fig 4.6 C). These

combined results therefore suggest that the changes in the plastoquinone pool size, and its

possibly altered redox state, are not the primary cause of the glucose toxicity observed for

the authentic tocopherol mutants in the presence of glucose.

Photosystem II Activity

Given the significant reduction in the Chl a, phylloquinone, and plastoquinone

contents in response to the presence of glucose, whole-chain and PS II-dependent

electron transport was assessed in the mutants (Fig 4.9). In cells grown in the absence of

glucose, the whole-chain and the PS II-dependent oxygen evolution rates of all three

mutants were similar to, or even slightly higher, than that of the wild type. Likewise,

144

somewhat higher O2-evolving activity (the whole-chain or the PS II-dependent) was

detected for the wild type when grown in the presence of glucose. In contrast, no O2-

evolving activity (the whole-chain and the PS II-dependent) was detected for the

tocopherol mutants when grown in the presence of glucose.

Loss of PS II activity is often seen when cells are exposed to oxidative stress

caused by exposure to extremely high light, to low temperature, or to an environment

containing a high salt concentration (Allakhverdiev et al., 1999; Hideg et al., 2000; see a

review by Aro et al., 1993, and references therein), and this phenomenon is known as

photoinhibition. Under such conditions, the D1 protein, a subunit that forms a part of the

PS II core structure (Ferreira et al., 2004), rapidly degrades, and, as a result, the activity

of PS II is lost. The possibility that the observed PS II inactivation is related to

photoinhibition in the tocopherol mutant in response to glucose was tested by

immunological assay. When cells were grown in the absence of glucose, the amount of

immunologically detectable D1 protein was virtually indistinguishable between the wild-

type and the mutant strains (Fig 4.10). After transfer to medium containing glucose, the

amount of immunologically detectable D1 protein was only slightly reduced in the

mutants compared to wild type, even though by 24 h a complete loss of PS II-mediated

oxygen evolution activity was observed (Fig 4.9). PsbO, or the 33 kDa protein, is a

subunit that forms a peripheral domain of PS II known as the oxygen-evolving complex

(Ferreira et al., 2004). This complex is responsible for the catalysis of water splitting and

the production of oxygen. The immunologically detectable PsbO protein was also largely

unaffected in response to glucose in both wild-type and three tocopherol mutant strains.

145

These results demonstrate that the loss of PS II activity in the tocopherol mutants in the

presence of glucose is not associated with structural damage that leads to a loss of the D1

protein and PsbO, and they suggest that oxidative stress is not the cause of the PS II

inactivation. The expression of the sodB gene, encoding superoxide dismutase, which is

known to accumulate several-fold in response to various reactive oxygen species

(Ushimaru et al., 2002), was analyzed by RT-PCR (Fig 4.11). The amounts of the sodB

transcript detected for the wild type and the slr1736 mutant were very similar when cells

were grown in the absence of glucose (Fig 4.11, shown at 0 h) and in the presence of

glucose (Fig 4.11, at 4, 8, and 24 h). The combined results therefore strongly suggest that

the loss of α-tocopherol does not cause oxidative stress in response to glucose and that

the inactivation of PS II is unrelated to oxidative stress.

Relative Phycobiliprotein Content

The relative content of phycobiliproteins (PBPs), the primary protein components

of the light-harvesting antennae known as phycobilisomes, was determined in response to

the transfer of the wild type and the “authentic” tocopherol mutants to medium

containing glucose (Fig 4.12 A). In the absence of glucose, all three tocopherol mutants

contained 20-25% less PBP than the wild type. After 24 h of growth in the presence of

glucose, the PBP content of wild-type cells decreased to ~60% of the level found in the

absence of glucose, while the PBP content of the three tocopherol mutants decreased

even more sharply to 18-26% of the level in their respective controls obtained for the

cells grown in the absence of glucose. Time-course analyses revealed that the reduction

of PBPs initiates by 4 h after transfer to medium containing glucose (Fig 4.12 B). In

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comparing the decreased PBP levels of the wild type and the three tocopherol mutants

during growth in the presence of glucose, it is obvious that the loss of PBPs is much more

severe in the three mutants than in the wild type.

These data are consistent with the pale, chlorotic (yellow-green) coloration of the

mutant colonies in the presence of glucose. The mutants had significantly lower

absorption in the orange-red region of their visible spectra, and as a result were less blue-

green in coloration than control cells. This bleaching, due to a decrease in the cellular

content of PBPs, is well known to be associated with nutrient starvation, and can be

caused by carbon (Miller and Holt, 1977), nitrogen (Allen and Smith, 1969; Foulds and

Carr, 1977; Wood and Haselkorn, 1980; Yamanaka and Glazer, 1980; Stevens et al.,

1981; Elmorjani and Herdman, 1987), phosphorous (Ihlenfeldt and Gibson, 1975), sulfur

(Schmidt et al., 1982; Jensen and Rachlin, 1984; Warner et al., 1986), or iron limitation

(Sherman and Sherman, 1983). The nblA gene, encoding a factor essential for the

controlled degradation of phycobilisomes, is known to accumulate in Synechocystis sp.

PCC 6803 cells in response to nitrogen deprivation (Richaud et al., 2001). RT-PCR

analyses showed that, in the wild-type samples, the nblA transcript was barely detectable

when cells were grown in the absence of glucose (Fig 4.11, shown at 0 h) and that a small

induction occurred by 4 h after transfer to medium containing glucose. In the slr1736

mutant sample, the wild-type level of the nblA transcript was detected in the absence of

glucose and a dramatic increase followed by 4 h after transfer to medium containing

glucose. This correlates well with the observed behavior of PBP contents in the

tocopherol mutants (Fig 4.12, also see above) and suggests that the slr1736 mutant is

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experiencing nutrient limitation in response to glucose. Because the BHEPES medium

used in culturing these mutants contains an appropriate mixture of all micronutrients and

macronutrients to support the growth of Synechocystis sp. PCC 6803 (Stainer et al., 1971;

see Materials and Methods), the observed macronutrient starvation response of the

mutants in the presence of glucose is most likely to be due to an impairment in an

essential biosynthetic or regulatory process, which involves carbon, nitrogen, and

glucose.

Involvement of CO2 in the Glucose Toxicity

The observed glucose-sensitive phenotype of the “authentic” tocopherol mutants

resembled that of the icfG mutant reported in a previous study (Beuf et al., 1994). The

icfG gene encodes a putative protein phosphatase similar to RsbU (anti anti-sigma factor

B) in Bacillus subtilis (Price, 2000; Woodbury et al., 2004). This mutant showed a

glucose sensitive-phenotype only at low CO2 [air level, 0.035% (v/v)] levels. The

presence of succinate, fumarate, pyruvate, and glutamate did not cause lethality at low

CO2, while the presence of ornithine caused a lethal effect. The striking behavioral

resemblance between the icfG mutant and the “authentic” tocopherol mutants in their

response to various carbon sources suggested a possible involvement of CO2 in the

glucose toxicity observed for the tocopherol mutants. Fig 4.13 shows the growth curves

of wild type and the “authentic” slr1736 mutant in the presence of glucose in air and 3 %

(v/v) CO2. The slr1736 mutant grew similarly to the wild type in air even in the presence

of glucose, and in fact, the growth of the two strains was virtually indistinguishable under

these conditions. When cells were grown in the presence of glucose at 3% (v/v) CO2,

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however, the slr1736 mutant exhibited severe growth defects as described above.

Therefore, these results demonstrated that the glucose toxicity occurs only at high CO2

levels. This led to the hypothesis that α-tocopherol is involved in the global regulation of

organic and inorganic carbon metabolism in Synechocystis sp. PCC 6803, possibly

through a signal transduction cascade involving IcfG.

Involvement of pH in Glucose Toxicity

The observed dependence of the glucose toxicity on the CO2 concentration was,

however, questioned after discussion with Prof. Aaron Kaplan of the Hebrew University,

Israel. In the presence of 3% (v/v) CO2, the growth medium was found to acidify from

the initial pH 8.0 to about pH 7 (see above). Acidification of cultures can be caused by a

high concentration of CO2, such as 3% (v/v), since the in organic carbon (Ci ) supplied

into medium as dissolved CO2 is rapidly hydrated and reaches equilibrium with HCO3-.

Thus, the possibility that the high-CO2 requirement of the glucose toxicity was actually

because of medium acidification was tested directly. The wild-type and the “authentic”

tocopherol mutants were grown at various pH values at 1% CO2 (v/v) in medium

BHEPES40, which is a modified BHEPES medium supplemented with 40 mM HEPES

for higher pH buffering capacity (see Materials and Methods). After culturing for 2 days

in this medium at 1% (v/v) CO2, the pH remained near the original value within a

deviation of ≤ 0.05 pH unit. In the absence of glucose, both the wild type and the

tocopherol mutant strains grew well at all pH values tested between 6.8 and 8.0, and no

significant difference was observed among the four strains (Fig 4.14). This establishes

that the loss of α-tocopherol does not affect the growth rates of the cells at all pH values

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between 6.8 and 8.0 in the absence of glucose. When the cells were transferred to media

containing glucose, the wild type grew faster as compared to the growth in the absence of

glucose (Fig 4.14), which confirms that the glucose is taken up and metabolized in the

wild-type cells. The tocopherol mutants grew similarly to wild type at pH 8.0 and 7.6

(Fig 4.14). However, they grew very poorly or not at all at pH 7.2, 7.0, and 6.8. In

comparing the pH-independent growth phenotype in the absence of glucose and the pH-

dependent growth in the presence of glucose, it is obvious that the observed glucose

toxicity of the tocopherol mutants occurs at pH 7.2 or lower. Therefore, the glucose-

sensitive phenotype characterized in detail in the earlier section of this chapter is also due

to the lowering of the pH caused by the acidification of the growth media during growth

in the presence of 3% (v/v) CO2.

Fig 4.15 shows that growth curves of the wild-type and the “authentic” slr1736

mutant cultures at various pH values in air and 1% (v/v) CO2. The slr1736 mutant grew

similarly to wild type at pH 8.0 and 7.6 both in air and 1% (v/v) CO2 (Fig 4.15 A and B).

At pH 7.2, the slr1736 mutant grew slightly slower than wild type during the initial 20 h

and grew even more slowly after 20 h (Fig 4.15 C). At pH 7.0 and 6.8, the mutant grew

substantially slower than wild type during the first 20 h and then stopped growing after

20 h (Fig 5.2 D and E). The growth behavior of the slr1736 mutant was virtually

indistinguishable either when grown with air or with 1% (v/v) CO2 (Fig 4.15 A-E). These

results demonstrate it is the pH of the medium and not the extracellular CO2

concentration that causes the glucose-sensitive growth phenotype of the slr1736 mutant.

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It is noteworthy that the pH-dependent glucose toxicity occurs after 20 h, which

suggests that the cells were accumulating a toxic substance during the initial 20 h. 3-O-

methyl-glucose is known to be transported into the cells through the glucose transporter;

however, it is not phosphorylated and therefore is not metabolized further (Beuf et al.,

1994). Both the wild type and the slr1736 mutant grew well in the presence of this

glucose analogue in the medium at pH 7.0 (Fig 4.16, top). The growth rates were very

similar to those in the absence of glucose (based on visual inspection). The fact that the

slr1736 mutant was able to grow similarly to wild type in the presence of this glucose

analog suggests that the cause of the glucose toxicity is not the presence of glucose per se

that accumulates inside the cells. The presence of 5 mM pyruvate, succinate, or fumarate

in growth medium at pH 7.0 caused the tocopherol mutant to grow slightly slower yet

very similarly to wild type (Fig 4.16, top). These results strongly suggest that the

causative agent for the glucose toxicity at pH 7.0 in the tocopherol mutants is likely to be

metabolites accumulated either during glucose metabolism through the glycolytic

pathway or the pentose phosphate pathway. The glucose toxicity was observed when

cells were grown at RT (ca. 25 °C). The growth experiments described so far were

conducted under constant pH values; in other words, the cells were grown at pH 7.0 in

the absence of glucose before being transfer to medium containing glucose at pH 7.0 in

order to assess the effect of glucose on the growth of these cells. Likewise, the cells were

grown at pH 8.0 in the absence of glucose before transferred to medium containing

glucose at pH 8.0. Fig 4.16 (bottom) shows a similar, yet somewhat different experiment.

In this experiment, the cells were initially grown at pH 8.0 in the absence of glucose and

then were transferred to medium containing glucose at pH 7.0. The purpose of this

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experiment was to test whether the incubation at pH 7.0 (in the absence of glucose)

“sensitizes” the tocopherol mutant cells to glucose or its by-product. The results showed

that the tocopherol mutants were unable to grow after transfer from medium at pH 8.0

(containing no glucose) to medium containing glucose at pH 7.0. This suggested that the

pH value of the medium before transfer to medium containing glucose has nothing to do

with the observed glucose toxicity in the tocopherol mutant cells.

Photosynthetic Activity

Fig 4.17 shows the activities of PS II measured for cells grown at various pH

values in the absence and the presence of glucose. In the absence of glucose, PS II

activities of the slr1736 cells (filled squares) were higher those that of wild type (filled

circles) at all pH values. Although the reason for this higher activity for the slr1736

mutant is not known, this result is reproducible, as can be also seen in Fig 4.9. In the

presence of glucose, the PS II activities of the wild-type cells were higher than those in

the absence of glucose at all pH values (open circles). On the contrary, no PS II activity

was detectable for the slr1736 mutant at pH 6.8 and 7.0. Higher activity was observed as

the pH value was increased, and at pH 8.0 the PS II activity was virtually

indistinguishable from that of wild type. Therefore, the inactivation of the PS II is also

dependent on the presence of glucose, the pH of the surrounding environment, and the

absence of tocopherols.

The PBP contents

When the cells were grown at pH 7.0 in the absence of glucose and with 1% CO2

(v/v), the PBP contents of the wild type and the “authentic” tocopherol mutants were

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virtually indistinguishable (Fig 4.18A). When cells were grown in the presence of

glucose at pH 7.0, however, the PBP contents in the tocopherol mutants decreased to

about one-third of the wild type level (Fig 4.18A). This is consistent with the results

described in Fig 4.12, and demonstrates that the tocopherol mutants are experiencing

macronutrient starvation in response to glucose at pH 7. The effect of pH on the PBP

content was analyzed for the wild-type and the slr1736 mutant cells grown in the

presence of glucose (Fig 4.18B). At all pH values tested, the PBP content in the wild-type

cells remained constant. The PBP content in the slr1736 mutant cells was significantly

lower, less than 40 % of the wild-type level at pH 7.0 and below, which confirms the

reproducibility of the pH-dependent macronutrient starvation response in the tocopherol

mutant as described above. As the pH of the medium increased, higher PBP contents

were detected. At pH 8.0, the PBP content of the slr1736 mutant was 70% of the wild-

type level. The observed reduction of the PBP content over a wide range of pH values

strongly indicates that the presence of glucose alone induces a macronutrient starvation

response in the tocopherol mutants and that the macronutrient starvation response is

severely exacerbated at pH values below 7.2.

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DISCUSSION

In this study, it was determined that the previously reported Synechocystis sp.

PCC 6803 mutants of genes slr1736, sll0418, and slr0089, encoding homogentisate

phytyltransferase, MPBQ methyltransferase and γ-tocopherol methyltransferase,

respectively, are probably genotypically heterogeneous mixtures of secondary suppressor

mutants. These mutants with defects in tocopherol biosynthesis were originally selected

and maintained in the presence of glucose, and although the targeted genes were

completely disrupted, a variety of colony growth morphologies have now been observed,

which is consistent with genetic heterogeneity. When these mutant lines were

reconstructed by a second round of transformation, followed by selection and

maintenance in the absence of glucose, the resulting mutant lines (here denoted as

“authentic” mutants to distinguish them from the original mutants previously selected in

the presence of glucose) were also found to be completely disrupted at each respective

locus, but the observed colony morphology and growth characteristics were now uniform.

It was found that the “authentic” mutants are highly sensitive to the presence of glucose

at pH values less than 7.2, and that the growth of each mutant strain ceased within 24 h

following transfer to medium containing glucose under those conditions. These results

clearly show that α-tocopherol is conditionally essential for the survival of Synechocystis

sp. PCC 6803 in the presence of glucose. It is likely that the selection and culturing of the

original transformants in the presence of glucose caused the selection of secondary

suppressor mutations that permitted survival under these otherwise suboptimal or lethal

conditions. Although the exact nature of the suppressor mutations has yet to be

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determined, one can propose a few possibilities that enable the glucose-sensitive cells to

become glucose tolerant: inactivation of the glucose transporter, alteration of flux through

the glycolytic and/or oxidative pentose phosphate pathways, or the alteration of the

regulatory pathway(s) that controls glucose metabolism (Beuf et al., 1994, Hihara et al.,

1997, also see below).

The glucose toxic phenotype was found to be dependent on the pH of medium and

occurs at pH ~7.2 or lower. PS II inactivation and the reduction of PBP contents were

also found to be pH-dependent when glucose is present in the medium and α-tocopherol

synthesis is interrupted. Similar pH-dependent growth defects and reduction of

photosynthetic activities were observed for the cotA mutant (Katoh et al., 1996). CotA is

involved in light-induced proton extrusion, and its absence has been shown to lead to

severe growth defects at pH 7.0 or lower. Therefore, it is possible that the glucose

toxicity observed for the “authentic” tocopherol mutants are related to defects in proton

extrusion. However, a simple comparison between the cotA mutant and the tocopherol

mutants is not appropriate, because of the significant phenotypic differences between

them. The cotA mutant shows pH-dependent growth defects in the absence of glucose,

whereas the tocopherol mutant does not under the same conditions. Growth rate of the

cotA mutant is slower than wild type (60-70% of the wild-type level) at pH 8.0 in the

absence of glucose, whereas the tocopherol mutants grow similarly to wild type under the

same conditions (visual inspection). Therefore, the pH-dependent glucose toxicity of the

tocopherol mutants seems to be very complex and involves one or more factors that are

associated with the pH homeostasis of the cells. Further studies are required to resolve

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the molecular mechanism of the pH-dependent glucose toxicity in the tocopherol

mutants.

Trebst and co-workers reported that the herbicide-mediated loss of α-tocopherol

led to the complete loss of PS II activity with concomitant degradation of the D1 protein

following high light treatment in the green alga Chlamydomonas reinhardtii (Trebst et

al., 2002). This was certainly not the case in the tocopherol mutants of Synechocystis sp.

PCC 6803, as the level of the D1 protein remained similar to the wild type even when the

PS II activity was completely lost. The growth light intensity (50 µE m-2 s-1) used in this

study was less than one half of the saturating intensity, and this value seems unlikely to

promote a photoinhibitory oxidative stress. In fact, the glucose toxicity observed for these

mutants is light-independent, since it occurs over a wide range of light intensity, from < 5

to 300 µE m-2 s-1, as described in the Results. Given that the transcript level of the sodB

gene is largely unaffected in the mutants under these lethal conditions, it is highly

unlikely that the inactivation of PS II and the death of the cells are directly associated

with an oxidative stress. Furthermore, the deleterious effects of glucose on the slr1736,

sll0418, and slr0089 mutants were virtually indistinguishable despite the varying

compositions and contents of tocopherols in each mutant. Should α-tocopherol function

solely as an antioxidant, an increasing susceptibility to glucose with decreasing

tocopherol content would reasonably be expected among the various tocopherol mutants,

but this is not observed. Therefore, it appears most likely that α-tocopherol plays a role

other than, or at least in addition to, that as a bulk antioxidant in the survival of

Synechocystis sp. PCC 6803 in the presence of glucose. Taking all of these considerations

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together, it was concluded that the observed metabolic perturbation, PS II inactivation,

and cell death of the tocopherol mutants grown in the presence of glucose at pH values

below 7.2 are unlikely to be directly associated with the antioxidant activity of α-

tocopherol in Synechocystis sp. PCC 6803. Moreover, it appears most likely that α-

tocopherol plays a role other than as a bulk antioxidant in the survival of Synechocystis

sp. PCC 6803 in the presence of glucose.

PBPs are the major constituent of the light harvesting antennae in cyanobacteria,

and they can constitute nearly half of the total soluble protein (Grossman et al., 1994).

When cyanobacteria are subjected to macronutrient deprivation conditions for carbon,

nitrogen, phosphorus, sulfur, and iron, the synthesis of PBPs ceases and their active

degradation begins (Allen and Smith, 1969; Ihlenfeldt and Gibson, 1975; Foulds and

Carr, 1977; Yamanaka and Glazer, 1980; Wood and Haselkorn 1980; Stevens et al.,

1981; Schmidt et al., 1982; Sherman and Sherman, 1983; Jensen and Rachlin, 1984;

Elmorjani and Herdman, 1987). It is generally believed that the degradation of these

proteins provides amino acids for energy production and maintenance protein synthesis,

while reducing the potential for photooxidative stress due to light stress at PS II.

Therefore, it is highly likely that the observed, dramatic decrease in the PBP contents in

the “authentic” tocopherol mutants during growth in the presence of glucose represents a

response to a perceived state of nutritional deprivation. Supporting this interpretation is

the observation that transcription of the nblA gene, known to accumulate in response to

nitrogen deprivation (Richaud et al., 2001), also accumulated in the tocopherol-deficient

mutant in response to glucose. The combined results hence suggest that the complete loss

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of, or a substantial decrease of, α-tocopherol perturbs metabolic processes related to

nitrogen, carbon, and sulfur, and that this disruption ultimately leads to phycobilisome

degradation and cell death.

Recent studies in mammalian systems have demonstrated the involvement of α-

tocopherol in modulating the production of signaling molecules and pathways (Chan et

al., 2001; Azzi et al., 2002; Ricciarelli et al., 2002). For example, α-tocopherol has been

shown to bind to phospholipase A2 specifically at the substrate-binding pocket and act as

a competitive inhibitor, thereby decreasing the release of arachidonic acid for eicosanoid

synthesis (Chandra et al., 2002). α-Tocopherol has also been suggested to modulate the

phosphorylation state of protein kinase Cα in rat smooth-muscle cells, possibly via

phosphorylation of protein phosphatase 2A (Ricciarelli et al., 1998). α-Tocopherol is also

directly involved in transcriptional regulation in animals, including the expression of the

genes encoding liver collagen αI, α-tocopherol transfer protein, and α-tropomyosin

collagenase (Yamaguchi et al., 2001; Azzi et al., 2002). Therefore, it seems possible that,

in addition to acting as a bulk lipid-soluble antioxidant, α-tocopherol could also play a

regulatory role in Synechocystis sp. PCC 6803, possibly in the regulation of metabolic

processes such as those involved in carbon and nitrogen metabolism.

Two glucose-tolerant mutants reported previously appear to be relevant to the

observations reported here. The pmgA (sll1986) and icfG (slr1680) loci have been shown

to be determinants for glucose sensitivity (Hihara and Ikeuchi, 1997; Beuf et al., 1994).

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Inactivation of the pmgA locus has been shown to result in a glucose-sensitive phenotype

and enhanced photoautotrophic growth capability (Hihara and Ikeuchi, 1997).

Inactivation of the icfG locus, on the other hand, results in a glucose-sensitive phenotype

under low CO2 conditions (Beuf et al., 1994). The “authentic” tocopherol mutants, whose

phenotypes are characterized in this chapter, are glucose-sensitive both under high and

low CO2 environments but only at pH values less than 7.2. The amino acid sequences

deduced from the pmgA and icfG coding regions show weak similarity to RsbT/W (a

serine/threonine protein kinase) and RsbU/X (a protein phosphatase and a negative

regulator of sigma factor B) in Bacillus subtilis. These proteins form a signal transduction

network with other Rsb proteins and regulate the activity of sigma factor B through

phosphorylation/dephosphorylation interactions upon perception of environmental stress

(Price 2000; Woodbury et al., 2004). Homologous protein sequences of other Rsb

proteins are also found in the Synechocystis sp. PCC 6803 genome: Slr2031 (RsbU/X),

Ssr1600 (RsbS/V), Slr1856 (RsbS/V), and Slr1861 (RsbT/W). It is therefore plausible

that an Rsb-like pathway is present in this organism and may participate in the regulation

of glucose metabolism. In fact, studies have shown that recombinant Slr1861

phosphorylates Slr1856 and IcfG dephosphorylates the phosphorylated Slr1856 (Shi et

al., 1999). Whether the role of α-tocopherol is related to this hypothetical pathway is an

open question and is a subject of continuing investigation.

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SUMMARY

The tocopherol mutants (slr1736, sll0418, and slr0089), which had been

previously isolated in the presence of glucose, were shown to be phenotypically and

genotypically heterogeneous populations. Newly isolated “authentic” tocopherol mutants

were glucose-sensitive and were not able to grow in the presence of glucose after 24 h.

This glucose toxicity was shown to occur when the mutants were grown at pH values

below 7.2. Under these conditions, the PS II activities were significantly reduced or not

detected at all in the tocopherol mutants.

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Growth Conditions and Strains

The slr1736, sll0418, and slr0089 mutants were generated by targeted insertion

with the aphII gene, which confers kanamycin resistance, as described previously

(Shintani and DellaPenna, 1998; Collakova and DellaPenna, 2001; Shintani et al., 2002).

The wild-type strain used for transformation and further analysis was a glucose-tolerant

strain of Synechocystis sp. PCC 6803 (Williams, 1988). Medium BHEPES, pH 8.0, was

used for selection, maintenance, and growth measurements of wild type and mutants.

This medium is prepared by supplementing BG-11 medium (Stainer et al., 1971) with 4.6

mM HEPES [4-(2-hydroxyethyl)piperazine-N’-(2-ethnesulfonic acid)]-KOH and 18 mg

L-1 ferric ammonium citrate. Medium BEHEPS40, modified medium BEHEPS after

supplementing with 40 mM HEPES for higher buffer strength, was used for pH-

dependent or pH-controlled growth analyses. Wild-type cells were maintained on solid B-

HEPES medium containing 1.5 % (w/v) agar and 5 mM glucose, and the “authentic”

tocopherol mutants were maintained on solid medium BHEPES containing 1.5 % (w/v)

agar, 50 µg kanamycin ml-1, and importantly, no glucose. Prior to all growth

measurements and preparations for subsequent analyses, cells were transferred from solid

to liquid medium BHEPES and allowed to grow exponentially for several generations in

the absence of glucose. For determination of growth characteristics, late-exponential

phase cultures were diluted in fresh liquid medium BHEPES to an OD730 nm of

approximately 0.05 cm-1. The diluted cultures were grown at 32 °C with continuous

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bubbling with air containing 1% or 3% (v/v) CO2. Growth was monitored by measuring

the optical density at 730 nm. For growth in the presence of glucose, the medium was

supplemented with 5 mM glucose. The growth light intensity was 50 µE m-2 s-1, unless

otherwise noted. For growth measurements in the presence of inhibitors of the electron

transport chain, the following concentrations were used: 0.03 µM DCMU (3-(3,4-

dichlorophenyl)-1,1-dimethyl-urea); 2, 20, or 200 µM potassium cyanide. Pre-

conditioned medium was prepared from a culture of an authentic tocopherol mutant that

had been grown in the presence of glucose for 72 h. The cells were removed by

centrifugation at 8000 × g, and the supernatant was filtered through a sterile 0.22 µm

pore-size membrane. The filtrate was then mixed with five-fold strength medium

BHEPES in a four-to-one ratio (v/v) to achieve a normal, one-fold-strength medium.

Isolation of Tocopherol Mutants

Synechocystis sp. PCC 6803 wild type was transformed with genomic DNA

extracted from the slr1736, sll0418, and slr0089 mutants selected in the presence of

glucose as previously described (Williams, 1988). Segregation of mutant alleles from the

wild type allele was carried out in the absence of glucose and in the presence of 50 µg

kanamycin ml-1. Oligonucleotide primers used for PCR analysis were as follows; slr1736

forward primer (5’-GGCTTCTCCTACCCGGAATTCTACTTCCTG-3’), slr1736 reverse

primer (5’-GCTTTCTAAGTGTACATCTAGACTCCGCCA-3’), sll0418 forward primer

(5’-ATGCCCGAGTATTTGCTTCTGCC-3’), sll0418 reverse primer (5’-

GCACTGCTTTGAACATACCGAAG-3’), slr0089 forward primer (5’-

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TCTACCGGAAATTGCCAACTACCA-3’), and slr0089 reverse primer (5’-

CCTAGGAGATTGTGGACTTCAA-3’).

Oxygen Evolution and Consumption Measurements

Cells grown in the absence and in the presence of glucose for 24 h were harvested

by centrifugation at 8,000 × g at room temperature and resuspended in a 25-mM HEPES

buffer, pH 7.0, to obtain an optical density of 1.0 cm-1 at 730 nm. For whole-chain

oxygen evolution and consumption measurements, the cell suspensions were incubated in

the dark in the presence of 10 mM NaHCO3 for at least 15 min prior to measurements.

For the Photosystem II (PS II)-dependent oxygen evolution measurements, 1 mM 1,4-

benzoquinone and 0.8 mM K3Fe(CN)6 were added instead of NaHCO3 and measurements

were performed immediately. The excitation light intensity was approximately 3 mE m-2

s-1. The oxygen concentration was measured polarographically with a Clark-type

electrode as previously described (Sakamoto and Bryant, 1998).

Quantification of Pigments and Quinones by High Performance Liquid Chromatography

Chl a in whole cells was extracted with methanol, and its molar content per unit

optical density at 730 nm was determined using the molar absorption coefficient 82 L g-1

cm-1 (MacKinney, 1941). For quinone analyses, cells were harvested by centrifugation at

8,000 × g, at 4 °C for 6 min, washed in 1 ml of 50 mM Tris-HCl buffer, pH 8.0, and

centrifuged at 18,000 × g for 1 min to obtain tight cell pellets. Pigments and quinones

were extracted by ultrasonication in 400 µl of cold acetone-methanol (7:2, v/v) in the

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dark. High performance liquid chromatography (HPLC) analysis was performed with a

Agilent 1100 system equipped with a diode array detector (Agilent Technologies,

Wilmington, DE, USA). One hundred microliters of extract was mixed with 10 µl of 1 M

ammonium acetate and injected onto a NovaPak C18 column (3.9 × 300 mm, 4 µm

packing, Waters, Milford, MA). Analytes were eluted by a linear gradient program

consisting of solvent A (100% methanol) and solvent B (100% isopropanol) using the

following protocol: [100% A] for 10 min, [100% A] to [20% A : 80% B] in 20 min, [20%

A: 80% B] for 5 min, and [20% A : 80% B] to [100% A] for 5 min. The flow rate was

0.75 ml min-1. The Chl a, phylloquinone, and plastoquinone contents of each sample

were determined based on integrated peak areas and their molar absorption coefficients,

which are 17.4 mM-1 cm-1 at 618 nm, 18.9 mM-1 cm-1 at 270 nm (Dunphy and Brodie,

1971), and 15.2 mM-1 cm-1 at 254 nm (Crane and Dilley, 1963), respectively. The

absorption coefficient of Chl a at 618 nm was calculated from the ratio of the absorption

peaks at 618 nm and 666 nm in methanol-isopropanol (6:4, v/v) and from its absorption

coefficient at 666 nm in methanol (MacKinney, 1941). The quinone content per unit

optical density at 730 nm was calculated by multiplying the molar ratio of quinone to Chl

a by the molar Chl a content per optical density at 730 nm.

Estimation of Relative Content of Phycobiliproteins

Cells were harvested by centrifugation at 8000 × g for 6 min, and pellets were

resuspended in the 25 mM HEPES buffer, pH 7.0, to obtain 2 ml of the cell suspensions

with an optical density of 0.5 cm-1 at 730 nm. One milliliter of this suspension was heated

164

at 100 °C for 1 min. A modification was made in the method previously described (Zhao

and Brand, 1989) for estimation of the relative PBP content. The absorbance at 635 nm

and 730 nm were recorded for unheated and heated samples, and the data were then

inserted into the following equation: the relative PBP content = (∆OD635nm –

∆OD730nm)/OD730nm·unheated, where ∆ indicates ODunheated sample minus ODheated sample

SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting of the D1 protein

Cells were grown in the absence of glucose to mid-exponential phase or in the

presence of glucose for 24 h, and harvested by centrifugation at 8,000 × g at 4 °C for 6

min. The resulting cell pellets were immediately frozen in liquid N2 and stored at –80 °C

until being used. Frozen cells were thawed on ice and resuspended in the 25 mM HEPES

buffer, pH 7.0, at an optical density of 100 cm-1 at 730 nm. An equal volume of glass

beads was added to the cell suspension, which was allowed to stand on ice for 5 min.

Using a home-built bead-beater, the samples were vigorously shaken 4 times for 30 s at 4

°C with 30 s intervals on ice. An aliquot (10 µl) of each sample was mixed with an equal

volume of loading buffer, and the mixture was incubated at 65 °C for 20 min, and applied

onto a discontinuous SDS-polyacrylamide gel with 10% (w/v) acrylamide in the

separating gel as described (Schägger and van Jagow, 1987). Electrophoresis was carried

out at 50 V for 12 h. The antibodies raised against the amino acids 234-242 of the D1

protein of Synechocystis sp. PCC 6803 were a kind gift from Prof. Eva-Mari Aro of the

University of Turku, Finland. The antibodies raised against the PsbO protein of

Synechocystis sp. PCC 6803 were a kind gift from Prof. Robert Burnap of the Oklahoma

165

State University, USA. After electrophoresis, proteins were transferred to a nitrocellulose

membrane. Proteins were detected by immunoblotting by using an enhanced

chemiluminescence reagents and analysis system (Amersham Biosciences, Piscataway,

NJ) according to manufacturer’s specifications.

Isolation of total RNA and RT-PCR analyses

Total RNA was isolated from cells using the Mini-to-Midi RNA isolation kit

(Invitrogen Corp., Carlsbad, CA) followed by an RNase-free DNase treatment, and

repurification by using the same kit. The RNAs obtained were adjusted to a final

concentration of 50 ng mL-1 in RNase free double distilled water and store at -80°C until

being used. RT-PCR reactions were carried out by using the One-Step RT-PCR kit

(Qiagen Inc., Valencia, CA) for detecting the rnpB transcript. The reaction volume was

25 µL, and 4 ng µL-1 of the total RNAs was used. For detecting the nblA and sodB

transcripts, 400 ng of total RNA was first converted to cDNA by the RT reaction using

M-MLV reverse transcriptase (Promega, Madison, WI). The reaction volume was 20 µL

for each sample. The resulting cDNA was diluted with 80 µL of TE buffer, and stored at

–20 °C until being used. Two microliters of cDNA stock were used for PCR (final

volume, 25 µL) using Hot-Start Taq polymerase (Qiagen Inc., Valencia, CA). RNasin

(Promega, Madison, WI), an RNase inhibitor, was present in one-step RT-PCR reactions

and during the cDNA synthesis.

166

ACKNOWLEDGEMENTS

The authors thank Prof. Eva-Mari Aro (University of Turku) for kindly providing the D1

antibodies, Prof. Robert Burnap (Oklahoma State University) for kindly providing the

PsbO antibodies, and Prof. Aaron Kaplan, Hebrew University for discussion, critical

comments, and suggestions.

167

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Fig 4.1: Biosynthetic pathway of α-tocopherol in Synechocystis sp. PCC 6803. HPPD, 4-hydroxyphenylpyruvate dioxygenase; HPT, homogentisate phytyltransferase; MPBQ MT, 2-methyl-6-phytyl-1,4-benzoquinone methyltransferase; TC, tocopherol cyclase; γ-TMT, γ-tocopherol methyltransferase.

177

Fig 4.2: Colony morphologies of three tocopherol mutants previously isolated in the presence of glucose. Cells were grown in the absence of glucose (A-D) and in the presence of glucose (E-H). Wild type (A, E), the slr1736 mutant (A, E), the sll0418 mutant (C, G), and the slr0089 mutant (D, H), on solid BHEPES medium.

178

Fig 4.3: Growth curves of wild type and cell lines derived from small (A, C) and large colonies (B, D) of tocopherol mutants selected in the presence of glucose (see Fig 4.2) at 32 °C, 50 µE m-2 s-1 with 3% (v/v) CO2. The data shown for each strain are averages of at least three independent cultures, and standard error bars are shown. Growth in the absence of glucose (A, B) and in the presence of glucose (C, D). (A, C) Wild type (filled circles) and a cell line derived from a small colony slr0089 mutant selected in the presence of glucose (open circles) (see Fig 4.2). (B, D) Wild type (filled circles) and cell lines derived from large colonies the slr0089 (open circles), sll0418 (open triangles), and slr1736 mutants (open squares) selected in the presence of glucose (see Fig 4.2).

179

Fig 4.4: PCR analysis of the genomic DNAs extracted from the newly isolated tocopherol mutants selected in the presence of glucose. (A) The slr1736 mutant, (B) the sll0418 mutant, and (C) the slr0089 mutant. Lane 1 and lane 2 in each panel show PCR products amplified from the wild-type and mutant genomic DNA templates, respectively. The DNA fragments amplified from the mutant templates are 1.3-kb longer than those from the wild-type template because of the insertion of the aphII cassette encoding resistance to kanamycin.

180

Fig 4.5: Growth curves of wild type and the “authentic” tocopherol mutants (A) in the absence and (B, C) in the presence of glucose at 32 °C, 50 µE m-2 s-1, 3% (v/v) CO2. Closed circles indicate the wild-type strain; open squares, triangles, and circles indicate the authentic slr1736, sll0418, and slr0089 mutant strains, respectively. The data shown for each strain are averages of three independent cultures, and standard error bars are shown.

181

Fig 4.6: Growth curves of wild type and the “authentic” slr1736 mutant in the presence of glucose at 32 °C, 3% (v/v) CO2. Growth (A) at 300 µE m-2 s-1, (B) at < 5 µM m-2 s-1, (C) in the presence of 0.03 µM DCMU at 50 µE m-2 s-1, and (D) in the presence of potassium cyanide (2 µM and 20 µM) at 50 µE m-2 s-1. Closed circles and open squares indicate the wild-type and the “authentic” slr1736 mutant strains, respectively. Data shown for each strain are averages of three independent cultures, and standard error bars are shown. Dotted lines in (D) and (E) indicate the presence of DCMU and potassium cyanide, respectively.

182

Fig 4.7: Growth analyses of wild type and the “authentic” slr1736 mutant of Synechocystis sp. PCC 6803 at 32 °C, 50 µE m-2 s-1, 3% CO2 (v/v), in the presence various carbon sources. (A) 5 mM glucose, (B) 5 mM succinate, (C) 5 mM pyruvate, (D) 5 mM fumarate, (E) 15 mM glutamate, and (F) 15 mM ornithine. Filled circles and open squares indicate the wild-type and the slr1736 mutant strains, respectively. Data shown here average of three independent measurements, and standard error bars are shown. In panel F, the tube on the left is the wild-type strain grown in the presence of 15 mM ornithine, while the tube on the right is the slr1736 mutant grown under the same conditions.

F

183

Figure 4.8: Chlorophyll a (Chl a) contents, and quinone contents in wild type and the “authentic” tocopherol mutants grown in the absence and in the presence of glucose. (A) Chl a content, (B) phylloquinone (PhyQ) content, and (c) plastoquinone (PQ) content. The data shown are averages of three determinations, and standard error bars are shown. Cultures grown in the absence of glucose (shows at 0 h) were transferred to media containing glucose for 24, 48, and 72 h. Wild type, the slr0089, the sll0418, and the slr1736 mutants are shown in black, diagonally slashed, checked, and open columns, respectively.

184

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��

��

��

��

0

300

600

900

1200

Fig 4.9: Oxygen-evolution activities of the whole-chain and the Photosystem II-dependent electron transport chains in wild type and the “authentic” tocopherol mutants of Synechocystis sp. PCC 6803. O2-evolution activities of whole-chain electron transport (H2O HCO3

-) were measured for cells grown in the absence of glucose (black columns) and in the absence of glucose (diagonally slashed columns). O2-evolution activities of PSII-dependent electron transport (H2O 1,4-BQ/K3Fe(CN)6) were measured for cells grown in the absence of glucose (checked columns) and in the presence of glucose (open columns). Data shown here average of five independent measurements, and standard error bars are shown. The cells were grown for 24 h under the designated conditions before measurements. N.D., not detectable.

WT slr1736 sl0418 slr0089

N.D. N.D.

µmol

O2 (

OD

730n

m h

L)-1

N.D. N.D. N.D. N.D.

185

Fig 4.10: Immunoblotting analysis for the D1 and PsbO proteins in whole cells of wild type and three “authentic” tocopherol mutants grown in the absence of glucose (Photoautotrophic) and in the presence of glucose (Photomixotrophic) at 32 °C, 50 µE m-2 s-1, with 3% (v/v) CO2. Proteins from equal amount of cells (10 µL of cell suspension with OD730 nm =100) was loaded in each lane. After electrophoresis, proteins were transferred to a nitrocellulose membrane. Proteins were detected by immunoblotting as described in the Materials and Methods.

186

Fig 4.11: Time-dependent RT-PCR analysis of the sodB and nblA transcripts in wild type and the “authentic” slr1736 mutant of Synechocystis sp. PCC 6803 The cells were initially grown in the absence of glucose (shown at 0 h) and transferred to media containing 5mM glucose and grown for 4, 8, 24 h at 3% CO2 (v/v), 32°C, 50 µE m-2 s-1. rnpB, encoding the catalytic RNA subunit of RNase P, was used as control.

187

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��

0

0.2

0.4

0.6

0.8

1

0

0.4

0.8

1.2

Fig 4.12: Relative phycobiliprotein contents in wild type and the “authentic” tocopherol mutants of Synechocystis sp. PCC 6803 grown at 3% (v/v) CO2, 32°C, 50 µM E m-2 s-1. (A) Cells grown in the absence of glucose (black columns) were transferred to glucose-containing BHEPES media and grown for 24 h (diagonally slashed columns). (B) Time-course analyses in wild type (closed circles) and the slr1736 mutant (open squares) grown in the absence of glucose (shown at 0 h), and transferred to medium containing glucose and grown for 4, 8, and 24 h. The values represent the ratio relative to wild type grown in the absence of glucose. Data shown here are the average of six independent measurements, and standard error bars are shown.

Rel

ativ

e ph

ycob

ilipro

tein

con

tent

WT slr1736 sll0418 slr0089

Rel

ativ

e ph

ycob

ilipro

tein

con

tent

0 4 8 24 Time (h)

B

A

188

Fig 4.13: Growth curves of wild type and the “authentic” slr1736 mutant in the presence of glucose at 3% (v/v) CO2 and air. Closed circles and open squares indicate the wild-type and the slr1736 mutant strains, respectively. Solid and dotted lines indicated the presence of 3% (v/v) CO2 and air in medium, respectively. Cells were grown at 32°C, 50 µE m-2 s-1.

189

Fig 4.14: Cultures of wild type and the “authentic” tocopherol mutants of Synechocystis sp. PCC 6803 grown at various pH for 2 days at 1% (v/v) CO2, 32°C, 50 µE m-2 s-1. Cells were grown in the absence (above) or in the presence (below) of glucose in medium BHEPES40 at pH 6.8, 7.0, 7.2, 7.6, and 8.0.

190

Fig 4.15: Growth curves of wild type and the “authentic” slr1736 mutant of Synechocystis sp. PCC 6803 at various pH values in the presence of glucose in air and 1% (v/v) CO2. Cells were grown in BHEPES40 medium at (A) pH 8, (B) pH 7.6, (C) pH 7.2, (D) pH 7.0, (E) pH 6.8, in 1% (v/v) CO2 (circles) and air (squares). Wild type and the “authentic” slr1736 mutant strains are indicated with filled symbols and open symbols, respectively. Growth analyses were carried out at 50 µE m-2 s-1, 32°C.

191

Fig 4.16: Cultures of wild type and the “authentic” slr1736 mutant of Synechocystis sp. PCC 6803 grown at various conditions for 48 h at pH 7. (Top) Growth in the presence of 5 mM pyruvate, fumarate, succinate, and 3-O-methyl-glucose at pH 7. (Bottom) Growth in the presence of 5 mM glucose at pH 7 with 5 mM NaCl (left), at room temperature (middle), and cultures pre-conditioned at pH 8. Growth analyses were carried out at 50 µE m-2 s-1, 32°C, 1% (v/v) CO2 using medium BHEPES40.

192

Fig 4.17: PS II-dependent O2-evolution activities of wild type and the “authentic” slr1736 mutant grown in the absence (closed symbols) and in the presence (open symbols) of glucose. Wild type and the slr1736 mutant are indicated by circles and squares, respectively. Cells were grown in medium BHEPES40 at the designated pH. O2 evolution was measured for 2 mL of cell suspension (OD 730nm = 1.0) in 50 mM HEPES buffer, pH 7.0, using 1mM 2,6-dimethyl-1,4-benzoquinone as the electron acceptor. Excitation light intensity was ca. 3 mE m-2 s-1. Temperature of the measurement chamber was maintained at 30 °C. A Clark-type electrode equipped with a magnetic stirrer was used for the assay.

193

Fig 4.18: Phycobiliprotein contents in wild type and the tocopherol mutants of Synechocystis sp. PCC 6803 grown at 1% (v/v) CO2, 32°C, 50 µE m-2 s-1. (A) Wild type, and the slr1736, sll0418, and slr0089 mutants were grown for 24 h at pH 7 in the absence (black columns) and in the presence (open columns) of glucose. (B) Wild type and the slr1736 mutant were grown for 24 h at various pH values in the presence of glucose.

194

Chapter 5

Transcriptional Regulation of Metabolic and Sigma Factor Genes in the Vitamin E-

Deficient Mutant of Synechocystis sp. PCC 6803

Publication:

Yumiko Sakuragi, Dean DellaPenna, Donald A. Bryant, manuscript in preparation

195

ABSTRACT

Transcriptional regulation of metabolic genes and sigma factor genes was studied in the

vitamin E (α-tocopherol)-deficient mutant (slr1736) of Synechocystis sp. PCC 6803.

When glucose was present in growth medium at pH 8.0, the transcripts of nblA (a factor

essential for the controlled degradation of PBP), sbpA (the periplasmic sulfate binding

protein), and sigB, sigC, sigD, sigE, sigH, sigI (alternative sigma factors) were shown to

accumulate in the tocopherol-less mutant cells. These results indicate that the α-

tocopherol mutant responds to the presence of glucose as though nitrogen, carbon, and

sulfur were limiting in the environment. When glucose is present in the growth medium

at pH 7.0, the tocopherol mutant ceased growth after 20 h. Under these conditions, the

amounts of the sigA (the principal sigma factor), rbcL (the RuBisCO large subunit), and

ccmK1 and ccmL (carboxysome shell proteins) transcripts diminished after 4 h. Because

the amounts of these transcripts remained unaltered when cells were grown in the

absence of glucose or in the presence of glucose pH 8.0, it was concluded that the pH-

dependent, glucose-induced misregulation of the sigA and rbcL are largely responsible

for the glucose-induced lethality of the tocopherol mutants under these conditions. Thin-

section electron micrographs revealed the absence of carboxysomes in the tocopherol

mutant grown in the presence of glucose at pH 7.0. The transcription of slr2031 (a

putative protein phosphatase that shows sequence similarity with RsbU and that is

essential under sulfur- and nitrogen-limiting conditions) and ndhF3, ndhR, sbtA

(inorganic carbon uptake mechanisms) were shown to be constitutively down-regulated

under all conditions tested in the tocopherol mutant. This result demonstrates that α-

196

tocopherol is involved in the transcriptional regulation of these metabolic genes in

Synechocystis sp. PCC 6803. Thin-section electron micrographs of the tocopherol-less

mutant showed that a large number of glycogen granules accumulated in the cells when

grown even in the absence of glucose. The targeted interruption of the pmgA gene (a

putative anti-sigma factor homologous to RsbW) resulted in a similar pH-dependent,

glucose-induced lethal phenotype. Based on these results, it is concluded that α-

tocopherol acts as a global regulatory molecule in carbon, sulfur, and nitrogen

metabolism in Synechocystis sp. PCC 6803.

197

ABBREVIATIONS

CCM inorganic carbon concentrating mechanisms

DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea

HEPES 4-(2-hydroxyethyl)-piperazine-N’-(2-ethanesulfonic acid)

PBP phycobiliprotein


RT-PCR reverse transcriptase-polymerase chain reaction

RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase

198

INTRODUCTION

α-Tocopherol is synthesized only in oxygenic phototrophs including

cyanobacteria, green algae and higher plants (Threlfall and Whistance 1971; Collins and

Jones, 1981). Given that α-tocopherol constitutes an essential component of our diet,

most of our knowledge concerning the roles and functions of this molecule has been

obtained from studies in animal systems. Based on results gathered in animals, animal

cell cultures, and artificial membranes, it has been shown that tocopherols function as

antioxidants and scavenge or quench various reactive oxygen species and lipid oxidation

byproducts that would otherwise propagate lipid peroxidation chain reactions in

membranes (Kamal-Eldin and Appelqvist, 1996). This antioxidant role of α-tocopherol

has now been demonstrated in eukaryotic phototrophs owing to studies in the past several

years. The herbicide-mediated interruption of the α-tocopherol biosynthesis pathway has

been shown to render PS II more susceptible to oxidative stress under extremely high

light illumination in the green alga Chlamydomonas reinhardtii (Trebst et al., 2002). The

presence of α-tocopherol has been shown to suppress the propagation of lipid

peroxidation during seedling germination in Arabidopsis thaliana (Sattler et al., 2004).

Thus, there is little doubt that α-tocopherol functions as a bulk antioxidant in the

eukaryotic phototrophs as well as in animals. The antioxidant role of α-tocopherol is also

being investigated in the cyanobacterium Synechocystis sp. PCC 6803, and the results

obtained so far support an antioxidant role of α-tocopherol in this organism (H. Maeda,

Y. Sakuragi, D. A. Bryant, and D. DellaPenna, personal communication)

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Perhaps the most novel and exciting functions of α-tocopherol have been

uncovered by various research groups during the last few years. In addition to the

antioxidant functions, other “non-antioxidant” functions related to modulation of

signaling and transcriptional regulation in mammals have also been reported (Chan et al.,

2001; Azzi et al., 2002; Ricciarelli et al., 2002). For example, α-tocopherol has been

shown to bind to phospholipase A2 specifically at the substrate-binding pocket and to act

as a competitive inhibitor, thereby decreasing the release of arachidonic acid for

eicosanoid synthesis (Chandra et al., 2002). α-Tocopherol is also directly involved in

transcriptional regulation in animals, including the expression of the genes encoding liver

collagen αI, α-tocopherol transfer protein, and α-tropomyosin collagenase (Yamaguchi

et al., 2001; Azzi et al., 2002). The presence of α-tocopherol resulted in a modulation of

the phosphorylation state of protein kinase Cα in rat smooth-muscle cells, possibly via

phosphorylation of protein phosphatase 2A (Ricciarelli et al., 1998). None of these non-

antioxidant functions of α-tocopherol has yet been experimentally demonstrated in

oxygenic phototrophs.

In the previous studies described in Chapter 4, the role(s) of α-tocopherol in

cyanobacteria was investigated by using three mutants, slr1736, sll0418, and slr0089,

deficient in enzymes involved in the α-tocopherol biosynthetic pathway (see Fig 4.1).

The tocopherol mutants are able to grow similarly to wild type at pH values between 6.8

and 8.0 in the absence of glucose (control conditions), under which conditions the PS II

activities and the phycobiliprotein (PBP) contents were comparable to those of wild type.

After transfer to a medium containing glucose at pH 8.0, the tocopherol mutants also

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grew similarly to wild type and the PS II activity remained unaltered, although the PBP

content decreased by ~30 % (permissive conditions). After transfer to medium containing

glucose at pH 7.0, however, the tocopherol mutants ceased growth by 24 h, and the PS II

activity was completely lost. Under these conditions the PBP content was reduced by ≥

60 % (lethal conditions). The involvement of oxidative stress in the PS II inactivation

was largely ruled out, because the levels of the D1 protein and the sodB transcript were

comparable between wild type and the tocopherol mutants under these conditions. Based

on the observed reduction in the PBP content and the accumulation of the nblA transcript,

which is known to accumulate in response to nutrient limitation (Collier and Grossman,

1994; Richaud et al., 2001), it was suggested that the tocopherol mutants are experiencing

macronutrient starvation. This led to the hypothesis that α-tocopherol plays a role as a

regulatory molecule in macronutrient metabolism in Synechocystis sp. PCC 6803.

In this chapter, a study of the transcriptional regulation of metabolic genes and

sigma factor genes in a tocopherol-deficient mutant (slr1736) is described. Of 39 genes

initially analyzed, the amounts of the transcripts of 10 genes were shown to respond

either to the presence of glucose alone or to the presence of glucose and pH in the

medium, whereas the amounts of the transcripts of 4 genes were shown to be

constitutively down regulated upon loss of α-tocopherol. Based on these observations, it

is concluded that α-tocopherol is involved in transcriptional regulation of some key

metabolic genes and plays a role in the global coordination of macronutrient metabolism

in Synechocystis sp. PCC 6803.

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RESULTS

Transcription of nblA and sbpA

Given the significant reduction of the PBPs in response to glucose and lowered

pH (see Chapter 4), the nblA transcript was analyzed in wild type and slr1736 mutant

cells grown at pH 7.0 and pH 8.0 (Fig 5.1). In the absence of glucose, the wild-type strain

barely accumulated detectable nblA transcripts both at pH 7.0 and pH 8.0, and only a

small increase followed in response to glucose in the growth medium. This appeared

somewhat contradictory to the observed behavior of the PBP content in the wild type, in

which the PBP content increased by 20% after transfer to medium containing glucose

when cells were grown at pH 7.0 (see Chapter 4, Fig 4.18). Furthermore, the medium

used for culturing these cells contains a growth-rate saturating concentration of nitrate

(ca. 18 mM) (Stainer 1971; see Materials and Methods). Thus, it is inconceivable that

these cells were actually limited by the availability of nitrogen. The presence of glucose

in the medium might lead to an increase in the intracellular carbon pool and, as a result,

facilitate the faster growth of this organism. These “carbon-saturated”, fast-dividing cells

perhaps are not capable of metabolizing nitrogen at an equivalent rate, and thus respond

as though they are limited by nitrogen in their environment.

The slr1736 mutant accumulated a higher amount of nblA transcript in the

absence of glucose as compared to the wild-type control (Fig 5.1, shown at 0 h), which

suggests that the slr1736 mutant is “perceiving” nitrogen limitation even in the absence

of glucose. In response to glucose, the amount of the nblA transcripts increased

dramatically in the slr1736 mutant after 4 h when cells were grown in medium at pH 7.0

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(Fig 5.1). Similar although more moderate increases were observed when cells were

grown in the presence of glucose at pH 8.0. These results indicate that the slr1736 mutant

is “perceiving” a severe nitrogen limitation at pH 7.0 and moderate nitrogen limitation at

pH 8.0. This is consistent with the dramatic reduction of PBP contents at these pHs (ca.

40% of the wild-type level at pH7.0 and ca. 70% of the wild-type level at pH 8.0).

The sbpA transcript, encoding the periplasmic sulfate binding protein, is known to

accumulate in response to sulfate limitation (Laudenbach et al., 1991). No detectable

sbpA transcripts were present in wild-type cells grown at pH 7.0 and pH 8.0 in the

absence or presence of glucose (Fig 5.1). In the slr1736 mutant cells grown at pH 7.0, a

small yet discernible amount of the sbpA transcripts was detected in the absence of

glucose, and these transcripts dramatically increased by 8 h after transfer to medium

containing glucose. When the slr1736 mutant cells were grown at pH 8.0, no sbpA

transcripts were detectable in the absence of glucose, and a gradual increase was

observed in 24 h after transfer to a medium containing glucose. These results indicate that

the slr1736 mutant is “perceiving” sulfur limitation even in the absence of glucose at pH

7.0, and the response is exacerbated after transfer to a medium containing glucose. It is

noteworthy that the amounts of the sbpA and nblA transcripts accumulated at pH 7.0 and

8.0 coincided well with the degree of reduction of the PBP contents at these pHs in the

presence of glucose (See Chapter 4, Fig 4.18, and Fig 5.1). The combined results hence

demonstrate that the reduction of the PBP content is associated with “perceived” nitrogen

and sulfur limitation in the slr1736 mutant.

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The cpcA transcripts, encoding the phycocyanin α-subunit, which is a subunit of

the most abundant PBPs, were found to decrease gradually in the tocopherol mutant in

response to glucose when the slr1736 mutant cells were grown at pH 7.0. At pH 8.0 its

level remained largely unaltered both in wild type and the slr1736 mutant before and

after addition of glucose. Therefore, it was concluded that the mechanism of the

reduction of the PBP content at pH 7.0 and 8.0 in the slr1736 mutant grown in the

presence of glucose are different. At pH 8.0, the reduction of the PBP content primarily

involves active degradation of the PBP proteins, while at pH 7.0 it involves both the

active degradation, as characterized by the accumulation of the nblA transcript, and the

transcriptional down regulation of the genes encoding PBPs, which further supports the

conclusion that the cells are perceiving nutrient limitation and responding to it (Grossman

et al., 1994; also see references therein)

Transcription of Alternative Sigma Factor Genes

The Synechocystis sp. PCC 6803 genome encodes eight σ70-type alternative sigma

factors (SigB through I) in addition to the principal sigma factor (SigA), and they play

roles as transcription factors that are activated in response to various kinds of stresses

(Caslake et al., 1997; Gruber and Bryant, 1998; Huchauf et al., 2000; Muro-Pastor et al.,

2001; Imamura et al., 2003a, b). Fig 5.2 shows the results of the RT-PCR analysis of

sigB, sigC, sigD, sigE, sigH, and sigI in wild type and the slr1736 mutant in the absence

of glucose and in the presence of glucose. SigB has been suggested to be involved in a

variety of stress responses, including dark response (Imamura et al., 2003a) and heat-

shock response (Huchauf et al., 2000) in Synechocystis sp. PCC 6803, and carbon and

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nitrogen starvation responses in Synechococcus sp. PCC 7002 (Caslake et al., 1997).

When cells were grown at pH 7.0, the amount of the sigB transcripts was largely

unaltered in wild type before and after addition of glucose to the medium (Fig 5.2). In the

slr1736 mutant, the amount of the sigB transcript was somewhat higher in the absence of

glucose as compared to the wild-type control, followed by a large increase by 4 h after

transfer to medium containing glucose. A similar response was observed for the slr1736

mutant grown at pH 8.0. Therefore the amount of the sigB transcripts responded to

glucose alone and was not influenced by the pH of the medium. Similar accumulation

patterns of transcripts were also observed for sigC, sigH, and sigI. SigH is involved in the

heat-shock response (Hackauf et al., 2000), while SigC, which is related to SigE in

Synechococcus sp. PCC 7002, is likely to be involved in the post-exponential phase of

growth (Gruber and Bryant, 1998). The role of SigI has not yet been elucidated. The

results suggest that the slr1736 mutant is experiencing a mixture to stresses in response of

glucose.

Transcription of sigE is under the control of NtcA, a global nitrogen regulator,

which binds to its characteristic binding site located in the upstream flanking sequence of

the sigE transcription initiation site and induces transcription of sigE in response to

nitrogen deprivation (Muro-Pastor et al., 2001). Interestingly, the amount of the sigE

transcripts increased in response to glucose in wild type both at pH 7.0 and 8.0 (Fig 5.2).

This is consistent with the pattern of the accumulation observed for the nblA transcripts

(Fig 5.1, see above). Although the reason for this glucose-induced nitrogen limitation

response has not yet been defined, it is possible that this is due to an altered intracellular

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carbon-nitrogen balance caused by increased carbon flux induced by the glucose

metabolism (see also above). In the slr1736 mutant, the amount of the sigE transcripts

increased more dramatically than for the wild type in response to glucose. This glucose-

induced accumulation of the sigE transcripts occurred both at pH 7.0 and pH 8.0, which

is consistent with the results obtained for the nblA transcripts (Fig 5.1, see above) and

which indicates that the slr1736 mutant is “perceiving” a nitrogen limitation in response

to glucose.

In contrast to sigE, the amount of the sigD transcripts was largely unaffected by

the presence of glucose in wild type (Fig 5.2). In the tocopherol mutant, the amount of

the sigD transcript was lower than that of wild type in the absence of glucose and

increased by several-fold in response to glucose (by visual inspection). SigD is highly

expressed during exposure to high light (Imamura et al., 2003a,b). It has been suggested

that this light-dependent behavior of sigD expression is regulated by the redox state of PS

II (Imamura et al., 2003a). Because the growth conditions used in this study employ

continuous illumination of the cultures with a moderate light intensity (50 µE m-2 s-1),

and because the PS II activity is nearly at the wild-type level for the slr1736 mutant in the

absence of glucose at pH 7.0 and 8.0 or in the presence of glucose at pH 8.0, the observed

alteration of the sigD transcription can not be explained simply by the previously

suggested light-responsive role of SigD.

Taking these results together, it was firmly concluded that the transcription of

these alternative sigma factor genes is modulated in the slr1736 mutant in response to

glucose and that pH plays little role in the glucose-induced response of the transcription

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of the alternative sigma factors. This indicates that α-tocopherol plays a role in regulating

the stress response related at least to carbon, nitrogen and sulfur metabolism in

Synechocystis sp. PCC 6803.

Transcription of sigA and Ci Genes

The sigA gene encodes the principal sigma factor, which is responsible for the

transcription of genes encoding proteins essential for growth and reproduction in

eubacteria including cyanobacteria (Gruber and Bryant, 1997; Caslake and Bryant, 1996).

The amount of the sigA transcripts was analyzed for wild type and the slr1736 mutant

grown at pH 7.0 and 8.0 (Fig 5.3). At pH 7.0, the sigA transcripts in wild type remained

constant after addition of glucose to medium, which is consistent with the fact that the

wild-type cells grow normally under these conditions. The sigA transcript level in the

slr1736 mutant at pH 7.0 was virtually identical to that for the wild type in the absence of

glucose, which is also consistent with the fact that the slr1736 mutant grows similarly to

wild type in the absence of glucose at pH 7.0. The level of the sigA transcripts remained

unaltered for 4 h when the slr1736 mutant was transferred to medium containing glucose

at pH 7.0. However, after 8 h the sigA transcript level significantly diminished. Such a

dramatic reduction in the amount of the sigA transcript is likely to cause a reduction of

the content of SigA available for transcription of house-keeping genes, which would in

turn severely impair the growth capacity of the cells. Indeed, the tocopherol mutants are

unable to grow in the presence of glucose at pH 7.0 (see Chapter 4, Fig 4.14 and Fig

4.15). In contrast, when the slr1736 mutant was grown in the presence of glucose at pH

8.0, the amount of the sigA transcript was virtually unaltered and was very similar to that

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in the wild type control. This is consistent with the fact that the slr1736 mutant grew

similarly to wild type under these conditions (see Chapter 4, Fig 4.14 and Fig 4.15). The

direct correlation between the amounts of the sigA transcripts and the growth behavior of

the slr1736 mutant in the absence and in the presence of glucose at pH 7.0 and 8.0

strongly indicate that the pH-dependent glucose toxicity is caused by the pH-dependent,

glucose-induced alteration of the sigA transcript level in the slr1736 mutant.

Accompanying the reduction of the amounts of the sigA transcript, the amounts of

the transcripts of carboxysome-related genes (rbcL, ccmK1, ccmL), a carbon-

concentrating mechanism (CCM) gene (ndhF4), and sigG were also decreased in a pH-

and glucose-dependent manner in the slr1736 mutant (Fig 5.3). rbcL encodes the large

subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which is

responsible for the fixation of CO2 and is essential for the survival of the cells, because

this gene cannot be completely inactivated in cyanobacteria (Pierce et al., 1989). ccmK1

and ccmL encode carboxysome shell proteins, which form the outer shell of

carboxysomes and encapsulate RuBisCO within the carboxysomal structure (Price et al.,

1993). ndhF4 encodes a subunit of a multi-subunit complex that is a low-affinity

constitutive CO2 uptake transporter (Shibata et al., 2001). The reduction in the levels of

the carboxysome transcripts most likely leads to the reduction in the amount of

carboxysomes in the cells; as a result, the ability to fix CO2 would be severely impaired

(Price et al., 1993).

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Intracellular Structures

Thin-section electron micrographs of wild-type and slr1736 mutant cells grown at

pH 7.0 in the absence and the presence of glucose were recorded. Fig 5.4 shows the

electron micrographs of wild type grown in the absence (Fig 5.4A) and in the presence of

glucose (Fig 5.4B). In the wild-type cells grown in the absence of glucose, the space

between the thylakoid membranes was filled with electron-opaque material, which most

likely represents the protein-rich phycobilisomes (Fig 5.9A). In the cytoplasm, electron-

opaque polyhedral bodies are visible, which are characteristic of carboxysomes. Slightly

larger electron-transparent bodies with round or oval shapes are most likely due to poly-

β-hydroxybutyrate (Stainer, 1988). In the wild-type cells grown in the presence of

glucose (Fig 5.4B), on the other hand, the intermembrane space is filled with small

electron-transparent objects with irregular shapes, which are characteristic of glycogen

granules. Glycogen granules serve as general carbohydrate reserves (Stainer, 1998). The

cellular content of glycogen accounts for 10-20 % of the dry cell weight when cells are

growing photoautotrophically and exponentially, whereas it accounts for up to 60 % of

dry cell weight when cells are subjected to nitrogen-limiting conditions (Stainer, 1988;

Smith 1982). That few glycogen granules are observed in the wild-type cells grown in the

absence of glucose is consistent with the fact that these cells grow in a balanced manner

and optimally under these conditions. The observed accumulation of glycogen in the

wild-type cells grown in the presence of glucose most likely reflects a carbon-saturated,

or nitrogen-limited metabolic state of the cells.

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Fig 5.5A shows an electron micrograph of an slr1736-mutant cell grown in the

absence of glucose at pH 7.0. The intracellular structures, such as the presence of

thylakoid membranes, carboxysomes, and poly-β-hydroxybutyrate, are very similar to

those of the wild-type control grown under the same conditions (Fig 5.4 A). However, in

the slr1736 mutant, the intermembrane space was filled with glycogen granules. This

clearly contrasts with the situation in the wild-type cells, in which only a small number of

glycogen granules was detectable (Fig 5.4A). Rather, this feature of the slr1736 mutant

cell resembles that of the wild-type cell grown in the presence of glucose (Fig 5.4B). This

suggests that the slr1736 mutant accumulates an unusually large amount of carbohydrate

even in the absence of glucose. Therefore, the slr1736 mutant cells seem either to be

experiencing nitrogen limitation, or to be performing CO2 fixation or glycogen synthesis

much more efficiently than the wild-type cells. The former is not likely to be the case as

the mutant cells grow similarly to wild type under these conditions. Therefore, it is most

likely that the mutant is overproducing photosynthate. In either case, it is obvious that the

“metabolic state” of the slr1736 mutant is different from that of the wild type even in the

absence of glucose.

When the slr1736 mutant was grown in the presence of glucose, the appearance of

the intracellular structures changed dramatically (Fig 5.5B). The intermembrane space is

no longer electron-opaque, which is consistent with the significant reduction in the PBP

content. No carboxysomes were observed in the majority of the cells, which supports the

results that the transcripts of the carboxysome genes are substantially reduced in the

slr1736 mutant under these conditions. The number of layers formed by the thylakoid

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membranes, however, does not seem to be affected significantly. Interestingly, new oval

structures with an electron-opaque appearance are visible between the thylakoid

membranes; these structures were absent in wild type grown in the absence and in the

presence of glucose and in the slr1736 mutant grown in the absence of glucose.

Constitutive Alteration of the CCM Genes and slr2031 Expression

Given the alteration of the “metabolic state” in the slr1736 in the absence of

glucose, further transcription analyses were carried out in order to understand the

involvement of α-tocopherol in macronutrient metabolism. Of 39 genes analyzed for

their transcript level, 4 genes (ndhF3, ndhR, sbtA, slr2031) showed constitutive down-

regulation both in the presence and in the absence of glucose (Fig 5.6). ndhF3, ndhR, and

sbtA encode components of high-affinity CO2 uptake transporters (Klughammer et al.,

1999; Shibata et al., 2001, 2002), a repressor of the ndhF3 operon, and the sodium-

dependent bicarbonate transporter, respectively, and all participate in inorganic carbon

concentrating mechanisms (CCM) under limited inorganic carbon environments. The

transcripts of these genes were detected in wild type both in the absence and the presence

of glucose at pH 7.0 and 8.0. On the contrary, the transcript levels for these genes were

significantly reduced in the slr1736 mutant both in the presence and in the absence of

glucose both at pH 7.0 and 8.0. Thus, the constitutive alteration of these CCM genes is

directly linked to the loss of α-tocopherol.

These CCM genes are known to be upregulated in response to low inorganic

carbon environments, and are hardly detectable under high CO2 conditions as analyzed

by Northern-blot hybridization (Figge et al., 2001). This raises a question as to whether

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the detection of the CCM genes in this study [at 1% (v/v) CO2] is due to an artifact such

as contamination with genomic DNA during the RNA isolations. The RNA preparation is

carried out through three steps, including isolation, RNase-free DNase treatment, and

finally purification; thus, it is unlikely that genomic DNA is present in the preparation.

Indeed, PCR analyses on the same RNA templates used for RT-PCR analyses using

primers targeted to the rnpB gene resulted in no product formation, which confirms that

the RNA preparations are free of genomic DNA contamination. Currently, we do not

have an explanation other than attributing the discrepancy to a difference in the

sensitivity of the detection methods. Typically one-step RT-PCR analyses using the

Qiagen One-Step RT-PCR kit (Qiagen inc., Valencia, CA) requires 50 pg µL-1 to 5 ng

µL-1 of total RNA in order to detect the target transcripts, whereas typical Northern-blot

hybridization reactions require several micrograms of RNA. Indeed, when more sensitive

methods were employed, other groups have also detected the presence of the ndhF3 and

sbtA transcripts in RNA preparations obtained from cells grown at high CO2 level [3-5%

(v/v) CO2] (Shibata et al., 2002; McGinn et al., 2003).

slr2031 encodes a putative protein phosphatase (similar to RsbU) involved in the

regulation of SigB in B. subtilis. In wild-type cells, the amount of slr2031 transcripts

were low yet detectable in the absence of glucose, and transcript levels substantially

increased by 8 h in response to glucose both at pH 7.0 and pH 8.0 (Fig 5.6). In the

slr1736 mutant, the slr2031 transcript is hardly detectable in the absence of glucose, and

only a slight induction was observed in response to glucose at pH 7.0. At pH 8.0, the

slr2031 transcript behaved similarly at pH 7.0, although a moderate increase was

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observed in the presence glucose by 24 h. In Synechocystis sp. PCC 6803, Slr2031 has

been shown to be somehow involved in sulfur and nitrogen metabolism, and its absence

is deleterious under sulfur- and nitrogen-limiting conditions (Huchauf et al., 2000). An

independent study has shown that the presence of Slr0231 is also essential when the cotA

gene is inactivated (Katoh et al., 1995). CotA has been suggested to be involved in light-

induced proton extrusion and CO2 transport in Synechocystis sp. PCC 6803 (Katoh et al.,

1996a,b). Thus, it is possible that Slr2031 is involved in the transcriptional regulation of

genes that are involved in regulating processes including carbon, nitrogen, and sulfur

metabolism. The fact that the expression of slr2031, as well as ndhF3, ndhR, and sbtA, is

constitutively down-regulated in the absence of α-tocopherol demonstrates that α-

tocopherol plays a role in the transcriptional regulation of metabolic genes in


Is the significant reduction of slr2031 transcription directly related to the cause of

the pH-dependent glucose toxicity of the tocopherol mutant? In order to answer this

question, an slr2031 disruption mutant was generated by insertion of the aadA gene

cassette, conferring spectinomycin resistance, into the unique MfeI site of the slr2031

coding region. Complete segregation of the mutant allele from the wild-type allele was

demonstrated by PCR analysis (Fig 5.7). Using the primers designed to target and

amplify a 0.9-kb N-terminal portion of the slr2031 loci resulted in the amplification of a

0.9-kb DNA fragment when the genomic DNA isolated from wild type was used as the

template. No 0.9-kb product was detectable when the genomic DNA isolated from the

slr2031::addA transformant was used as the template. Instead, a product of 2 kb was

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detected. The size difference between the two products corresponds to the size of the

addA gene cassette, which demonstrates that the slr2031::addA transformant is

homozygous and free from the slr2031 wild-type allele.

A preliminary growth analysis was carried out at pH 7.0 and pH 8.0. The slr2031

mutant (slr2031::aadA) was able to grow both at pH 7.0 and pH 8.0 in the absence of

glucose. When the cells were transferred to medium containing glucose, the slr2031

mutant was able to grow similarly to wild type both at pH 7.0 and pH 8.0 (Fig 5.8 A and

B). An slr1736 slr2031 double mutant behaved similarly to the slr1736 mutant at both

pHs in the presence of glucose (Fig 5.8 A and B). Therefore, it was concluded that the

constitutive down-regulation of slr2031 transcription is not the direct cause of the pH-

dependent glucose toxicity observed for the tocopherol mutant.

As mentioned in Chapter 4, the pmgA mutant of Synechocystis sp. PCC 6803 is

unable to grow in the presence of glucose, which indicates that PmgA plays a role in the

regulation of photomixotrophic growth (growth on CO2 and glucose as the carbon source)

(Hihara and Ikeuchi, 1997). To test whether the role of PmgA is related to the role of α-

tocopherol, a pmgA inactivation mutant was constructed by inserting the accC1 gene

cassette, conferring gentamicin resistance, into the unique SpeI site within the pmgA

coding region. The complete segregation of the mutant allele from that of wild type was

demonstrated by PCR analysis (Fig 5.7). PCR using the primers designed to target and

amplify a 0.8-kbp of the pmgA locus resulted in the amplification of a 0.8-kbp fragment

when genomic DNA isolated from wild-type cells was used as the template. No 0.8-kb

product was detectable for the PCR product when genomic DNA isolated from the

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pmgA::aacC1 transformant was used as the template. Instead, a product of 1.9 kb was

detected. The size difference between the two products corresponds the size of the aacC1

gene cassette, which demonstrates that the pmgA::aacC1 transformant is homozygous

and free from the pmgA wild-type allele.

A preliminary growth analysis was carried out in the absence of glucose and in

the presence of glucose at pH 7.0 and pH 8.0. The results showed that the pmgA mutant

grew similarly to wild type in the absence of glucose at pH 7.0 and at pH 8.0 (by visual

inspection). In the presence of glucose, the pmgA mutant grew similarly to wild type in

medium at pH 8.0; however, its growth was found to be severely impaired in the presence

of glucose at pH 7.0. This apparent similarity in the growth behavior between the

tocopherol and pmgA mutants in response to glucose and pH suggests that α-tocopherol

and PmgA play roles in the same regulatory pathway. PmgA shows similarity to

RsbW/RsbT, protein serine/threonine kinase, in B. subtilis (Price CW, 2000; Woodbury

et al., 2004). RsbW functions as an anti-SigB factor, while RsbT functions as an activator

of RsbU, which in turns activates SigB in the signal transduction cascade that regulates

the activity SigB. They act together with other protein phosphatases and kinases

including RbsU, an Slr2031 homologue (Price CW, 2000; Woodbury et al., 2004). More

than 6 Rsb-like proteins were found to be conserved in the genome of Synechocystis sp.

PCC 6803, and PmgA, Slr2031, and Ssr1600 are also found to be present in the genomes

of Thermosynechococcus elongatus BP-1, Nostoc sp. PCC 7120, Nostoc punctiforme,

Trichodesmium erythraeum, Synechococcus sp. PCC 7002, Prochlorococcus marinus sp.

MED4, P. marinus sp. MIT9313, P. marinus sp. SS120, and Synechococcus sp.

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WH8102. Thus, it is highly plausible that a pathway similar to the Rsb pathway in B.

subtilis is present in cyanobacteria and regulates the balance between carbon, nitrogen

and sulfur metabolism.

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DISCUSSION

The potential involvement of α-tocopherol in macronutrient metabolism was

investigated. RT-PCR analyses performed on the cells grown at pH 7.0 and 8.0 in the

absence and in the presence of glucose demonstrated that the transcripts of the alternative

sigma factors accumulate in the slr1736 mutant in response to glucose, which indicated

that the slr1736 mutant is experiencing a variety of stresses when grown in the presence

of glucose (Caslake et al., 1997; Gruber and Bryant, 1998; Huchauf et al., 2000; Muro-

Pastor et al., 2001; Imamura et al., 2003a,b). The nblA and sigE transcripts, known to

accumulate in response to nitrogen limitation (Mulo-Pastor, 2001; Richaud 2001), also

accumulated in response to glucose in the slr1736 mutant. The sbpA transcript, known to

accumulate in response to sulfur limitation (Laudenbach and Grossman, 1992), also

accumulated in response to glucose in the slr1736 mutant. In combination with the results

obtained for the PBP content as described in Chapter 4, these results demonstrate that

slr1736 mutant is experiencing macronutrient starvation when grown in the presence of

glucose, and that α-tocopherol is somehow influencing carbon, nitrogen, and sulfur

metabolism in Synechocystis sp. PCC 6803.

When cells were grown at pH 7.0 in the presence of glucose, the level of

transcripts of sigA, ndhF4, cpcA, and carboxysome genes (rbcL, ccmK1, ccmL) decreased

substantially. These genes were constitutively expressed, and their products play

housekeeping roles for the survival of cyanobacteria. Thus, it is possible that the

reduction of the ndhF4, and cpcA, and carboxysome transcripts is caused by the reduction

of the cellular content of SigA (Pierce et al., 1989; Caslake and Bryant, 1996; Gruber and

217

Bryant, 1997). Because the functions of SigA and RuBisCO are absolutely indispensable

for the survival of cyanobacteria, the observed pH-dependent, glucose-induced

transcriptional misregulation of sigA and rbcL is a likely cause of the cell death of the

tocopherol mutant in the presence of glucose in medium at pH 7.0.

The mechanism of the pH-dependent, glucose-induced misregulation of sigA

transcription has yet to be determined. A study has suggested that sigA transcription is

regulated by the activity of the photosynthetic electron transport chain, and in the

presence of DCMU or in the dark the amount of sigA transcripts is rapidly lowered

(Tuominen et al., 2003). Interestingly, the activity of PS II, and thus the activity of the

photosynthetic electron transport chain, is reduced in a pH-dependent manner in the

tocopherol mutant in response to glucose, and PS II activity was completely lost at pH

7.0 (see Chapter 4, Fig 4.17). The PS II content in the tocopherol mutant under these

conditions appeared to be largely unaltered based on the contents of the D1 and PsbO

proteins (see Chapter 4, Fig 4.10). Thus, the inactivation of PS II is thought to be related

either to the loss of electron transfer cofactors or to the generation of metabolites that

inhibit the activity of PS II. Decades of studies and recent X-ray crystallographic

structures of the PS II complexes from the cyanobacterium Synechococcus elongatus

have confirmed the presence of one bicarbonate molecule in the vicinity of the non-heme

iron located between the two bound plastoquinone molecules (QA and QB) (Blubaugh and

Govindjee, 1986; Petrouleas et al., 1994; Ferreira et al., 2004, see also reviews by

Blubaugh and Govindjee, 1988; Govindjee and van Rensen, 1993; van Rensen et al.,

1999, and references therein ). It has been demonstrated that this bicarbonate molecule is

218

displaced in vitro by other molecules such as formate, NO, glycolate, glyoxylate, and

oxalate (Petrouleas et al., 1994). Glycolate is a metabolite generated by the oxygenase

activity of RuBisCO, which occurs when the intracellular CO2 concentration is

substantially reduced. Although, there is no evidence suggesting a significant reduction in

the intracellular Ci pool in the tocopherol mutant, this possibility needs to be examined

nevertheless.

Perhaps the most exciting outcome of the RT-PCR analyses is the discovery that

the expression of slr2031 and CCM genes (ndhF3, ndhR, sbtA) is constitutively down-

regulated in the tocopherol mutant in the presence and absence of glucose both at pH 7.0

and 8.0. This direct effect of the loss of α-tocopherol on gene expression is a strong

indication that α-tocopherol is involved in transcriptional regulation in cyanobacteria.

Because the function of these products are directly related to macronutrient metabolism,

this further leads to the conclusion that α-tocopherol is involved in the regulation of the

macronutrient metabolism as a transcriptional regulator in cyanobacteria. It is noteworthy

that, in mammalian systems, it has been demonstrated that α-tocopherol is directly

involved in transcriptional regulation of the genes encoding liver collagen αI, α-

tocopherol transfer protein, and α-tropomyosin collagenase (Yamaguchi et al., 2001;

Azzi et al., 2002). The mechanisms of these transcriptional regulation processes are not

completely understood; however, it is a reasonable assumption that it involves a

modulation of a signal transduction cascade that regulates the expression of these genes.

Indeed, α-tocopherol has been suggested to modulate the cell signaling pathway by

modulation of the phosphorylation state of protein kinase Cα in rat smooth-muscle cells,

219

possibly via phosphorylation of protein phosphatase 2A (Ricciarelli et al., 1998). In light

of the observed involvement of α-tocopherol in cell signaling in the animal system, it is

intriguing to recognize the phenotypic similarity between the tocopherol mutant and the

pmgA mutant in Synechocystis sp. PCC 6803. As mentioned earlier, the pmgA gene has

been demonstrated to be a determinant of glucose tolerance in this organism (Hiahara and

Ikeuchi, 1997), and its deduced amino acid sequence shows similarity to a

serine/threonine protein kinase, which has been characterized as the anti-sigma factor

(RsbW) or the activator for the anti-RsbW (RsbT) in the regulatory pathway of SigB in B.

subtilis (Price, 2000; Woodbury et al., 2004). Thus, it is highly plausible that α-

tocopherol plays a regulatory role through this Rsb-like pathway in Synechocystis sp.

PCC 6803 as summarized in Fig 5.9. This is the first time, to the best of my knowledge,

that the involvement of α-tocopherol in transcriptional regulation has been

experimentally demonstrated in the oxygenic phototrophs. This newly discovered role of

α-tocopherol in Synechocystis sp. PCC 6803 thus strongly indicates that α-tocopherol

plays a regulatory role in addition to a role as a bulk antioxidant in some cyanobacteria.

The next question is whether such a regulatory role is evolutionarily

conserved among green algae and plants. Recent studies have shown that a mutant

in maize, originally isolated because of its deficiency in sucrose transport through

plasmodesmata, is also deficient in tocopherol cyclase (Porfirova et al., 2002;

Sattler et al., 2003). This suggests that α-tocopherol plays a role in sugar

metabolism in maize. It is therefore highly plausible that α-tocopherol has similar

regulatory functions in macronutrient metabolism in plants.

220

SUMMARY

It was shown that a α-tocopherol mutant responds to the presence of glucose as

though nitrogen and sulfur were limiting in the environment. This conclusion is based on

the accumulation of stress-responsive gene transcripts. When glucose is present in growth

medium at pH 7.0, the amounts of the sigA and rbcL transcripts diminished after 4 h.

Because the amount of these transcripts remained unaltered when cells were grown in the

absence of glucose or in the presence of glucose pH 8.0, it was concluded that the pH-

dependent glucose-induced misregulations of the sigA and rbcL are largely responsible

for the glucose-induced lethality of the tocopherol mutants under these conditions. The

transcription of metabolic genes (slr2031, ndhF3, ndhR, and sbtA) is shown to be

constitutively down regulated under all conditions tested in the tocopherol mutant. This

result demonstrates that α-tocopherol is involved in transcriptional regulation of these

metabolic genes in Synechocystis sp. PCC 6803. Thin-section electron micrographs of the

tocopherol mutant showed that large amounts of glycogen granules accumulated in the

cells. The targeted interruption of the pmgA gene (a putative anti sigma factor

homologous to RsbW) resulted in the similar pH-dependent glucose-induced lethal

phenotype. Based on theses results, it was concluded that α-tocopherol is involved as a

regulatory molecule in the global regulation of carbon, sulfur, and nitrogen metabolism in


221


Strains and Growth Conditions

The wild-type strain used for transformation and further analyses was a glucose-

tolerant stain of Synechocystis sp. PCC 6803 (Williams 1988). Medium BHEPES, pH

8.0, was used for maintenance of the wild type and mutant strains. This medium is

prepared by supplementing BG-11 medium (Stainer et al., 1971) with 4.6 mM of HEPES

[4-(2-hydroxyethyl)piperazine-N’-(2-ethnesulfonic acid)]-KOH and 18 mg L-1 ferric

ammonium citrate. Medium BHEPES40 is prepared by supplementing the medium

BHEPES with 40 mM HEPES. Prior to all growth measurements and the preparation of

cells for subsequent analyses, cells were transferred from solid to liquid medium

BHEPES and allowed to grow exponentially for several generations in the absence of

glucose. For the determination of growth characteristics, late-exponential phase cultures

were diluted in fresh liquid medium BHEPES40, pH 7.0 or 8.0, to an OD730 nm of

approximately 0.05 cm-1. The diluted cultures were grown at 32 °C with continuous

bubbling with air containing 1% (v/v) CO2. For growth in the presence of glucose, the

medium was supplemented with 5 mM glucose. The light intensity was 50 µE m-2 s-1,

unless otherwise noted. For isolation of RNA, late-exponential phase cultures were

diluted in fresh liquid medium BHEPES40 to an OD730 nm of approximately 0.3 cm-1 in a

total volume of 80 mL. The diluted cultures were grown under the same conditions as

described above.

222

Generation of the slr2031 and the pmgA Mutants

The 5’-terminal portion of the slr2031 locus was amplified by PCR using primers

designed to target this genomic region [slr2031-F, 5’-

AGGAGTTGGTGGCTAAGCTTTACCGGGAACAAA-3’, including an engineered

HindIII cleavage site (underlined); slr2031-R, 5’-

TGACAAAGCGATGGGAATTCTCCAGGTCGGCA-3’, including an engineered

EcoRI cleavage site (underlined)]. The pmgA locus was amplified by PCR using primers

designed to target this genomic region [pmgA-F, 5’-

TTCTCTGTGCCGAAAGCTTCTATG-3’, including an engineered HindIII cleavage site

(underlined); pmgA-R, 5’-CACCATGGTGGCGAATTCAGCC-3’, including an

engineered EcoRI cleavage (underlined)]. The amplified DNA fragments were digested

with HindIII and EcoRI and ligated with pUC19 obtained after digestion of pUC19 with

HindIII and EcoRI. An EcoRI fragment of pSRA2, containing the aadA gene conferring

spectinomycin resistance, was inserted into the unique MfeI site within the 5’ slr2031

coding region. An XbaI fragment of pMS266, containing the accC1 gene conferring

gentamicin resistance, was inserted into the unique SpeI site within the pmgA coding

region. The resulting constructs were linearized after digestion with EcoRI and used to

transform wild-type Synechocystis sp. PCC 6803 cells. Selection of transformants was

carried out on solid medium BHEPES, pH 8.0, in the presence of 50 µg mL-1

spectinomycin or in the presence of 20 µg mL-1 gentamicin at 27 °C, under a moderate

light intensity (~50 µE m-2 s-1). PCR analyses were carried out using the same sets of

primers used for the cloning.

223

Isolation of Total RNA and RT-PCR Analyses

Total RNA was isolated from cells using the Mini-to-Midi RNA isolation kit

(Invitrogen corp., Carlsbad, CA) followed by an RNase-free DNase treatment and

repurification by using the same kit. The final concentration of the purified RNA

preparations was 50 ng mL-1 in RNase free double-distilled water; the RNA samples were

stored at -80°C until being used. RT-PCR reactions were carried out by using the One-

Step RT-PCR kit (Qiagen Inc., Valencia, CA) for detecting the rnpB, spbA, sigC, sigE,

sigI, ndhF4, ndhF3, ndhR, sbtA and slr2031 transcripts. The reaction volume was 25 µL,

and 4 ng µL-1 of the RNA preparation was used for each reaction. For detecting the other

transcripts, 400 ng of total RNA was first converted to cDNA by a reverse transcription

reaction using M-MLV reverse transcriptase (Promega, Madison, WI). The reaction

volume was 20 µL for each sample. The resulting cDNA was diluted with 80 µL of TE

buffer, and stored at –20 °C until being used. Two microliters of cDNA stock were used

for each PCR reaction (final volume, 25 µL) using Hot-Start Taq polymerase (Qiagen

Inc., Valencia, CA). RNasin (Promega, Madison, WI), an RNase inhibitor, was present

in the one-step RT-PCR reactions and during the cDNA synthesis.

Transmission Electron Microscopy

Cells grown in the absence or in the presence of glucose for 24 h were harvested

and immediately fixed in a 2.5 % glutaraldehyde solution prepared in a 0.1 M cacodylate

buffer, pH 7.4, over night at 4°C. The fixed cells were washed in the 0.1 M cacodylate

buffer for three times at room temperature, and subjected to the second fixation in a 1%

osmium tetroxide solution prepared in the 0.1 M cacodylate buffer for 1. 5 h at room

224

temperature. The cells were washed twice in the 0.1 M cacodylate buffer and then once in

double distilled water. After incubation in a 2 % uranyl acetate solution for 2 h at room

temperature, the cells were washed in the following concentration of ethanol: 50% (v/v),

70% (v/v), 90% (v/v), 95% (v/v) ethanol in water followed by two washes in 100% (v/v)

ethanol. Final washes were performed three times in 100% ethanol (EM grade) and then

three times in acetone at room temperature before infiltration. The washed cells were

incubated in the presence of the following concentration of the resin Spurr (Spurr 1969)

over night at room temperature: 50% (v/v) and 72 % (v/v) in acetone and twice in 100%

(v/v) Spurr. The samples were then polymerized at 60°C over night. Thin sections (ca.

50-60 nm thickness) were stained with 2 % (v/v) uranyl acetate before examination under

a JEM 1200 EXII transmission electron microscope (Joel USA Inc., Peabody, MA,

USA).

225

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233

Fig 5.1: Time-course RT-PCR analysis of the cpcA, nblA, and sbpA transcripts in wild type and slr1736 mutant grown at pH 7.0 and pH 8.0. Cell were grown in the absence of glucose (shown at 0 h) and in the presence of glucose for 4, 8, and 24 h, at 1% (v/v) CO2, 32 °C at 50 µE m-2 s-1

.

234

Fig 5.2: Time-course RT-PCR analysis of alternative sigma factor transcripts in wild type and slr1736 mutant grown at pH 7.0 and pH 8.0. Cell were grown in the absence of glucose (shown at 0 h) and in the presence of glucose for 4, 8, and 24 h, at 1% (v/v) CO2, 32 °C at 50 µE m-2 s-1

.

235

Fig 5.3: Time-course RT-PCR analysis of transcripts for sigA, sigG and genes involved in Ci uptake and fixation in wild type and slr1736 mutant grown at pH 7.0 nd pH 8.0. Cell were grown in the absence of glucose (shown at 0 h) and in the presence of glucose for 4, 8, and 24 h, at 1% CO2 (v/v), 32 °C at 50 µE m-2 s-1

.

236

Fig 5.4. Thin-section electron micrographs of the wild-type cells grown (A) in the absence and (B) in the presence of glucose for 24 h Cells were grown in medium BHEPES40, pH 7.0, 1% (v/v) CO2, 32 °C, 50 µE m-2 s-1, at 32 °C. Cells in the mid-exponential growth phase (A) were transferred to medium containing glucose and allowed to grow for 24 h (B).

237

Fig 5.5: Thin-section micrographs of the slr1736 mutants grown (A) in the absence and (B) in the presence of glucose for 24 h Arrows in (B) show the novel, proteinaceous structure (see text). Cells were grown in medium BHEPES40, pH 7.0, 1% (v/v) CO2, 32 °C, 50 µE m-2 s-1, at 32 °C. Cells in the mid-exponential growth phase (A) were transferred to medium containing glucose and allowed to grow for 24 h (B).

238

Fig 5.6: Time-course RT-PCR analyses of the slr2031 and various CCM gene transcripts in wild type and slr1736 mutant grown at pH 7.0 and pH 8.0, 1% (v/v) CO2, 32 °C at 50 µE m-2 s-1. Cell were grown in the absence of glucose (shown at 0 h) and in the presence of glucose for 4, 8, and 24 h.

239

Fig 5.7: PCR analysis of the genomic DNAs extracted from the slr2031::aadA and pmgA::accC1 mutants. Reaction s were carried out using (A) the primer set slr2031-F and slr2031-R, and (B) the primer set pmgA-F and pmgA-R (see Materials and Methods). Left and right lanes in each panel shows PCR products amplified from the wild type (WT) and mutant genomic DNAs.

240

Fig 5.8: Cultures of wild type, slr1736, slr2031, and pmgA mutants of Synechocystis sp. PCC 6803 grown in the presence of glucose (A) at pH 8.0 and (B) at pH 7.0 Cell were grown at 1% (v/v) CO2, 32 °C at 50 µE m-2 s-1

.

241

Fig 5.9: Summary and a model of the non-antioxidant role of α-tocopherol in Synechocystis sp. PCC 6803 A Rsb-like regulatory pathway, which involves PmgA, appears to exist in Synechocystis sp. PCC 6803 and possibly in other cyanobacteria (see text). In this model, α-tocopherol either directly or indirectly interacts with the Rsb-like pathway, possibly via the function of PmgA. Through such interaction, it is hypothesized that α-tocopherol affects the transcriptional regulation of some metabolic genes, which may be required for photomixotrophic growth of this organism.

242

Chapter 6

Comparative Genome Analyses and Genetic Studies of the Plastoquinone

Biosynthesis Pathway in Cyanobacteria

Publication:

Yumiko Sakuragi and Donald A. Bryant, manuscript in preparation

243

ABSTRACT

The biosynthetic pathway of plastoquinone-9 (PQ-9) was studied by means of

comparative genome analyses in 14 cyanobacteria. The presence of genes homologous to

ubiA, ubiC, ubiD, ubiE, ubiH, and ubiX involved in ubiquinone-8 (UQ) biosynthesis in

Escherichia coli was detected in cyanobacterial genomes, which suggests that PQ-9

biosynthesis in cyanobacteria occurs from 4-hydroxybenzoate through a pathway similar

to that for the UQ biosynthesis in proteobacteria. Attempted insertional inactivation of

the ubiA, ubiX, and ubiH homologs in Synechocystis sp. PCC 6803 resulted in an

incomplete segregation of the alleles, indicating that the products of these genes are

essential for the survival of this cyanobacterium. Of the several homologs of ubiE genes

found in the cyanobacterial genomes, only two are conserved among 14 species.

However, inactivation mutagenesis of these two conserved ubiE homologs, sll1653 and

sll0418, as well as others, including sll0829, slr1039, sll0487, slr0407, and slr1618 had

little effect on the cellular PQ-9 content. These results therefore demonstrate that these

UbiE homologs are probably not involved in PQ-9 biosynthesis in cyanobacteria. After

exhaustive database searching, the presence of ubiC homologs, which are closely related

to the plastid ycf21 gene, was detected in many cyanobacterial genomes. Taking these

observations together, a hypothetical PQ-9 biosynthetic pathway in cyanobacteria is

proposed.

244

ABBREVIATIONS


UQ-8 ubiquinone-8

MSBQ 2-methyl-6-solanyl-1,4-benzoquinone

MPBQ 2-methyl-6-phytyl-1,4-benzoquinone

ORF open reading frame

4-HBA 4-hydroxybenzoate


HPLC high performance liquid chromatography

SAM S-adenosyl-L-methionine

245

INTRODUCTION

Plastoquinone (PQ-9, 2-3-dimethyl-5-solanyl-1,4-benzoquinone) is an isoprenoid

quinone that is only synthesized in oxygenic phototrophs including cyanobacteria, algae,

and higher plants (Threlfall and Whistance, 1971; Collins and Jones, 1981). It serves as a

one-electron carrier (QA) as well as a two-electron carrier (QB) within Photosystem II and

stabilizes the charge separation between P680+ and QB- upon photoexcitation (Barry et

al., 1994; Diner and Babcock, 1994; Bricker and Ghanotakis, 1996; Britt, 1996). PQ-9

also shuttles electrons from NADH dehydrogenase to cytochrome b6f complex, therefore

serving as an essential membrane-associated, lipid-soluble electron carrier both in

photosynthetic and respiratory electron transport chains.

Studies of PQ-9 biosynthesis have been carried out since the 1960’s in higher

plants by using isotopic tracers and direct enzyme activity assays (see a review by

Threlfall and Whistance, 1970) as well as by genetic methods (Collakova and

DellaPenna, 2001; Shintani et al., 2002; Cheng et al., 2003). The combined results have

shown that PQ is synthesized from homogentisate after two enzymatic reactions:

condensation of solanyl diphosphate and homogentisate by a solanyl transferase resulting

in 2-methyl-6-solanyl-1,4-benzoquinone (MSBQ), and a methylation reaction at the C3

position of the benzoquinoid ring system by MSBQ methyltransferase. This MSBQ

methyltransferase is also responsible for methylation of 2-methyl-6-phytyl-1,4-

benzoquinone (MPBQ) in α-tocopherol biosynthesis, and therefore it is a bifunctional

enzyme in higher plants (Shintani et al., 2002; Cheng et al., 2003). Given the frequent

similarity of biochemical pathways in plants and cyanobacteria, it might be assumed that

246

the same pathway functions in cyanobacteria. However, recent studies (Dänhardt et al.,

2002; Cheng et al., 2003) showed that neither the synthesis of homogentisate nor the

presence of MPBQ methyltransferase activity is required for PQ-9 biosynthesis in the

cyanobacterium Synechocystis sp. PCC 6803. These results suggested that PQ-9

biosynthesis in cyanobacteria is different from that in higher plants and that the PQ-9

biosynthetic pathways convergently evolved in cyanobacteria and higher plants (Cheng et

al., 2003).

How is PQ synthesized in cyanobacteria? A preliminary inspection of the whole

genome sequence of Synechocystis sp. PCC 6803 has shown the presence of ORFs that

show sequence similarity to enzymes responsible for ubiquinone-8 (UQ-8, 2,3-

dimethoxy-5-methyl-6-octaprenyl-1,4-benzoquinone) biosynthesis. UQ-8 and PQ-9 are

both members of the alkyl-substituted benzoquinone family and contain a 1,4-

benzoquinoid ring with a polyprenyl side chain (45 carbon units for PQ-9 and 40 carbon

units for UQ-8) at the C6 position of the benzoquinoid ring system. UQ-9 biosynthesis

occurs as follows (Fig 6.1, see a review by Meganathan, 2001, and references therein).

The first committed step of the pathway is the aromatization of chorismate to 4-

hydroxybenzoate (4-HBA), which is catalyzed by chorismate-pyruvate lyase (UbiC).

Prenylation by 4-HBA octaprenyltransferase (UbiA) and decarboxylation by 3-

octaprenyl-4-HBA decarboxylase (UbiD and UbiX) results in the synthesis of 2-

octaprenyl phenol, which is converted to UQ after three hydroxylation reactions that

alternate with three methylation reactions. A hydroxyl group is first introduced by 2-

octaprenyl-phenol monooxygenase (UbiB) (at the position ortho to the C1 OH), which is

247

subsequently methylated by an O-methyltransferase (UbiG) resulting in 2-methoxy-6-

octaprenyl phenol. Secondly a hydroxyl group is introduced by a monooxygenase (UbiH)

at the position para to the C1 hydroxyl group, resulting in the synthesis of 2-methoxy-6-

octaprenyl-1,4-benzoquinol. UbiE, a C-methyltransferase, delivers a methyl group to a

position ortho to the C6 prenyl side chain, resulting in the synthesis of 2-methoxy-5-

methyl-6-octaprenyl-1,4-benzoquinol. Further hydroxylation by a monooxygenase (UbiF)

and methylation by UbiG at the C3 position of the ring system results in UQ-8. In the

structure of PQ-9, the two methoxy groups are replaced by two methyl groups (see Fig

6.1), and no additional methyl group is present at the ring position ortho to the prenyl

side chain. Therefore, it is possible that the PQ-9 biosynthesis occurs by a pathway that is

similar to the UQ-8 biosynthesis pathway in proteobacteria, at least in that part of the

pathway that leads to the formation of the prenyl benzoquinone.

To investigate the PQ-9 biosynthesis pathway in cyanobacteria, comparative

genome analyses using 14 cyanobacterial genome sequences were performed in

combination with a reverse genetic approach. The results suggest that the PQ-9

biosynthesis occurs by a pathway similar to that for the UQ-8 pathway in proteobacteria.

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RESULTS

Comparative Genome Analyses

As of April 26, 2004, largely completed genomic sequences of 14 cyanobacteria

are available: eight complete sequences exist for Synechocystis sp. PCC 6803 (Kaneko et

al., 1996), Nostoc sp. PCC 7120 (Kaneko et al., 2001), Thermosynechococcus elongatus

BP-1 (Nakamura et al., 2002a,b), Gloeobacter violaceus (Nakamura et al., 2003),

Prochlorococcus marinus MED4 and Prochlorococcus marinus MIT9313 (Rocap et al.,

2003), Prochlorococcus marinus SS120 (Dufresne et al., 2003), Synechococcus sp.

WH8102 (Palenik et al., 2003); and incomplete sequences exist for Synechococcus sp.

PCC 7002 (J. Marquardt, T. Li, J. Zhao, and D. A. Bryant, unpublished), Nostoc

punctiforme (Gene Bank accession number: NZ_AAAY00000000), Trichodesmium

erythraeum (Gene Bank accession number: NZ_AABK00000000), Synechococcus

elongatus PCC 7942 (Gene Bank accession number: NZ_AADZ01000001), Anabaena

variabilis ATCC 29413 (Gene Bank accession number: NZ_AAEA01000001), and

Crocosphaera watsonii WH8502 (Gene Bank accession number: NZ_AADV01000004).

Database searches using the E. coli UbiA, UbiB, UbiC, UbiD, UbiE, UbiF, UbiG, UbiH,

and UbiX protein sequences against these genomes revealed the presence of homologs

for UbiA, UbiD, UbiX, UbiE, and UbiH. Similarities between the sequences of E. coli

Ubi proteins and the cyanobacterial homologs were about 50% for UbiA (Table 6.1), 30-

50% for UbiH (Table 6.2), 60% for UbiD (Table 6.3), and 50% for UbiX (Table 6.4).

Multiple UbiE/UbiG homologues were detected, and the number varied depending on the

species (Table 6.5). The observed conservation of these Ubi proteins among

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cyanobacteria and between cyanobacteria and E. coli suggests that the part of the UQ-8

biosynthesis pathway involving prenylation, decarboxylation and the introduction of a

hydroxyl group is probably functioning in cyanobacteria. The only exception to this is P.

marinus MED4, in which no UbiD and UbiX homologs were detected. Reverse-phase

HPLC analyses of the whole cell extracts confirmed that P. marinus MED4 synthesizes

about two molecules of PQ-9 per 100 chlorophyll a molecules. Similar value was

obtained for the whole cell extracts from Synechocystis sp. PCC 6803. Therefore, it is

safely concluded that P. marinus MED4 synthesizes PQ-9 and it is suggested that the

carboxylation reaction is probably catalyzed by yet unknown enzymes.

UQ-8 biosynthesis in E. coli involves eight enzymes and two of these were absent

in cyanobacteria: UbiB, and UbiF. The absence of UbiB and UbiF, the monooxygenases

that introduce hydroxyl groups into ring system (which are subsequently methylated by

UbiG), is consistent with the absence of the methoxy substituents in PQ-9. In

combination with the results described above, it was hypothesized that biosynthesis of

PQ-9 in cyanobacteria occurs as shown in Fig. 6.2. In this pathway, 4-hydroxybenzoate is

converted by UbiA to 2-solanyl-4-hydroxybenzoate, which is decarboxylated by

UbiD/UbiX giving rise to 2-solanyl phenol. It is proposed that 2-solanyl phenol is first

hydroxylated by UbiH, followed by two methylation reactions performed by one or more

of the UbiE homologs, although it is entirely possible that one or both of the methylation

reactions occurs before hydroxylation, as is the case for O-methylation by UbiG, which

precedes hydroxylation by UbiH during UQ-8 synthesis (see Meganathan, 2001, and Fig

6.1).

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UbiC, chorismate lyase, is responsible for the first committed step in the pathway

(Fig 6.1). The ubiC gene in some β-proteobacteria and γ-proteobacteria, including

Neisseria meningitis Z2491, Pseudomonas aeruginosa PA01, Salmonella enterica subsp.

enterica serovar Typhi, Shigella flexneri str. 301 setotype 2a, Yersinia pestis C092 and E.

coli, is located immediately upstream of the ubiA gene based on genome analyses

performed on these organisms. In cyanobacteria, no ubiC homolog was found in the

vicinity of the ubiA gene, and no obvious ubiC homologs were found in their genomes.

These observations appeared to indicate that homologs of this gene were absent in the

cyanobacteria, and that synthesis of 4-hydroxybenzoate occurs through an alternative

pathway (see discussion). After exhaustive database searching, however, ORFs that

showed very weak similarity to the UbiC sequences of E. coli (< 30 %) were detected

(Table 6.6). These ORFs were highly conserved among cyanobacteria (50 –80%

similarity) and showed amino acid sequence similarity to Ycf21 proteins, which are also

found in the plastid genomes of eukaryotic phototrophs including Porphyra purpurea and

Cyanophora paradoxa. Fig 6.3 shows an alignment of sequences of the UbiC protein in

E. coli and the Yfc21 homologs in the cyanobacteria and eukaryotic phototrophs. In the

alignment, a glutamate residue at the position 155 (UbiC) was found to be conserved

among the Ycf21 homologs in both the cyanobacteria and eukaryotic phototrophs. This

residue constitutes a portion of the catalytic site and is hydrogen-bonded to the hydroxyl

oxygen of 4-hydroxybenzoate as previously shown in the 1.4-Å resolution X-ray crystal

structure of the UbiC protein (Gallagher et al., 2001). An arginine residue at the position

79, which has also been shown to constitute a portion of the active site, was conserved

among many cyanobacteria, although not all. Therefore, it is possible that the Ycf21

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homologs catalyze the conversion of chorismate to 4-hydroxybenzoate in the PQ-9

biosynthesis in cyanobacteria.

Mutagenesis of ubiA, ubiX, and ubiH

An attempt was made to inactivate the ORFs slr0926 (ubiA), slr1099 (ubiX), and

slr1300 (ubiH) in Synechocystis sp. PCC 6803 by insertion of the aphII gene conferring

kanamycin resistance. These attempts, however, failed to lead to homozygous mutants

and resulted in incomplete segregation of the alleles under photoautotrophic conditions

even under selective conditions in the presence of 50 µg mL-1 kanamycin (data not

shown). Addition of glucose to growth medium, which provides electrons to the electron

transport chains through NADH (thereby bypassing Photosystem II), likewise did not

lead to complete segregation. Addition of various quinoid compounds to the growth

medium either to inhibit the Photosystem II activity (atrazine) or to compensate the loss

of PQ-9 (2,6-dimethyl-1,4-benzoquinone, PhyQ, UQ-0, UQ-1, and UQ-2) did not lead to

complete segregation (data not shown). Merodiploid mutants were unstable both under

photoautotrophic and photomixotrophic conditions under illumination at a normal light

intensity (20 - 50 µE m-2 s-1), as no further growth was observed after several transfers on

solid media. These results indicates that the ubiA, ubiX, and ubiH genes are indispensable

for the survival of Synechocystis sp. PCC 6803, which is consistent with the anticipated

essential role of plastoquinone in cyanobacteria.

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Phylogenetic Reconstruction with Ubi proteins of Cyanobacteria and Proteobacteria

Evolutionary relationships between the Ubi homologues of cyanobacteria and

proteobacteria (E. coli, Rhodopseudomonas palustris, Rhodospirillum rubrum and

Rhodobacter sphaeroides) were analyzed by constructing phylogenetic trees based on

deduced amino acid sequences and by comparing the results to the phylogenetic tree

based on 16S ribosomal RNA (rRNA) (Fig 6.4). 16S rRNA is universally present and

highly conserved among prokaryotes and thus is often used as a molecular marker to

reflect the evolutionary clock (Woese, 1987). Phylogenetic trees based on the nucleotide

sequence of 16S rRNA showed that the cyanobacteria and proteobacteria formed

independent groups, which is consistent with previous results (Woese, 1987). Within the

cyanobacteria, two major clades were observed: clade A consisted of Synechocystis sp.

PCC 6803, Synechococcus sp. PCC 7002, Nostoc sp. PCC 7120, N. punctiforme, and T.

erythraeum, and clade B consisted of Synechococcus sp. WH8102 and the three

Prochlorococcus species (Fig 6.4). Phylogenetic trees based on the amino acid sequences

of UbiA, UbiD, and UbiX showed similar branching orders and topologies to the tree for

16S rRNA. The trees were supported by high bootstrap values (Fig 6.4), suggesting that

the ubiA, ubiD, and ubiX genes have evolved through vertical heritage from the last

common ancestor of both cyanobacteria and the proteobacteria. The topology of the tree

based on the UbiH sequences appeared different, however. Within the group formed by

all cyanobacteria, the proteobacteria formed a cluster with clade A, which may indicate

the lateral transfer of the ubiH gene between these two groups of organisms. However,

the bootstrap value assigned to the branch connecting a node where cyanobacteria in

clade A and clade B bifurcate and a node where the proteobacteria deviate from the

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cyanobacteria in clade A is only 52, which only poorly supports the likeliness of this

branching order. Alternatively, it is possible that the out-group used to construct the tree

(Mlr2092, a putative UbiH homologue in Mesorhizobium loti) may be distorting the

topology and thereby gives rise to an artifact. This happens if the sequence of the out-

group is more closely related to the cyanobacterial sequences than to the proteobacterial

sequences. As shown in Table 6.2, the amino acid sequence of Mlr2092 that was used as

an out-group is equally distantly related to any of the UbiH sequences analyzed. The

replacement of Mlr2092 by another protein sequence, NahG (salicylate hydroxylase in

Pseudomonas putida ) or Bsu1050 (a putative monooxygenase in Bacillus subtilis), did

not alter the topology of the tree. Therefore, it is possible that the proteobacteria form a

group within the cyanobacteria and a lateral exchange of ubiH took place between

proteobacteria and the cyanobacteria in clade A. This evolutionarily close relationship of

UbiH homologs between the two groups of organisms provides further support for the

involvement of these proteins in the PQ-9 biosynthesis in cyanobacteria.

UbiE

Database search of the genomes of cyanobacteria showed the presence of multiple

UbiE homologs (Table 6.5, Fig 6.5). There are two ORFs that are conserved in all 14

cyanobacteria: these are homologs of Sll1653 and Sll0418 of Synechocystis sp. PCC

6803. Note that the incomplete genome sequence of N. punctiforme only contained a

homolog of Slr0089, which encodes γ-tocopherol methyltransferase. Whether a homolog

of Sll0418 is present in this organism is yet to be determined. Homologs of Sll0829 and

Sll0487 are conserved in all cyanobacteria except P. marinus MED4 and P. marinus

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SS120. Slr1618 and Slr0407 seem to be specific to Synechocystis sp. PCC 6803 and C.

watsonii, or Synechocystis sp. PCC 6803, A. variabilis, and G. violaceus, respectively

(Table 6.5). Amino acid sequences of all these homologs contain the conserved S-

adenosyl-L-methionine binding motif (data not shown). UbiE in E. coli is a bifunctional

enzyme and is responsible for the C-methylation reactions in both UQ-8 and

menaquinone biosyntheses (Lee et al., 1997). Phylogenetic analyses conducted on the

UbiE homologues of cyanobacteria showed that Sll1653 forms a cluster with the E. coli

UbiE (Fig 6.5). Furthermore, as described in Chapter 2 and Chapter 3 (also see Sakuragi

et al., 2002), this ORF is responsible for transferring a methyl group to 2-prenyl-1,4-

naphthoquinone in phylloquinone and menaquinone-4 biosyntheses in cyanobacteria.

Therefore, it was expected that Sll1653 would be responsible for at least one of the

methylation reactions in PQ-9 synthesis. Whole-cell extracts from the sll1653 mutant

were analyzed by reverse-phase HPLC. PQ-9 eluted at 36 min using the current analytical

system (see “Materials and Methods”, see also Chapter 2). Whereas the chemically

synthesized 2-methyl-6-solanyl-1,4-benzoquinone (a kind gift from Prof. DellaPenna in

the Michigan State University, see Shintani et a., 2000), a predicted intermediate in the

PQ-9 synthesis, eluted at 33 min as shown in Fig 6.6A. The HPLC profile of the whole-

cell solvent extracts from the sll1653 mutant showed a peak at 35.5 min. The absorption

spectrum of the component eluting at this retention time showed a maximum at 256 nm

(Fig 6.6B, solid line), which is characteristic of PQ-9 (Dunphy and Brodie, 1971) and is a

few nanometers red shifted as compared to the absorption maximum observed for the

MPBQ standard (Fig 6.6B, dotted line). The results therefore demonstrate that PQ-9 is

present in the sll1653 mutant cells. The contents of PQ-9 were analyzed based on the

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integrated peak area in the HPLC chromatograms and were expressed as relative to the

cellular contents of chlorophyll a (Fig 6.7). The results showed that the PQ-9 content in

the sll1653 mutant is comparable to that in wild type. Therefore, it was concluded that

Sll1653 is not involved in the PQ-9 biosynthesis or that a redundant activity conferred by

another methyltransferase(s) is present.

An in vitro study has previously demonstrated that recombinant Sll0418, MPBQ

methyltransferase, can catalyze methylation of MSBQ and lead to the synthesis of PQ-9

(Shintani et al., 2002). Because this ORF is also highly conserved among the

cyanobacteria (Table 6.5, Fig 6.5), it was predicted that Sll0418 is either responsible for

transferring of one or two of the methyl groups in the PQ-9 biosynthesis or that it

provides a redundant activity with Sll1653. Targeted inactivation and complete

segregation of the mutated allele was performed and confirmed by PCR (see Chapter 3).

The sll0418 mutant was, however, capable of synthesizing the wild-type level of PQ-9 as

shown by HPLC analysis on the whole-cell lipid extracts (Fig 6.7).

Double and triple mutants of the UbiE homologs were constructed in

Synechocystis sp. PCC 6803 in further attempt to identify methyltransferase(s) involved

in the PQ-9 biosynthesis. Combinations of mutated loci are as follows: double mutations

were constructed for sll1653 and sll0418, sll1653 and slr0407, sll1653 and sll0829,

sll0418 and sll0829, sll0418 and slr0407, and sll0418 and slr0089; and triple mutations

were constructed for of sll1653, sll0418 and sll0829; and sll1653, slr0407, and sll0829.

Antibiotic resistance cartridges were used to insertionally inactivate these genes (Fig 6.8).

The full segregation of the mutated alleles from the respective wild-type alleles was

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analyzed by PCR as shown in Fig 6.9. PCR using the designed primers (Table 6.7) that

are targeted to the sll0829, sll01653, sll0418, slr0407, slr0089, slr1618 resulted in

products of 0.84 kbp, 0.3 kbp, 0.92 kbp, 0.94 kbp, 1.2 kbp, and 1.8 kbp, respectively (Fig

6.9, lane 1), which are expected based on the restriction maps as shown in Fig 6.8. No

product with corresponding wild-type size was detected for the mutants (Fig 6.9, lanes 2);

instead products with larger sizes were detected in all cases. The difference between the

mutants and the wild-type corresponds either to the size of antibiotic resistance cartridges

or to the size of antibiotic resistance cartridges minus the deleted regions (Fig 6.8).

Therefore, it was concluded that all the mutants are free of respective wild-type alleles

and were homozygous in the allele for which the drug cartridges had been introduced.

Reverse-phase HPLC analyses on whole-cell lipid extracts showed that none of

the double and triple mutants showed a significant difference from the wild type in the

amount of PQ-9 accumulated in the cells (Fig 6.7). The fact that the sll1653 sll0418

double mutant did not affect the PQ-9 content strongly indicates that Sll1653 and

Sll0418, despite their conservation among all the cyanobacteria and their predicted

functioning in PhyQ/MQ synthesis (or the demonstration of function in vitro PQ-9

synthesis), do not play major roles in the PQ-9 biosynthesis. This is further supported by

the construction of the sll1635 sll0418 double mutant, which also synthesized a wild-type

level of PQ-9 (Fig 6.7). The possibility that another UbiE homolog is providing an

additional activity to Sll01653 and/or Sll0418 was tested by analyzing the PQ-9 contents

in the sll0418 slr1618, sll0418 slr0407, sll0418 sll0829, sll1653 sll0829, and sll1653

slr0407 double mutants by reverse-phase HPLC. The results showed that all of the double

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mutants contained essentially the wild-type level of PQ-9, which indicates that Sll0829,

Slr0407, and Slr1618 are not involved in the PQ-9 biosynthesis. This is further supported

by the result that the two triple mutants, sll1653 sll0418 sll0829 and sll1653 slr0407

sll0829, contained a wild-type level of the PQ-9 in the whole-cell solvent extracts.

Participation of Slr0089 in the PQ synthesis was also ruled out as the sll0418 slr0089

double mutant synthesized the wild-type level of PQ-9. Slr1039 and Slr0487 are two

hypothetical proteins with an S-adenosyl-L-methionine binding motif, and they are well

conserved among most of the cyanobacteria (Fig 6.5, Table 6.5). Targeted inactivation of

these ORFs, however, did not affect the PQ-9 biosynthesis. In combination, these results

strongly suggest that the UbiE homologs do not play a major role in the PQ-9

biosynthesis in Synechocystis sp. PCC 6803.

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DISCUSSION

Database searches of the whole genome sequences of 14 cyanobacteria (8

complete and 6 incomplete) revealed the presence of the UbiA, UbiC, UbiD, UbiE, UbiH,

and UbiX homologs, which are proteins involved in the UQ-8 biosynthetic pathway in

proteobacteria. Attempted insertional inactivation of the ubiA, ubiH, and ubiX genes in

Synechocystis sp. PCC 6803 resulted in the generation of unstable merodiploids, which

indicates that these genes are essential for the survival of this organism and which is

consistent with the presumed essential role of PQ-9 in cyanobacteria. Based on these

findings, it is proposed that the PQ biosynthesis occurs in cyanobacteria as shown in Fig

6.2. In this proposal, UbiA catalyzes the condensation of 4-hydroxybenzoate and

solanyldiphosphate resulting in 3-solanyl-4-hydroxybenzoate, which is decarboxylated by

UbiD/UbiX resulting in 2-solanyl phenol. Hydroxylation by UbiH is expected to take

place at the C4 position of the ring system, resulting in 2-solanyl-1,4-benzoquinol.

Whether the two methylation reactions at the C2 and C3 position take place before or after

the hydroxylation is an open question. These methylation reactions, however, are not

catalyzed by the UbiE homologs (Sll1653, Sll0418, Sll0829, Slr0407, Sll0487, Slr1618

and Slr1039) in Synechocystis sp. PCC 6803 as demonstrated by insertional mutagenesis

and subsequent HPLC analyses. Phylogenetic analyses have indicated that the presence

of the UbiA, UbiD, and UbiX homologs in cyanobacteria are the result of vertical

heritage from the common ancestor of cyanobacteria and proteobacteria, suggesting a

possible common evolutionary origin of the UQ-8 and PQ-9 biosyntheses. UbiE

(Sll1653) is probably inherited from the common ancestor and evolved in cyanobacteria

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to be specific to the methylation reaction in the PhyQ/MQ biosynthesis, while another

methyltransferase(s) has been recruited to function for the PQ-9 biosynthesis.

The methyltransferase(s) that function in the PQ-9 biosynthesis is/are still a

mystery. The fact that mutants of 7 UbiE homologs contained wild-type levels of PQ-9

suggests i) that a SAM (S-adenosyl-L-methonine)-dependent methyltransferase(s) that is

only very distantly related to UbiE is responsible for these reactions, or ii) that a SAM-

independent methyltransfrase(s) is responsible for the reactions. Of the few SAM-

independent methyltransferases detected in the Synechocystis sp. PCC 6803 genome,

none is conserved among cyanobacteria except for slr0212, which is annotated as

encoding the methionine synthase and uses 5-methyltetrahydrofolate as the methyl donor

([EC 2.1.1.13], see http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/1/1/13.html). This

ORF is, however, highly conserved among other bacteria and eukaryotes and is present in

the genome as a single copy. Therefore, this gene product most likely functions as

methionine synthase and is unlikely to be involved in the PQ biosynthesis. When the

genome was searched for SAM-binding proteins, ORFs sll1407, slr1115, slr0303,

slr1071, sll1693, slr1815 were detected. Of these, slr0303 is the only ORF that is

conserved among all the cyanobacteria. The possible involvement of this hypothetical

protein in PQ-9 biosynthesis needs to be tested.

The presence of conserved Ycf21 homologs in cyanobacteria, which are weakly

similar to UbiC in E. coli, suggested that 4-hydroxybenzoate derives from chorismate by

the action of chorismate-lyase activity possibly conferred by these Ycf21 homologs. This

was thought to be a highly plausible hypothesis because catalytic residues of the UbiC

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protein are largely conserved among the Ycf21 homologs in cyanobacteria. However, the

poor amino acid sequence similarity between the Ycf21 homologs and UbiC proteins

leaves a possibility that an alternative pathway exists that leads to the synthesis of 4-

hydroxybenzoate from a compound other than chorismate. Genome analyses have

revealed that many organisms that synthesize UQ lack UbiC, including higher plants,

animals, and even some proteobacteria such as Agrobacterium tumefaciens, Brucella

melitensis, Rhodopseudomonas palustris, Chromobacterium violaceum, and

Xanthomonas axonopodis. There are three pathways that are known to lead to the

synthesis of the intermediate 4-hydroxybenzoate in these organisms: i) the UbiC-

dependent chorismate pathway in E. coli and some β- and γ-proteobacteria; ii) the 4-

hydroxyphenyllactate pathway in rat (see review by Meganathan, 2001, and references

therein); and iii) the aromatic amino acid pathway in higher plants (see a review by

Threlfall and Whistance, 1971, and references therein). In UQ biosynthesis, 4-

hydroxybenzoate is synthesized from the aromatic amino acid tyrosine or phenylalanine

via p-coumarate (Loscher and Heide, 1994). It has also been reported that the

cyanobacterium Anacystis nidulans converts the aromatic amino acid tyrosine to 4-

hydroxybenzoate, and that this conversion may be carried out by thylakoid-bound

enzyme complexes consisting of tyrosine ammonia-lyase and 4-hydroxybenzoate

synthase (Löffelhardt, 1976). Therefore, it is plausible that a similar aromatic amino acid

pathway may exist in other cyanobacteria and could provide 4-hydroxybenzoate for PQ-9

biosynthesis as proposed in Fig 6.10.

261

Lastly, the fact that the sll0418 mutation did not affect the cellular PQ content in

Synechocystis sp. PCC 6803 indicates that Sll0418 is only involved in α-tocopherol

biosynthesis. However, this ORF is conserved in nearly all cyanobacteria whose genome

sequence is so far available, including those that are predicted not to be capable of α-

tocopherol synthesis (Synechococcus sp. WH 8102 and three Prochlorococcus species,

see Chapter 5). What is the function of the Sll0418 homologs in these cyanobacteria? Is it

possible that the Sll0418 homologs are involved in PQ biosynthesis in these

cyanobacteria, given that the recombinant Sll0418 can catalyze the methylation of MSBQ

and generate PQ-9. Genetic analyses of the role of Sll0418 needs to be performed in these

other organisms.

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SUMMARY

The PQ-9 biosynthesis pathway was studied by means of comparative genome

analyses in 14 cyanobacteria. The presence of UbiA, UbiD, UbiE, UbiH, and UbiX

homologs, which are required for UQ-8 biosynthesis in E. coli, was detected in the

cyanobacteria. The presence of Ycf21 homologs, which show weak similarity to UbiC,

was detected after exhaustive database searching in cyanobacteria. The results suggest

that PQ-9 synthesis occurs similarly to UQ-8 biosynthesis pathway. Targeted inactivation

of six UbiE homologs (methyltransferases), however, did not affect PQ-9 biosynthesis in

Synechocystis sp. PCC 6803. This suggested that enzymes that are not evolutionarily

related to UbiE in E. coli catalyze the methylation reactions in PQ-9 biosynthesis in

cyanobacteria.

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Growth Conditions

The wild-type strain used for transformation and further analysis was a glucose-

tolerant stain of Synechocystis sp. PCC 6803 (Williams, 1988). Medium BHEPES, pH

8.0, was used for selection, maintenance, and growth measurements of wild type and

mutants. This medium is prepared by supplementing BG-11 medium (Stainer et al., 1971)

with 4.6 mM of HEPES [4-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid)]-KOH

and 18 mg L-1 ferric ammonium citrate. The wild-type and ubiA, ubiH, and ubiX mutant

strains were grown on solid medium BHEPES containing the following additions: 5 mM

glucose alone; 5 mM glucose and various concentrations (5 µM, 50 µM, 500 µM, 1 mM,

and 10 mM) of ubiquinone-0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone), ubiquinone-1

(2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone), ubiquinone-2 (2,3-

dimethoxy-5-methyl-6-geranyl-1,4-benzoquinone), or phylloquinone; 5 mM glucose and

5 µM atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine); or 5 mM

glucose, 5 µM ubiquinone-2, and 20 µM 2,6-di-O-methyl-β-cyclodextrin. The cells on

these solid media were grown at room temperature at moderate light intensity (ca. 50 µE

m-2 s-1) or at low light intensity (ca. 5 µE m-2 s-1).

Constructions of Single and Double Methyltransferase Mutants

DNA fragments containing the sll0829, sll0418, slr0407, slr1618, slr1039, and

slr0089 genes were amplified from isolated genomic DNA by PCR using a set of forward

and reverse primers (see Table 6.7 for sequences and Fig 6.8 for positions). The

264

constructions of the insertional inactivation of these alleles are described in Table 6.8.

The aacC1 gene, conferring gentamicin resistance, derived from the pMS266 plasmid

(Becker et al., 1995) and the aadA gene, conferring spectinomycin resistance, derived

from the pSRA2 plasmid (Frigaard et al., 2004) were inserted into the selected site within

these gene coding regions (see Table 6.8 and Fig 6.8). The resulting p829Gm and

p1039Sp plasmids were linearized and used to transform the wild-type Synechocystis sp.

PCC 6803 to obtain the homozygous sll0829::accC1 and slr1039::aadA mutants.

Genomic DNA isolated from the sll0418::aphII mutant (Chapter 4) and the

sll1653::aphII mutant (Chapter 3) were used to transform the homozygous

sll0829::accC1 mutant to generate the sll0418::aphII sll0829::accC1 and sll1653::aphII

sll0829::accC1 double mutants. The linearized p418Sp and p407Sp were used to

transform the homozygous sll1653::aphII sll0829::accC1 double mutant to generate the

sll1653::aphII sll0418::aadA sll0829::accC1 and sll1653::aphII slr0407::aadA

sll0829::accC1 triple mutants. The linearized p418Sp was used to transform the

homozygous sll1653::aphII mutant to generate the sll0418::aadA sll1653::aphII double

mutant. The linearized p1618Sp, p407Sp, and p89Gm were used to transform the

homozygous sll0418::aphII mutant to generate the sll0418::aphII 1618::aadA,

sll0418::aphII slr0407::aadA, and sll0418::aphII slr0089::accC1 double mutants. A

homozygous sll0487::aphII mutant was constructed in a previous study (W. M.

Schluchter, G. Shen, and D. A. Bryant, unpublished results). Complete segregation of the

each mutated alleles from the corresponding wild-type allele was confirmed by PCR.

Primer used for the PCR analyses are summarized in Table 6.7.

265

Quinone and Chlorophyll Analysis

Cells were harvested from photoautotrophically grown cultures and recovered in a

tight pellet. Pigments and quinones were extracted with 400 µL of cold acetone:methanol

(7:2 v/v) after brief ultrasonication. After centrifugation and filtration through a PTFE

filter membrane with a 0.2-µm pore size (Whatman International Ltd., Maldstone, UK),

the extract in the organic phase were directly injected into an Agilent Technology 1100

series HPLC system (Agilent Technology, Palo Alto, CA, USA) equipped with

SUPELCO (Sigma-Aldrich Corp., St. Louis, MO, USA) Discovery® C18 column (25 cm

x 4.6 mm, 5 µm). Analyses were carried out using the following protocol: 80%A/20%B

from 0 to10 min, a linear change to 20%A/80%B in 40 min, and 20%A/80%B for 5 min,

where solvent A and solvent B are 100% methanol and 100% isopropanol, respectively.

The flow rate was 0.75 ml min-1. Detection of eluates was performed with a diode array

detector (Agilent 1100 series). The chlorophyll a, phylloquinone, and plastoquinone

contents of each sample were determined based on integrated peak areas and their molar

absorption coefficients, which are 17.4 mM-1 cm-1 at 618 nm, 18.9 mM-1 cm-1 at 270 nm

(Dunphy and Brodie, 1971), and 15.2 mM-1 cm-1 at 254 nm (Crane and Dilley, 1963),

respectively. The absorption coefficient of chlorophyll a at 618 nm was calculated from

the ratio of the absorption peaks at 618 nm and 666 nm in methanol-isopropanol (6:4,

v/v) and from its absorption coefficient at 666 nm in methanol (MacKinney, 1941).

266

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Methods Enzymol 167: 766-778

Woese CR (1987) Bacterial evolution. Microbiol.Reviews 51: 221-271

272 ab

le 6

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min

o ac

id se

quen

ce si

mila

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s bet

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hom

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m

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able

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ence

sim

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etw

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274 T

able

6.3

: Am

ino

acid

sequ

ence

sim

ilari

ties b

etw

een

Ubi

D h

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0

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84

84

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84

77

85

84

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76

78

100

E.

col

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60

60

60

60

-

59

58

61

61

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59

59

59

100

6803

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echo

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700

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4; M

IT, P

roch

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cocc

us m

arin

us M

IT93

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120,

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s mar

inus

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20; G

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lace

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. col

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sing

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plet

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nom

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275 T

able

6.4

: Am

ino

acid

sequ

ence

sim

ilari

ties b

etw

een

Ubi

X h

omol

ogs i

n 14

cya

noba

cter

ia a

nd E

. col

i (ex

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sed

as %

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rgan

ism

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cus

tag

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70

02

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t N

pun

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lon

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lo

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0

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1 85

83

10

0

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87

84

92

100

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0)

(64)

-

- -

-

T. e

long

atus

BP-

1 Tl

r222

5 80

78

80

79

-

100

Syne

choc

occu

s 81

02

Synw

1201

69

67

66

68

-

67

100

S. e

long

atus

794

2*

Selo

0211

96

79

76

78

77

- 78

64

10

0

A. v

aria

bilis

294

13*

(Ava

r334

101)

(9

1)

- -

- -

- -

- -

C. w

atso

nii 8

501*

-

- -

- -

- -

- -

- -

P. m

arin

us M

ED4

- -

- -

- -

- -

- -

- -

P. m

arin

us M

IT93

13

Pmt0

767

70

65

64

66

- 68

84

64

-

- -

100

P. m

arin

us S

S120

Pr

o098

9 71

66

66

65

-

68

78

66

- -

- 78

10

0

G. v

iola

ceus

742

1 G

ll052

9 78

77

80

80

-

78

77

74

- -

- 68

68

10

0

E. c

oli

Ubi

X 52

52

50

52

-

54

48

52

- -

- 50

55

52

10

0

68

03, S

ynec

hocy

stis

sp. P

CC

680

3; 7

002,

Syn

echo

cocc

us sp

. PC

C 7

002;

Nos

t, N

osto

c sp

. PC

C 7

120;

Npu

n, N

osto

c pu

nctif

orm

e; T

ery,

Tri

chod

esm

ium

er

ythr

aeum

; Tel

on, T

herm

osyn

echo

cocc

us e

long

atus

BP-

1; 8

102,

Syn

echo

cocc

us sp

. WH

8102

; Sel

o, S

ynec

hoco

ccus

elo

ngat

us P

CC

794

2; A

var,

Anab

aena

va

riab

ilis A

TCC

294

13; C

wat

, Cro

cosp

haer

a w

atso

nii W

H85

01; P

roch

loro

cocc

us m

arin

us M

ED4;

MIT

, Pro

chlo

roco

ccus

mar

inus

MIT

9313

; S12

0,

Proc

hlor

ococ

cus m

arin

us S

S120

; Glo

e, G

loeo

bact

er v

iola

ceus

PC

C 7

421;

Eco

, E. c

oli.

Pairw

ise

alig

nmen

ts w

ere

gene

rate

d w

ith B

LOSU

M30

usi

ng

Clu

stal

W. *

Inco

mpl

ete

geno

me

sequ

ence

s. Th

e N

-term

inal

107

and

62

resi

dues

wer

e tru

ncat

ed in

the

tery

2980

and

Ava

r334

101

sequ

ence

s wer

e 10

7 an

d 62

re

sidu

es a

s com

pare

d to

the

Slr1

099

sequ

ence

.

276 T

able

6.5

: Lis

t of t

he U

biE

hom

olog

s in

14 c

yano

bact

eria

Syne

choc

ystis

680

3 Sl

l165

3 Sl

l041

8 Sl

l082

9 Sl

r040

7 Sl

r161

8 Sl

r103

9 Sl

l048

7 Sy

nech

ococ

cus 7

002

* 89

-c70

244

80-1

6226

8 89

-467

31

- -

a AA

D26

590

a AA

C14

722

Nos

toc

PCC

712

0 A

lr525

2 A

ll212

1 A

ll375

0 -

- A

ll301

6 A

ll001

2 N

. pun

ctifo

rme

* N

pun3

758

- N

Pun0

871

- -

- N

pun5

885

T. e

ryth

raeu

m *

Te

ry33

21

Tery

4209

Te

ry12

23

- -

- Te

ry38

55

T. e

long

atus

BP-

1 Tl

l237

3 Tt

ll117

26

Ttll1

604

- -

- Tl

l194

8 Sy

nech

ococ

cus 8

102

Synw

1674

Sy

nw21

41

Synw

1749

-

- Sy

nw11

48

Sym

w04

87

S. e

long

atus

794

2*

Selo

2038

3 -

Selo

2321

01

- -

- Se

lo21

3301

A.

var

iabi

lis 2

9413

* A

var2

8710

1 A

var1

8460

1 A

var3

9520

1 A

var0

2574

1 -

Ava

r020

095

- C

. wat

soni

i 850

1*

Cw

at17

9201

C

wat

1941

01

Cw

at02

6375

-

Cw

at02

0748

-

Cw

at02

3036

P.

mar

inus

MED

4 Pm

m04

31

Pmm

1505

-

- -

Pmm

0662

Pm

m12

43

P. m

arin

us M

IT

Pmt0

276

Pmt1

785

Pmt1

242

- -

Pmt0

792

Pmt1

474

P. m

arin

us S

S120

Pr

o042

7 Pr

o166

1 -

- -

Pro0

816

Pro0

816

G. v

iola

ceus

742

1 G

ll012

7 G

lr303

9 G

ll148

3 G

lr204

2 -

Glr3

970

Gll1

180

*Inc

ompl

ete

geno

me

sequ

ence

s. Lo

cus t

ags w

ere

used

to id

entif

y th

e pr

otei

ns. F

or p

rote

ins i

n Sy

nech

ococ

cus s

p. P

CC

700

2, p

rote

ins a

re d

esig

nate

d w

ith a

co

ntig

num

ber a

nd tr

ansl

atio

n in

itiat

ion

site

sepa

rate

d w

ith a

hyp

hen,

or

a G

ene

Ban

k ac

cess

ion

num

bers

are

pro

vide

d if

avai

labl

e.

277

Table 6.6: Ycf21/UbiC homologs in cyanobacteria and eukaryotic phototrophs

Organism Locus tag

Synechocystis sp. PCC 6803 Sll1797

Crocosphaera watsonii WH 8501 Cwat020398

Nostoc sp. PCC 7120 All0938

Nostoc punctiforme Npun5760

Anabaena variabilis ATCC 29413 Avar303301

Trichodesmium erythraeum Tery3876

Thermosynechococcus elongatus Tll1562

Synechococcus elongatus sp. PCC 7942 Selo236201

Synechococcus sp. WH8102 Synw2384

Prochlorococcus marinus sp. MIT9313 Pmt2179

Prochlorococcus marinus sp. MED4 Pmm1676

Prochlorococcus marinus sp. SS120 Pro1837

Porphyra purpurea (red algae) PopuCp195

Cyanophora paradoxa (a photoautotrophic protist) CypaCp147

278

Table 6.7: Sequences of primers used for PCR analyses Lower case letters indicate heterologous nucleotides that are introduced in order to engineer restriction enzyme cleavage sites, which are indicated with under lines. Primer Sequence

sll0829-F 5’-GGTCCGCCAAGATTTAGCAGTAAG-3’

sll0829-R 5’-AGATTGGGACAGTAATCGAAGGC-3’

sll0418-F1 5’-CGACTGAGGAAACGGTTGAaTTCCCGCACC-3’ (EcoRI cleavage site).

Used for cloning the sll0418 coding region

sll0418-R1 5’-CTTACGTGGCAATTTAAGCTTGAGTGGCGT-3’

Used for cloning the sll0418 coding region

sll0418-F2 5’-ATGCCCGAGTATTTGCTTCTGCC-3’

Used for verification of segregation

sll0418-R2 5’-GCACTGCTTTGAACATACCGAAG-3’

Used for verification of segregation

slr0089-F 5’-TCTACCGGAAATTGCCAACTACCA-3’

slr0089-R 5’-CCTAGGAGATTGTGGACTTCAA

slr0407-F 5’-CTCTGGAAACATTTGAAAgCTTTATCCG-3’ (HindIII cleavage site)

slr0407-R 5’-TACGTTACTATTCCGCCGAaTTCCTCT-3’ (EcoRI cleavage site)

slr1618-F 5’-TCTATTCTGGATATGCTGGCACA-3’

slr1618-R 5’-TTGTTTGCCTTCGGCTTTAGC-3’

slr1039-F 5’-GGGTGTTTACAATAACAGGGC-3’

slr1039-R 5’-ACTGGTGGGAGGTTGGTGCC-3’

279

Table 6.8: Insertional inactivation constructs of the methyltransferase mutants in Synechocystis sp. PCC 6803. HindIII (blunt) indicates that the 3’-overhand generated after digestion with HindIII is filled by Klenow to yield a blunt end.

Target allele

(Construct)

Restriction digestions

Site of insertion in pUC19 Insertion of drug cartridge

sll0829 (p829Gm) HindIII, HincII HindIII, HincII aacC1 between the BxtXI

sites

sll0418 (p418Sp) EcoRI, HindIII EcoRI, HindIII aadA at the unique SmaI

site

slr0407 (p407Sp) EcoRI, HindIII EcoRI, HindIII aadA between tow HincII

sites

slr1618 (p1618Sp) HindIII, HincII HindIII, HincII aadA at the unique NheI

site

slr1039 (p1039Sp) EcoRI, HindIII EcoRI, HindIII aadA at the unique MfeI

site

slr0089 (p89Gm)

HindII (blunt), KpnI

HindII (blunt), KpnI

aacC1 between the two PstI sites

280

Fig 6.1 UQ biosynthesis pathway in E. coli

281

Fig 6.2. Hypothetical PQ-9 biosynthesis pathway in cyanobacteria The question mark indicates the possibility that an alternative route exists for 4-hydroxybenzoate synthesis.

282

Fig 6.3: An alignment of the Ycf21/UbiC homologs in cyanobacteria and eukaryotic phototrophs. Two catalytic residues (arginine at the position 76 and glutamate at position 155) of the UbiC protein are shown by black arrows.

283

284

Fig 6.4: Phylogenetic analyses based on nucleotide sequences of 16S rRNA and amino acid sequences of Ubi proteins in cyanobacteria and proteobacteria. Multiple alignments were generated with BLOSUM30 using ClustalW. Phylogenetic reconstruction was carried out using the Neighbor Joining method using PAUP* 4.0 beta. Bootstrap values were obtained after 1000 times repetitions. Ctep, Chlorobium tepidum; Cpar, Cyanophora paradoxa; Cwat, Crochosphaera watsonii WH 8501; Ecol, E. coli; Gvio, G. violaceus PCC 7421; Nostoc, Nostoc sp. PCC 7120; Npun, N. punctiforme; PMM, P. marinus MED4; PMT, P. marinus MIT 9313; S122, P. marinus SS120; Ppur, Porphyra purpurea; Rpal, Rps. palustris; Rrub, Rs. rubrum; Rsphe, Rb. sphaeroides; S6803, Synechocystis sp. PCC 6803; S7002, Synechococcus sp. PCC 7002; Synw, Synechococcus sp. WH 8102; Selon, Synechococcus elongatus PCC 7942; Tery, T.. erythraeum; Telon, T. elongatus. Aeropyrum pernix K1 (locus tag: Ape1674), C. tepidum (locus tag: CT1511), Bacillus halodurance (locus tag: Bh3930), and Mesorhizobium loti (locus tag: Mlr2092) were used as out-groups.

285

Fig 6.5: Phylogenetic trees based on amino acid sequences of UbiE homologs in cyanobacteria. Multiple alignments were generated with BLOSUM30 using ClustalW. This phylogenetic tree was constructed by the Neighbor Joining method using PAUP* 4.0 beta. Locus tags (Prefix followed by a 4-digit number) are used to indicate organisms and sequence identity (summarized in Table 6.6). Sll or Slr, Synechocystis sp. PCC 6803; All or Alr, Nostoc sp. PCC 7120; Npun, N. punctiforme; Tery, Td. erythraeum; Tll or Tlr, Ts. elongatus; Gll or Glr, G. violaceus; Pmm, P. marinus MED4; Pmt, P. marinus MIT9313; Pro, P. marinus SS120; Synw, Synechococcus sp. WH8102.

286

Fig 6.6: HPLC analysis of the sll1653 mutant of Synechocystis sp. PCC 6803 and the 2-phytyl-1,4-benezoquinone (MPBQ) standard. (A) Chromatograms of the whole-cell extract of the sll1653 mutant of Synechocystis sp. PCC 6803 (solid line) and the MPBQ standard (dotted line). (B) Absorption spectra of PQ-9 eluting at 35.5 min in the sll1653 mutant sample (dotted line) and of MPBQ eluting at 33 min.

287

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Fig 6.7: PQ-9 content in whole cells of the wild-type and methyltransferase mutant strains of Synechocystis sp. PCC 6803 An error bar for the wild-type strain represents the standard error derived from 6 determinations. At least two independent samples were analyzed for each mutant and, although somewhat varied, similar results were obtained.

mol

PQ

-9 /

mol

chl

orop

hyll

a (x

10-2

)

WT

sll0

418

sll1

653

sll0

829

sll0

418

sll1

653

sll0

418

sll0

829

sll1

653

sll0

829

sll0

418

slr0

089

sll0

418

slr0

407

sll0

418

slr1

618

sll1

653

sll0

418

sll0

829

sll1

653

slr0

407

sll0

829

sll0

487

slr1

039

288

Fig 6.8: Restriction maps of coding regions and their flanking sequences for the UbiE homologs in Synechocystis sp. PCC 6803. Primers used for PCR for both cloning and PCR verifications are shown by black arrows. F1 and R1 primers were used for cloning the sll0418 coding region, while F2 and R2 primers were used for verification of segregation.

289

Fig 6.9: PCR analyses of genomic DNA isolated from the single, double, and triple methyltransferase (UbiE homolog) mutants of Synechocystis sp. PCC 6803. Primers used for the analyses are shown by black arrows in Fig 6.5. The thin arrows indicate the relationship between parental strains and mutant strains. Identities of mutants are shown above the black lines and the primers used for the analyses are shown below. PCR reactions carried out using genomic DNA isolated from mutants are shown in lane 2 and reactions carried out using genomic DNA isolated from wild type are shown in lane 1 for comparison.

290

Fig 6.10: Aromatic amino acid biosynthesis and an alternative proposal for 4-hydroxybenzoate synthesis in cyanobacteria. CM, chorismate mutase; PD, prephenate dehydratase; PPAT, prephenate amino transferase; ADH, arogenenate dehydrogenase; TAL, tyrosine amino-lyase; PAT, phenylalanine aminotransferase; PAL, phenylalanine amino-lyase; CH, chorismate hydratase. The pathways leading to tyrosine and phenylalanine biosynthesis in cyanobacteria are as previously described (Bonner et al., 1995).

291

Appendix

A.1. Amino acid sequence alignments of phylloquinone (Men) and plastoquinone (Ubi)

biosynthetic enzymes.

A.1.1. MenA homologs in cyanobacteria and other organisms

A.1.2. MenB homologs in cyanobacteria and other organisms

A.1.3. MenD homologs in cyanobacteria and other organisms

A.1.4. UbiA homologs in cyanobacteria and other organisms

A.1.5. UbiD homologs in cyanobacteria and other organisms

A.1.6. UbiH homologs in cyanobacteria and other organisms

A.1.7. UbiX homologs in cyanobacteria and other organisms

A.1.8. UbiE homologs in Synechocystis sp. PCC 6803

A.1.9. Sll1653 homologs in cyanobacteria




A.1.13 Slr1039 homologs in cyanobacteria

A.2. Photosystem I (PS I) complexes

A.2.1. Polypeptide composition of PS I complexes isolated from the

menG, menF, and rubA menB mutants of Synechococcus sp. PCC

7002.

A.2.2. Illustration of genetically engineered PS I in cyanobacteria

292

A.1.1. An amino acid sequence alignment of MenA homologs in cyanobacteria and other

organisms

Slr1518MenA7002Alr0033Tlr2196Gll1578Synw2307Pmm0176At1g60600OJ1217B0918CymeCp095B3930Bsu38490CT1511AF2036

1 32

1 31

1 35

1 27

1 22

1 31

1 22

1 6

1 80

1 20

1 25

1 35

1 28

1 20

M T E S S P L A P S T A P A T R K L W L A A I K P P M Y T V A V

M L T E N P P T S S P K A T R R L W L A A I K P P I Y T V A V

M L N C L P Q L M T T K Q I S H P K I K L W M A A I K P P M Y S V A I

M T S S S E S I D R S R L W W A A I K L P L Y S V A V

M S G S P L R P W L N S F N P L I Y T A S V

M V E P Q A V A S R Y A D R R L L W K A A I K W P M Y S V A V

M N E R N K S L W K Q A I K W P L Y S V A I

M Y S V A L

M P L A G I A L A P L F V S H L A P P H H R R S S V A S A A A A A A R R R P R A V Q C S A T A T A A S G E G A G D D V E L S R G T L L W R A A K L P I Y S V A L

M N K I V T L F L A S R P K T L L V S F

M T E Q Q I S R T Q A W L E S L R P K T L P L A F

M N Q T N K G E G Q T A P Q K E S M G Q I L W Q L T R P H T L T A S F

M S V S S S S Q L S A F Q A W M L A I R P K T L P A G A

M E K L K A L I E V T K P K Q T F L L M

M P L A G I A L A P L F V S H L A P P H H R R S S V A S A A A A A A R R R P R A V Q C S A M S L W A I K P P Y . V A .


33 104

32 103

36 107

28 99

23 94

32 104

23 94

7 78

81 152

21 84

26 104

36 108

29 107

21 90

V P I T V G S A V A Y G L T G Q W H G D V F T I F L L S A I A I I A W I N L S N D V F D S D T G I D V R - - - - - - - - K A H S V V N L T G N R N L V F L I S N

T P V I V T T A A A Y G E F G V F N P V R F G L F L G A A I C L I A W M N L S N D V F D A D T G I D V N - - - - - - - - K A T S V V N L T G D R P L V F G I A L

M P I W V G G A V A F A G N K V F N L A V F F T F I V A A I L I L A W E N I S N D V F D S E T G I D V N - - - - - - - - K H H S L V N L T K K K T L V F W L G N

M P I W L G T A V A I A K T G R G H W W P F G L F L T A A V L I L A W L N L S N D V F D A E T G I D R H - - - - - - - - K Y H S V V N L T G K K Q L I F W L S N

I P I L V G T A F A W W Q G G V F D L Q R F A L T L A G L V F V H A W I N L T N D V F D W D T G V D R N - - - - - - - - K L N S L V R T T G S R S Q V L W V G N

M P V L L A A G W R I G A V G S L R W T Q L F G F L L A A I L L L L W E N L S N D L F D A D T G V D T T G - - - - - - - K P H S V V N L T G R R D R V A L A S L

L P V F I S G A Y T L N S F K N V K I Y N L I A F T I A A I L I L I W E N L T N D L F D S E T G I D E F - - - - - - - - K F H S I V N L V R S K T I V S I T A Y

V P L T V G A S A A Y L E T G L F L A R R Y V T Q L L S S I L I I T W L N L S N D V Y D F D T G A D K N - - - - - - - - K M E S V V N L V G S R T G T L A A A I

V P L T V G S A C A Y H H V G S F F G K R Y F V L L V A S V L V I T W L N L S N D V Y D S D T G A D K N - - - - - - - - K K E S V V N I V G S R T M T Q Y A A N

C C W L S V S S L Q H A F - - - - S I P A L L L A I G - - - - L Q I W A N W V N D Y L D F M N G I D H A - - - - - - - - L R Q G P T R W T S R L T P S F M K I I

A A I I V G T A L A W W Q - G H F D P L V A L L A L I T A G L L Q I L S N L A N D Y G D A V K G S D K P D R I G P L R G M Q K G V I T Q Q E M K R A L I I T V V

V P V L L G T V L A M F Y - V K V D L L L F L A M L F S C L W I Q I A T N L F N E Y Y D F K R G L D T A E S - - - - - - V G I G G A I V R H G M K P K T I L Q L

M P V V I G A A L A A A S - G V F K P L P A L V A L I C A L G I Q I A T N F I N E I Y D F R K G A D T A E R L G P T R T V A A G I I T E Q T M I R V S I V L G V

I T F L V S Y I V A R G G - A D F N F V I A A I S M F L A I S G T T A I N M W L D R - D I D A I M P R T - - - - - - - - R K R P V P A G I L K P S E C A A F G A

. P . . V G . A . A . G F . . . . L . . A I L . . . W N L . N D . F D . T G . D R G P R K S V V N . . . . . . . . .


105 176

104 174

108 179

100 171

95 162

105 177

95 169

79 154

153 231

85 146

105 177

109 181

108 174

91 157

F F L L A G V L G L M S M S W R A Q D - W T V L E L I G V A I F L G Y T Y Q G P P F R L G Y L G L G E L I C L I T F G P L A I A A A Y Y S Q S Q S - - - - - - -

G F L G L G I T G I A L I S Y L Q Q D - W M V F N L L L L A T A I A Y T Y Q G P P F R L G Y L G I G E F V C F L A Y W I T G Q G V F Y S - Q A Q T - - - - - - -

L C L I S G L L G I L A I A T W Q Q D - P T V I G I I L L C C G L G Y M Y Q G P P F R L G Y Q G L G E I L C F F A F G P L G V S A G Y Y S Q T Q T - - - - - - -

L C L L L G L L G I A A I S W L Q Q D - G T V L G L V L L C C A L G Y S Y Q G P P F R L G Y L G L G E P I C F I C F G P L A I A A A Y Y S Q V Q T - - - - - - -

I F L S I G Y V - - - - C F W A L Q D - W W L L A F G S L G I F L G Y A Y Q G P P F R L A Y K G L G E W I S S S C F G P I A V L T A T R A Q T G S - - - - - - -

A S L G S G L A L M A W L A W C S S - - V L V L A L V L L C C G L G Y I Y Q G P P F R L G Y R G L G E P L C W L A F G P C A T A A A L M V L E P Q - - - - - G A

T S L L I G L V V I A I I S I S T S - - I N V M L L V G A C C F L G Y L Y Q G P P F R L G Y Q G L G E P L C W L A F G P F A Y A A A L I A L N P S D - - - I Y M

T S L A L G V S G L V W T S L N A S N - I R A I L L L A S A I L C G Y V Y Q C P P F R L S Y Q G L G E P L C F A A F G P F A T T A F Y L L L G S - - - S S E M R

I S L L F G F M G L F W A F A Q A G D - A R F I L S V T C A I I C G Y V Y Q C P P F R L S Y R G L G E P L C F A A F G P L A T T A F Y F S N S S R N I S S G T A

L C L W C C L L F V C A S Y - - - - - - F K M K I L V G I L I L I A W F Y S - - - - - A W L G F A G E W L S F F A C G P L A T Y L F S S I Y Q V S - - - - - - -

L I C L S G L A L V A V A C H T L A D F V G F L I L G G L S I I A A I T Y T V G N R P Y G Y I G L G D I S V L V F F G W L S V M G S W Y L Q A H T - - - - - - -

A L A S Y G I A I L L G V Y I C A S S S W W L A L I G L V G M A I G Y L Y T G G P L P I A Y T P F G E L F S G I C M G S V F V L I S F F I Q T D K - - - - - - -

S V F V L G L Y L V A I G G - - - - - - W P I L L I G V L S L L F A W A Y T G G P F P I A Y S G L G D V F V F I F F G L V A V G G T Y Y V Q A L S - - - - - - -

A I F A I G Q V L A F L V S L E F G L V V F F G L F F D I V V Y T I L L K R K S P Y S I V L G G F A G A M P A L G - G W V A V Q G F T L - - - - - - - - - - - -

. L . . G . . . . . . . D . . . L . . . . . . . G Y . Y Q G P P F R L . Y G L G E . C . . . F G P . A . . . . . Q . I S S


177 252

175 250

180 255

172 247

163 238

178 256

170 248

155 234

232 311

147 223

178 253

182 257

175 249

158 231

- - - F S W N L L T P S V F V G I S T A I I L F C S H F H Q V E D D L A A G K K S P I V R L G T K L G S Q V L T L S V V S L Y L I T A I G V L C H Q A P W Q - T

- - - L T L N A V W A S A W V A L T T S I I L F C S H F H Q A E D D L A A G K K S P I V R L G T Y Q G A Q V L T Y S L G A V L V L T C L L V A V G T W S P W - M

- - - W S I T S L A A S V I V G I V T S L V L F C S H F H Q V D D D I K A G K R S P I V R L G T A N G A K V L T W F T A S I Y P F S L L F V L L G F F P V W - T

- - - F S A S I W P V A I L N G L T T T L I L F C S H F H Q V A D D L A A G K R S P V V R L G T A R S A Q L V Y G A C G L F Y G V L V A S V L W G L L P W P - T

- - - W E L G A L L A G I A V G S W T A T I L Y A H H F Y Q Y E D D R E F D K R T P V V R W G P E L A A K R F W W V I S P S Y V V L L L A V I G G F L P W T - V

N V I P W A T A W M L G A G P A V A T S L V L F C S H F H Q V S E D S A H G K R S P V V R L G T A R A A A L I P W L V A L T L A L E W L P M C R G D W P L T - V

I S I P W K E S L L L G S G S S L A T T L V L F C S H F H Q I K E D K E H G K N S P L V L L G A K K G A K I I P W I V F I I Y V F Q L F L I I N G F I P I L - C

H L P L S G R V L S S S V L V G F T T S L I L F C S H F H Q V D G D L A V G K Y S P L V R L G T E K G A F V V R W T I R L L Y S M L L V L G L T R I L P L P C T

L L P L S K T V I A S S I L V G L T T T L I L F C S H F H Q I D G D R A V G K M S P L V R I G T K T G S R L V T L G V V T L Y V L L A A F G M S K S L P S A C T

- - F W Q W D L L L L S I I N G S W S A A L L M A N N M R D R I S D T H S P K K S T C V W F G Q D W A S M Q Y Q M F I L M C I W C L F A W L I F R P S W F W - L

- - - L I P A L I L P A T A C G L L A T A V L N I N N L R D I N S D R E N G K N T L V V R L G E V N A R R Y H A C L L M G S L V C L A L F N L F S L H S L W - G

- - - I N M Q S I L I S I P I A I L V G A I N L S N N I R D I E E D K K G G R K T L A I L M G H K G A V T L L A A S F A V A Y I W V V G L V I T G A A S P W - L

- - - L P M E V L V A A A A P G A F S V C I L L V N N I R D I D T D R K V G K M T L P A R I G A P A A R A L Y V A L V V L A Y L V P - F Y M I S T G Y S L W - C

- - - - - P G F I I A A I V L L W I P S H I W Y I S M H Y E E D Y R M A N I P M Y P L V - V G M E R A S W A I V F A T A A M L V L A A S L Y V L L P L G I F Y L

. . . . . . . G . T . . I L F C S H F H Q . . . D . G K . S P . V R L G . . . . . . . . . Y . . . . . . . . . P C


253 307

251 317

256 309

248 298

239 295

257 310

249 305

235 287

312 346

224 269

254 308

258 311

250 307

232 281

L L I I A S L P W A V Q L I R H V G Q Y H D Q P E Q V S N C K F I A V N L H F F S G M L M A A G Y G W A G L G

L L T F A S A P F A L S L I N H V R L N H D Q P D K V S N C K F F A V K F H F A S G L L L A I A Y V L S Y Y N L E F L S V T G L G Y N

L L S W L S L P Y A F K L C R H V Q H N H N Q P E K V S N C K F I A V A V H F W S C L L L G L G F V I A G V

L L A L S S L P W A I Y L C Q R M A Q F H S V P E Q I K N S K F I A V Q L H F W S S L L L G L G Y L L

L A A L A T I P L A V S L V S F V R E N A T N P A V V V G A L P L A A R F H L V S G A L L T L G L L A G T L L P R

L L S G L G L P A G L A L I R L M Q R C H D Q P D R I S G S K F L A L R F Q A W N G L G L A L G L A L G R L

V L F L I S F P Q S L K L I N L L K Y S Y N K P E A I K N C K F I A I K F Q T L N G I G L I A G F I I N Y L I Y K

L M C F L T L P V G N L V S S Y V E K H H K D N G K I F M A K Y Y C V R L H A L L G A A L S L G L V I A R

V L C A L T L P V G K W V V D Y V L K N H E V T S D L T Y N L D A N L

L M M C V F L P Y A L R L C M M S L T T P T - - A L L Y P T L S F I F R Y C F L F S F C A S Y S

W L F L L A A P L L V K Q A R Y V M R E M D P - V A M R P M L E R T V K G A L L T N L L F V L G I F L S Q W A A

F V V F L S V P K P V Q A V K G F V Q N E M P - M N M I V A M K S T A Q T N T F F G F L L S I G L L I S Y F R

L L S L L S I P L A I G M V R T L Y A S E G - - Q A L N A V L A G T G K V L T V H G L L F S L G L V I P N I I S I F R P

V I S T S A V A F F L Y K A V K F A L S P D R - V K A R K M Y K L A S M T L G L V Y F S L L L G V F L

L L . . . . P . . L . . P . . A . . . G . L L . L G . . F V T G L G Y N

The multiple alignment was generated by BLOSUM30 using Clustal W. The locus tags used to indicate organisms are summarized in Table 2.1, except for the following: Cymecp095 in Cyanidiochyzon merolae and OJ1217B09.18 in Oryza sativa. AF2036, a hypothetical protein in the archaeon Archeoglobus fulgidis DSM4304, was used as an out-group in phylogenetic construction as shown in Fig 2.3.

293

A.1.2. An amino acid sequence alignment of MenB homologs in cyanobacteria and other

organisms

Sll1127MenB7002All2347Tll2458Synw0998Pmm0608At1g60550P0671D0118CymeCp092B2262CT1846Bsu3075AF0435

1 5

1 8 1 7

1 8

1 18

1 14

1 67

1 64

1 0

1 21

1 27

1 6

1 0

M D W H I

M E S L A W Q AM Q I D W Q S

M D S V H W R Q

M A E L R Q V L P G D P A A A W T A

M K I L P G E T N S N W T E

M A D S N E L G S A S R R L S V V T N H L I P I G F S P A R A D S V E L C S - - - A S S M D D R F H K V H G E V P T H E V V W K K T D F F G

M D A A G R R L A R V T A H L L P S S L P L P L A S A P T L A P S P A A S P A S D S Y R R V H G D V P S E P P E W R A A T D E S

M I Y P D E A M L Y A P V E W H D C S E G - - - -

M P V E P G Q R R F S S T T I E I S D M S T V N W I T

M A E W K T

M A D S N E A R R L V T H L P A L S P A A S . W


6 70

9 73

8 72

9 73

19 84

15 80

68 132

65 126

1 56

22 79

28 88

7 66

1 55

- - - A K H Y D D I L Y Y K A G - - G I A K I V I N R P H K R N A F R P Q T V F E L Y D A F C N A R E D N R I G V V L L T G A G P H S D G K

- - - V K A Y E D I L Y Q K L D - - G I A K I T I N R P H K R N A F R P Q T I F E M Y E A F C D A R E D Q D I G V I L L T G A G P H T D G K

- - - T K T Y E D I L Y D K A D - - G M A K I T I N R P H K R N A F R P K T V F E L Y D A F C D A R E D T S I G V V L F T G A G P H T D G K

- - - V H N Y T D I L Y H K T T - - G I A K I T I N R P H K R N A F R P Q T I V E M S Q A F E D A R N D P T I G V V L L T G A G P H T D G K

- - - W G T Y T D I L V D R C S E - G I A R V A I N R P A K R N A F R P Q T V V E L C D A F S R I R D D R S I G V V L F T G V G P A A D G G

- - - W K S Y E D I L F H N S G D - G I A R I A I N R P E K R N A F R P K T V C E M L D A F N L V R N D E K I G V V L L T G A G P D K K G V

E G D N K E F V D I I Y E K A L D E G I A K I T I N R P E R R N A F R P Q T V K E L M R A F N D A R D D S S V G V I I L T G K G - - - - - T

- - - G K G F V D I L Y D K A V G E G I A K I T I N R P D R R N A F R P L T V K E L M R A F E D A R D D S S I G V I I L T G K G - - - - - T

M D I K Y E K - K E - G I A T I T I N R P H V L N A F R P Q T V K Q M L E A M I D A R W D D D I G V V I L T G E G - - - - - T

- - - - - - F E D I R Y E K S T D - G I A K I T I N R P Q V R N A F R P L T V K E M I Q A L A D A R Y D D N I G V I I L T G A G - - - - - D

- - - A G E Y S D I L Y H K T E E - G I A K I T I N R P E R R N A F R P Q T V D Q M I E A L Q D A R N D S Q I G V I I L T G A G - - - - - D

- - - K R T Y D E I L Y E T Y N - - G I A K I T I N R P E V H N A F T P K T V A E M I D A F A D A R D D Q N V G V I V L A G A G - - - - - D

M G E R V K L E L D G - - E I A V A T L N R P E K L N A L D T K T R M E L A E V I E G I - - E E V A R V L I I T G S G - - - - - -

E G D K Y . D I L Y . K E G I A K I T I N R P K R N A F R P Q T V E M . A F D A R . D I G V . . L T G A G P H D G


71 139

74 142

73 141

74 142

85 153

81 149

133 201

127 195

57 125

80 149

89 157

67 135

56 123

Y A F C S G G D Q S V R G E G G - Y I D D Q G T P R L N V L D L Q R L I R S M P K V V I A L V A G Y A I G G G H V L H L V C D L T I A A D N

Y A F C S G G D Q S V R G E G G - Y V D Q A G V P R L N V L D L Q K L I R S M P K V V I A L V A G Y A I G G G H V L H L L C D L T I A A D N

Y A F C S G G D Q S V R G Q A G - Y V D D A G I P R L N V L D L Q R L I R S M P K V V I A L V A G Y A I G G G H V L H L I C D L T I A A D N

Y A F C A G G D Q S I R G E A G - Y L D E Q G V A R L N V L D L Q R Q I R S L P K V V I A L V A G Y A I G G G H V L H L V C D L T I A A E N

F A F C S G G D Q S V R G D G G - Y V G D D G L P R L N V L D L Q R I I R S L P K V V I A L V A G Y A M G G G Q V L H L L C D L S L A A D N

F S F C S G G D Q S I R G Q N G - Y E D D E G T Q R L N V L E L Q R L I R T L P K V V I A L V P G F A I G G G Q V L Q L V C D L S I A S E N

K A F C S G G D Q A L R T Q D G - Y A D P N D V G R L N V L D L Q V Q I R R L P K P V I A M V A G Y A V G G G H I L H M V C D L T I A A D N

Q S F C S G G D Q A L R D A D G - Y V D F D S F G R L N V L D L Q V Q I R R L P K P V I A M V A G Y A V G G G H V L H M V C D L T I A A D N

R A F C T G G D Q K V R G T K G - Y K D E N G K Q S L N V L Q L Q R E I R T I P K P V I A K V A G Y A I G G G Q I L Q M L C D L T I A A D N

K A F C S G G D Q K V R G D Y G G Y K D D S G V H H L N V L D F Q R Q I R T C P K P V V A M V A G Y S I G G G H V L H M M C D L T I A A D N

L A F C S G G D Q K I R G N A G - Y A D E K G V N K L N V L D F Q R D I R T C P K P I I A M V A G Y A I G G G H V L H M L C D L T I A A E N

K A F C S G G D Q K V R G H G G - Y V G D D Q I P R L N V L D L Q R L I R V I P K P V V A M V S G Y A I G G G H V L H I V C D L T I A A D N

K A F A A G A D I N E L L Q R D A I K A F E A T K L G - - T D L F S R I E E L E I P V I A A V N G Y T L G G G C E L A M A C D I R I A S E K

A F C S G G D Q V R G . G Y . D . G . R L N V L D L Q R I R . . P K P V I A . V A G Y A I G G G H V L H . . C D L T I A A D N


140 209 143 212

142 211

143 212

154 223

150 219

202 271

196 265

126 195

150 219

158 227

136 205

124 193

A I F G Q T G P K V G S F D G G F G S S Y L A R I V G Q K K A R E I W Y L C R Q Y S A Q E A E R M G M V N T V V P V D R L E E E G I Q W A KA I F G Q T G P K V G S F D G G F G A S Y M A R I V G Q K K A R E I W F L C R Q Y D A Q Q A L Q M G L V N Q V V P I A Q L E A E G V Q W A R

A I F G Q T G P K V G S F D G G F G A S Y L A R I V G Q K K A R E I W F L C R Q Y D A Q Q A L E M G L V N C V V P I E Q L E A E G I K W A G

A I F G Q T G P K V G S F D A G F G A S Y L A R I V G Q K K A R E I W F L C R Q Y T A Q E A L A M G L V N A V V P V E A L E A E G I R W A Q

A V F G Q T G P K V G S F D G G F G A G Y L A R V V G Q R K A R E I W F L C R R Y G A D E A L R M G L V N A V V P L D Q L E A E G V R W A R

A I F G Q T G P R V G S F D A G F G S S Y L A R L V G Q R K A R E I W F L C R K Y N S K E A L E M G L V N A I T K I E E L E A E G V I W A R

A I F G Q T G P K V G S F D A G Y G S S I M S R L V G P K K A R E M W F M T R F Y T A S E A E K M G L I N T V V P L E D L E K E T V K W C R

A I F G Q T G P K V G S F D A G Y G T S I M S R L V G P K K A R E M W F L S R F Y T A D E A D R M G L V N V V V P L A D L E R E T V K W C R

A I F G Q A G P R V G S F D G G Y G A S Y M A R I V G Q K R A R E I W F L C R Q Y S A Q E A L Q M G W I N A V V P L E E L D Q H T K A W A K

A I F G Q T G P K V G S F D G G W G A S Y M A R I V G Q K K A R E I W F L C R Q Y D A K Q A L D M G L V N T V V P L A D L E K E T V R W C R

A R F G Q T G P R V G S F D G G W G A S Y M A R L V G Q K K A R E I W Y L C R Q Y N A Q E A L D M G L V N T V V P L E K L E E E T I Q W C R

A I F G Q T G P K V G S F D A G Y G S G Y L A R I V G H K K A R E I W Y L C R Q Y N A Q E A L D M G L V N T V V P L E Q L E E E T I K W C E

A K F G Q P E I N L A I I P G A G G T Q R L P R L V G L G M A K K L V L T G E I I D A Q T A L R I G L V E E V V E H E R L M E R A K E V A A

A I F G Q T G P K V G S F D G G . G A S Y L A R I V G Q K K A R E I W F L C R Q Y A Q E A L M G L V N V V P L E L E E . . . W A R


210 275

213 279

212 277

213 278

224 289

220 285

272 337

266 331

196 263

220 285

228 293

206 271

194 256

E I L S K S P L A I R C L K A A F N A D C D G - - Q A G L Q E L A G N A T L L Y Y M T E E G S E G K Q A F L E K R P P D F S Q Y P W L P

E V L S K S P I A I R C L K A A L N A D C D G - - Q A G L Q E L A G N A T M L Y Y M T E E G R E G K Q A F L E K R S P N F R Q Y P W L P N

E I L E K S P I A I R C L K A A F N A D C D G - - Q A G L Q E L A G N A T L L Y Y M T E E G A E G K Q A F L E K R P P N F R D F P W L P

E I L S K S P L A I R C L K A A F N A D C D G - - Q A G L Q E L A G N A T L L Y Y L T Q E S A E G K T A F L E K R P P N F Q Q F P W R P

E V L Q H S P T A I R C L K A A F N A E T D G - - L A G I Q E L A G N A T H L F Y R T D E A L E G R N A F L E K R P P D F S E T G W L P

E I L R N S P T A I R I L K A S F N A E C D G - - I A G I Q E L S G Y T T Q L F Y S T D E A K E G R D A F L E K R P P D F S D Y G W T P

E I L R N S P T A I R V L K A A L N A V D D G - - H A G L Q G L G G D A T L L F Y G T E E A T E G R T A Y M H R R P P D F S K F H R R P

K I L R N S P T A I R V L K S A L N A A D D G - - H A G L Q E L G G N A T L I F Y G T E E A K E G K N A Y M E R R R P D F S K F P R K P

Q I L E H S P T A L S M L K A A L N A D C D G - - Q A G L Q E L A G L A T H L F Y G T E E A Q E G L N A F L E K R N P K F R R R K A L D E N

E M L Q N S P M A L R C L K A A L N A D C D G - - Q A G L Q E L A G N A T M L F Y M T E E G Q E G R N A F N Q K R Q P D F S K F K R N P

E I L A N S P L A I R C L K A A L N A D C D G - - Q A G L Q E L A G N A T L L Y Y M S E E G Q E G R N A F V E K R K P D F S K F P K R P

E M L E K S P T A L R F L K A A F N A D T D G - - L A G I Q Q F A G D A T L L Y Y T T D E A K E G R D S F K E K R K P D F G Q F P R F P

K I I E K S P L A V K V A K K A L N A S I N M P L K E G L R Y E A S L F A L L F S S - E D A K E G M R A F L E K R K P E F R G R

E I L . S P A I R C L K A A L N A D C D G P L Q A G L Q E L A G N A T L L F Y T E E A E G . . A F L E K R P D F S F P P N

The multiple alignment was generated by BLOSUM30 using Clustal W. The locus tags used to indicate organisms are summarized in Table 2.1, except for the following: Cymecp092 in Cyanidiochyzon merolae and P0671D01.18 in Oryza sativa. AF0435, a hypothetical protein in the archaeon Archeoglobus fulgidis DSM4304, was used as an out-group in phylogenetic construction as shown in Fig 2.3.

294

A.1.3. An amino acid sequence alignment of MenD homologs in cyanobacteria and other

organisms

Sll0603Alr0312Tll0130Synw0997Pmm0607At1g68890CymeCp094B2264ML2270

1 0

1 0

1 0

1 0

1 0

1 80

1 0

1 0

1 0

M R S S F L V S N P P F L P S L I P R Y S S R K S I R R S R E R F S F P E S L R V S L L H G I R R N I E V A Q G V Q F D G P I M D R D V N L D D D L V V Q V C V

M R S S F L V S N P P F L P S L I P R Y S S R K S I R R S R E R F S F P E S L R V S L L H G I R R N I E V A Q G V Q F D G P I M D R D V N L D D D L V V Q V C V


1 0

1 0

1 0

1 0

1 0

81 160

1 0

1 0

1 0

T R T L P P A L T L E L G L E S L K E A I D E L K T N P P K S S S G V L R F Q V A V P P R A K A L F W F C S Q P T T S D V F P V F F L S K D T V E P S Y K S L Y

T R T L P P A L T L E L G L E S L K E A I D E L K T N P P K S S S G V L R F Q V A V P P R A K A L F W F C S Q P T T S D V F P V F F L S K D T V E P S Y K S L Y


1 0

1 0

1 0

1 0

1 0

161 240

1 0

1 0

1 0

V K E P H G V F G I G N A F A F V H S S S V D S N G H S M I K T F L S D E S A M V T A Y G F P D I E F N K Y S T V N S K D G S S Y F F V P Q I E L D E H E E V S

V K E P H G V F G I G N A F A F V H S S S V D S N G H S M I K T F L S D E S A M V T A Y G F P D I E F N K Y S T V N S K D G S S Y F F V P Q I E L D E H E E V S


1 0

1 0

1 0

1 0

1 0

241 320

1 0

1 0

1 0

I L A V T L A W N E S L S Y T V E Q T I S S Y E K S I F Q V S S H F C P N V E D H W F K H L K S S L A K L S V E E I H P L E M E H M G F F T F S G R D Q A D V K

I L A V T L A W N E S L S Y T V E Q T I S S Y E K S I F Q V S S H F C P N V E D H W F K H L K S S L A K L S V E E I H P L E M E H M G F F T F S G R D Q A D V K


1 65

1 71

1 65

1 45

1 69

321 397

1 64

1 64

1 64

M V D F T N P N T L A A S V L V E T L F R L G L Q Q A V I C P G S R S S P L T V A L A R H G - - - G I D C V V S L D E R S A S F F A L G

M S N L M P I A Y K N I N Q L W A Y I F T E T L K R L G L A Y A V I C P G S R S T P L A V A F A Q Q A P - - N I E A I S I L D E R S A A F F A L G

M I L T E N L N V L W A S V L F E T L Y R L G L R T V V L S P G S R S G P L A I A A A A H P - - - H L E A L P I L D E R S A A F F A L G

M T R L V L C P G S R S G P L A A A A G L L A A R G D L A L T T A I D E R S A A F L A L G

M T S H I E C Q N F F R S L Q L L D L F T K I G V K N L I L C P G S R S G P L A I A A G E L N K R R V L N V F N S I D E R S A G F H S L G

E L L N R E T E V S N F L R D E A N I N A V W A S A I I E E C T R L G L T Y F C V A P G S R S S H L A I A A A N H P - - - L T T C L A C F D E R S L A F H A I G

M L L F C S F S L I D W S E L I I K L L A H Y G F Q Y K F I A P G A R S S L L A R A A L L H G - - - - - N C V V H F D E R S L A F A A L G

M S V S A F N R R W A A V I L E A L T R H G V R H I C I A P G S R S T P L T L A A A E N S - - - A F I H H T H F D E R G L G H L A L G

M N P S T T Q A R V V V D E L I R G G V R D V V L C P G S R N A P L A F A L Q D A D H A G R I R L H V R I D E R T A G Y L A I G

E L L N R E T M N N W A . . . E L R . G . . V . C P G S R S . P L A . A A A R G . . . . D E R S A A F A L G


66 135

72 141

66 135

46 115

70 139

398 467

65 144

65 134

65 134

H G K R - - - - - - - - - - T G Q P V A L V C T S G T A A A N F L P A I I E A H Y S Q V P L L V L T G D R P P R L R H C R A G Q T I D Q T K L Y G H Y P Q W Q T

I A K A - - - - - - - - - - T N R P V A I V C T S G T A G A N F Y P A V I E A Q E S R V P L L L L T A D R P P E L R D C H S G Q T I D Q M K L Y G S Y P N W Q A

L V Q Q - - - - - - - - - - Q G R P V A L L C T S G T A A A N F Y P A I I E A S L S H L P L I V L S A D R P P E L R F C Q A G Q A I D Q V H L Y G H A V R H Y R

M A T A - - - - - - - - - - G G R A V A V V T T S G T A V A N L L P A A V E A D R S T Q P L L L L T A D R P A R L K N C G A N Q T V N Q E Q F L Q P V C R W L G

I S T A - - - - - - - - - - S G D V S L V V T T S G T A V G N L L P A A I E A D K S C K S I V F I T A D R P L R L K N C G S N Q T V N Q E E F L N S V C R S N L

Y A K G - - - - - - - - - - S L K P A V I I T S S G T A V S N L L P A V V E A S E D F L P L L L L T A D R P P E L Q G V G A N Q A I N Q I N H F G S F V R F F F

V G K A S Q I Q T S K T N A S K T K A I L I V T S G T A V G N L W P A V M E A S L S Q T S L I L L T A D R P Y E L R D T S A N Q T L D Q V K L F N G Y V R W Q F

L A K V - - - - - - - - - - S K Q P V A V I V T S G T A V A N L Y P A L I E A G L T G E K L I L L T A D R P P E L I D C G A N Q A I R Q P G M F A S H P T H S I

L A V A - - - - - - - - - - A G A P V C V A M T S G T A V A N L G P A V V E A N Y A R V P L I V L S A N R P Y E L L G T G A N Q T M E Q L G Y F G T Q V R A A I

. A K A S Q I Q T S K T N A . G P V A . . T S G T A V A N L . P A . I E A S . P L . L L T A D R P P E L . C G A N Q T I . Q . G . R . .


136 206

142 211

136 204

116 186

140 209

468 539

145 205

135 203

135 210

E L A L P E P N M D Y C H Y L R Q T A L H S W Q K C F W P G L - - - - - - - G V V H L N C P F D E P L V P L E H A R - - E S L Q H L A E E F S K E H F Y R G L T

E L A L P V S D M G M L A Y L R Q T L V H S W Y R M Q A P T P - - - - - - - G P V H L N I P F R D P L A P I P D G - - - T D L S Y L L A K F H P E E F F A G I T

E L S L P - - E L P L L P Y L R Q T L C H S W Q T A L W P D P - - - - - - - G P V H L N I P L R D P L D L R S Q A N - - F H G E L P K G F F D Q V Q P F V P P R

H G A P E G L A S Q P P Q A L F D L A H E A W R H C H G R P P - - - - - - - G P V Q L N L P F E E P L H G A P E D Q - - L S L Q V S G L Q R S A P A P S S D S P

S T N L N G I H E N N D D D I L K I V E T V K K Q I L Q - S P - - - - - - - G P I H L N I A F E K P L D I S L K N K - - K K N F E V F E R F Y L N K T Y K F L K

N L P P P - T D L I P V R M V L T T V D S A L H W A T G S A C - - - - - - - G P V H L N C P F R D P L D G S P T N W S S N C L N G L D M W M S N A E P F T K Y F

D V P P P - H H E L D A D W L A S T L A Y A A F K A S D - - - - - - - - - - G P V H L N L M F R E P F A S Y P I P - - - - - - - - M P K K L V E Y V P A S T A T

S L P R P - T Q D I P A R W L V S T I D H A L G T L H A - - - - - - - - - - G G V H I N C P F A E P L Y G E M D D T G L S W Q Q R L G D W W Q D D K P W L R E A

S L G L A E D A P E R L D S L N A T W R S A T C R V L V A A T G S R T A N A G P V Q F D I P L R E P L I P D P E P - - - - H G A I T P Q G R P G G K P W T Y I P

L L P . . L T . A . . . P G S R T A N A G P V H L N . P F R E P L . P . . . P . .


207 279

212 274

205 259

187 240

210 274

540 609

206 253

204 257

211 254

T F T T M N R G E A I S L P V N F S I F S F P S P Q L D F S P L G L I L V G V M P G G E T P S - - L L T D I L A I A R G L G Y P V L C D A L C S L R N - - - - -

D T T P L P H H S P L S I P P - - - - - - - - - - E W L Q S Q R G I I I A G V A Q P Q Q P Q E - - Y C R A I A R L S Q T L Q W P V L A E G L S P V R N - - - - -

V V T S L P W Q T - - - - - - - - - - - - - - - - - W Q Q M Q R G L I I A G P S H G V D P L A - - E A A A I D R L S R F L Q W P V L A D A L S S A R - - - - - -

- L G A A P Q L D - - - - - - - - - - - - - - - - - P N Q - - A G V V V A G P W R G L A P A L P A Y Q Q A L L Q W L A R S G W P L L A D P L A A I P - - - - - -

N D N Q N K N I Q F S E K F F K R - - - - - - - - - I N L S N P G I I I V G P Y Q G S T K D L F S F N S A L K K L Q E I T G W P V F V D P V S G V S - - - - - -

Q V Q S H K S D G V T T G Q I T - - - - - E I L Q V I K E A K K G L L L I G A I H T E D E I W - - - - - A S L L L A K E L M W P V V A D V L S G V R L R K L F K

G F E W S L D W Q - - - - - - - - - - - - - - - - - - Q K - - - G L F I V A Q A M D D E S F H - - - - - L L L Q L A Q Q W S W P I L V E V F C H A R - - - - - -

P R L E S E K Q R D - - - - - - - - - - - - - - W F F W R Q K R G V V V A G R M S A E E G K - - - - - - K V A L W A Q T L G W P L I G D V L S Q T G - - - - - -

Q V T F D Q P L E - - - - - - - - - - - - - - - - - I D L S A D T V V I A G H G A G V H P N - - - - - - - - - - - - L T - E L P I V A E P T A P F G - - - - - -

S I F S S G . . I A G G . A . . L . L W P V L A D . L S . R N R K L F K

295 Sll0603Alr0312Tll0130Synw0997Pmm0607At1g68890CymeCp094B2264ML2270

280 355

275 350

260 332

241 312

275 347

610 688

254 326

258 329

255 318

- - Y D D G Q T A L I T N Y D F L I R C P R W A E Q L V - P E Q I I Q I G E L P T S K A L R H W L G - T I D C P R Y I L N F H G E N L D P L Q G Q T I Y L S A S

- - Y A D F N P Y L I S T Y D L I L R N Q Q L A T R L A - P D M V I Q I G D M P T S K E L R N W I D - T H Q P R R W V I D P S D Q N L D P L H G R T T H L R I R

- - - - - G L P H G I S H Y D L L L R D A H L R E Y L R - P E A V I Q L G P L P T S K A L R E W L S - A C D P L I W C L D P T G D N N N P L H G R C Q M L A I A

- - - - A D C P G Q L D G W E L Q L D R L Q L A P - - - - G S P V L R L G P L P A S R R L E A W L Q R Q T G P Q L L I S E G E P R G L D P L G L A D Q W S G G L

- - - - A E L K G L V E N W E L I L K - - K N K N I I Q - C D Q I L R F G P L S S S N Y L E D F L L N F E G L Q I L V K E N N I R K L D P I K K S L E Y D F G I

P F V E K L T H V F V D H L D H A L F S D S V R N - L I E F D V V I Q V G S R I T S K R V S Q M L E K C F P F A Y I L V D K H P C R H D P S H L V T H R V Q S N

- - - - - Y Q Y K H S C V I A Y H E T I C H K T M P F P D A N I I I Q F G E R L I S N H L I T I M K - - K V N Q Y V V V S S S T V R V D P L K A V T T R I C V T

- - - - - - - - Q P L P C A D L W L G N A K A T S E L Q Q A Q I V V Q L G S S L T G K R L L Q W Q A S C E P E E Y W I V D D I E G R L D P A H H R G R R L I A N

- - - - - - - - - P Y R P Y P L H P L A L P L L H - - - - P K Q V I M L G R P T L H R P V S A L L A - - - D P Q V P V Y A L T T G P C W P D V S G N S Q A A G T

P F Y . . D L . L . . L . V I Q . G T S K L W L . . . L D P L . . . .


356 430

351 425

333 404

313 383

348 427

689 764

327 401

330 398

319 386

V A Q V A E Y I H N - - Q G F I P D A E Q K N Y A H - - - S W L E K Q R Q S Q T I I I S A L A D A H T P L M V A Q L A H C L P P Q T N L F V A N S L P V R W L E

V E E L G Y Q G V E - - E D K S S V S E Y L Q L W C - - - N A E T K V R V N V D E T L D K M E D L V E C K A A W L L S Q M L P P E T P L F I A N S M P V R D V E

P Q A V D C P - - - - - P D P L P P N P Y L K D W Q - - - D Q D Q R V R E Q L K R T F E A I D W F S E V K L I Y H L P Q W L P S Q T A I F V A S S M P V R D V E

A A W W A E Q N Q D - - F P G A S T P E Q L M P Q S - - - G I A S L L R - - - - Q R L P L Q G A V N E P A L A H W L P L L L P P Q L P V M L A A S S P V R D W L

S N F V N Q L L G A F L N H K K K S K P L I N L G Q V L I K E G A K I K E I L K E Q L F S N T E I T E Y K L A N F V P K I W P E N Y P I M L S A S S P I R D W L

I V Q F A N C V L K S R F P W R R S K L H G H L Q A - - - - L D G A I A R E M S F Q I S A E S S L T E P Y V A H M L S K A L T S K S A L F I G N S M P I R D V D

N V Q S L L K F W F K T L R P T T K P Q W L S W W Q - - - H L N F C V G Y K L R Q Y F A Q E I K L S E P G V L R M C F Q Y A K P - - L I F V G N S M P I R D A N

I A D W L E L H P A E - - - - K R Q P W C V E I P R - - - - - - - L A E Q A M Q A V I A R R D A F G E A Q L A H R I C D Y L P E Q G Q L F V G N S L V V R L I D

R A V T T G T - - - - - - - - - P H P A W L Q R C A - - - E M N Q H A I S T V R S Q L A A H P L I T G L H V A A A V A D A L R P G D Q L V L G A S N P V R D M A

. . . L V L I . . . . . . . . E . A L L P P . L F . . N S P V R D . .


431 481

426 475

405 454

384 433

428 477

765 831

402 454

399 448

387 448

F F W P A N G D H - - - - - - - - - - - - - - - - - H R I F V N R G A N G I D G T L S T A M G I A H R S R G - - - - - - - - - - - - E T V L L T G D L S L L H D

F F W K P N N L R - - - - - - - - - - - - - - - - - V R S H F N R G A N G I D G T L S T A L G I S H R Q Q - - - - - - - - - - - - - S S V L I T G D L A L L H D

S V W Q G S D R R - - - - - - - - - - - - - - - - - H R F Y F N R G A N G I D G T L S T A L G V A H R G Q - - - - - - - - - - - - - P T L L I T G D L A C L H D

I W G G L Q A Q N - - - - - - - - - - - - - - - - - R R C F S F R G A S G I D G T L S L A M G L A M E Q G - - - - - - - - - - - - - P M V L V T G D L A L L H D

T F S E N A T L T - - - - - - - - - - - - - - - - - R E C F S F R G A S G I D G T L S L A L G I A R I T K - - - - - - - - - - - - - P L L L V T G D L A L I H D

M Y G C S S E N S S H V V D M M L S A E L P C Q W - I Q V T G N R G A S G I D G L L S S A T G F A V G C K K - - - - - - - - - - - - R V V C V V G D I S F L H D

H F V E C H H L H D P - - - - - - - - - - - - - - - V H V F A N R G V S G I D G N L A T S M G V S Y A L Q E - - - - - - - - - - - - P L M C I L G D L A F L H D

A L S Q L P A G Y - - - - - - - - - - - - - - - - - - P V Y S N R G A S G I D G L L S T A A G V Q R A S G K - - - - - - - - - - - - P T L A I V G D L S A L Y D

L V G L S T D G I - - - - - - - - - - - - - - - - - - Q V R S N R G V A G I D G T V S T A I G A A L A Y E R S P G Y A G S T D R P A R T V A L I G D L A F V H D

. . V V D M M L S A E L P C Q W . . . N R G A S G I D G T L S T A . G . A . . S P G Y A G S T D R P A P . V L . T G D L A . L H D


482 557

476 551

455 530

434 507

478 554

832 894

455 523

449 522

449 528

S N G F L N Q S Q - - M R G N L T I I L L N N N G G G I F Q T L P I A Q C E D V - - F E T Y F A T P Q G V D F G Q L C R T Y G V E H K I I T N L W D L K E Q W P

T N G F L I R N K - - F V G H L T I I L I N N N G G G I F E M L P I A K F E P P - - F E E F F G T P Q D I D F A Q L C T T Y N V Q H E L I H S W V H L Q Q R L N

T N G W L I T P Q - - F Q G C L T V L L I N N R G G G I F E H L P I R Q F D P P - - F E A F F A T P Q Q V D F S Y L A A A Y G I P Y H C L K D W A D V E A Q L R

S N G W L H G A Q - - G N P P L L V L L I D N Q G G G I F Q Q L P I Q S K Q - - - - F D R L F A M P Q R V N P L A L A A A H G I D G R Q V A C L E D L P E A L E

I N G F L I E N A - - I E L N L T I L L I N N N G G N I F N N L Y K N N L D E E - E L K K L F I M P K S I N W E N L A K G Y Q V P I K N V S D L N K L R E A F E

T N G L A I L K Q R I A R K P M T I L V I N N R G G G I F R L L P I A K K T E P S V L N Q Y F Y T A H D I S I E N L C L A H G

L T S L H L L R Q - - L T K H I W V I V I N N Q G G G I F E L I P N V F N H - - - - - - - - Y F H H H D H - F E H V A K Q F D F H Y Y A V N N W Y E L A L H L Q

L N A L A L L R Q - - V S A P L V L I V V N N N G G Q I F S L L P T P Q S E - - - - R E R F Y L M P Q N V H F E H A A A M F E L K Y H R P Q N W Q E L E T A F A

S S G L L I G P T E P T P Q Q L K I V V S N D N G G G I F E L L E Q G D P R L S A V S S R I F G T P H D V D V G A L C R A Y H V E G R Q I E V D K L R E A L D E

. N G . L I . Q . . L T I . L I N N N G G G I F L P I . . V . . F . T P Q V F L A . Y . . . . L


558 595

552 587

531 562

508 545

555 588

895 894

524 561

523 556

529 556

S N N S - S P I R V L E I I G D R H Q E A Q W L K S L Q A Q F C C A D S L I Q

P L P N - T G I R V L E L R T N R K I D A Q W R R D N L S N F A A D N I I

Q T P W - P K I R L L E F R S D R H R N A Q W R Q Q V L A H L G G

W G L A Q G R P A L L R L A T D R E A D A R L R T Q L R S A A Q N A E P L L

W S L S M Q K S V I I K V D I N V E N E M K G R N L I L K K I L T S

N T K G - - - A N L I E I Q T N Q L E N A T L H K K L T Q Y T N L F S V S F H S D

D A W R T P T T T V I E M V V N D T D G A Q T L Q Q L L A Q V S H L

P G V G - - - M R V L E V K A D R S S L R Q L H A A I T A T L

. L E . R A Q . . . S D

The multiple alignment was generated by BLOSUM30 using Clustal W. The locus tags used to indicate organisms are summarized in Table 2.1, except for the following: Cymecp094 in Cyanidiochyzon merolae. ML2270, a MenD homolog in Mycobacterium leprae, was used as an out-group in phylogenetic construction as shown in Fig 2.3.

296

A.1.4. An amino acid sequence alignment of UbiA homologs in cyanobacteria and other

organisms

S6803S7002A7120NpuncTeryTelonS8102PmarMED4PmarMIT9313PmarSS122GvioRpalRrubEcolCT1511

10 20 30 40 50 60 70

M V A Q T P S S P P L W L T I I Y L L R W H K - P A G R L I L M I P A L W A V C L A A Q G L P P L - - - - P L L G T I A L G T L A T S G LM V S E P T W K K I I Y L L R W D K - P A G R L I L I I P A L W A V F L A A E A K P D W - - - - K L V L V I I L G A I T T S A A

M L E T H Q - E P I W L T I F R L L R W H K - P E G R L I L M I P A L W A V F L A A A G K P P L - - - - P L V G V I V L G T L A T S A AL R M P E R P Q - P P V W L V I I R L L R W H K - P E G R L I L M I P A L W A V F L A A S G K P P L - - - - P L V G V I I L G T L A T S A A

M T Q A A S P E S T L M K V I R L L R W D K - P A G R F I L M I P A L W A V F L A T H A M P P I - - - - P L V G V I V V G T L A T S A AN S Q V S R T S V S T W V A I A Q L L R W H K - P A G R L I L L I P A L W S L T L A S E G L P S L - - - - K L L L I I T M G A I A T S A A

M D S I R E R L G P W L E L L R W T K - P T G R L I L L I P A G W S L W L S P S A P P G L - - - - D L L L Q I V V G G L A V S G AM I K K D Q N S K I N I L F K L L R W N K - P T G R L I L L I P A G W S L Y L T P E S N P S I - - - - Y M L L K I L I G G L L V S G LM T S R S F S N G L R Q W L E L L R W H K - P S G R L I L L V P A G W S L W L T P D A P P L A - - - - G L V S L I V V G G L M V S G AM K S F L L T K K L L N I F H L L R W N K - P T G R L I L L I P A G W S L W L T P N A P P S K - - - - E L V T L I I A G A I S V S G A

M S T A P T D W R A K A D A V F R L L R W D K - P A G R L I L M I P A L W G V F A A A G G S P S P - - - - L L V L V M V V G T I A T S A AN W V D N H A P L W T R P Y L R L A R F D R - P I G S W L L L M P C L W S A A L A A G M A H D L S R L P L F C A L F F I G S F V M R G A

D G W V D R M A P G V I R P Y L K L M R L D R - P I G T W L L L F P C W W S Q S M A A Q G W P D L - - - - W L A A L F A A G A L V M R G AM E W S L T Q N K L L A F H R L M R T D K - P I G A L L L L W P T L W A L W V A T P G V P Q L - - - - W I L A V F V A G V W L M R A A

M S V S S S S Q L S A F Q A W M L A I R P K T L P A G A M P V V I G A A L A A A S G - V F K P L P - - - - - - A L V A L I C A L G I Q I AL . . . . L L R W K L P . G R L I L L I P A L W . . . L A . P L S R L P L . . . I . . G . L . S . A


80 90 100 110 120 130 140

G C V V N D L W D - R D I D P Q V E R T K - Q R P L A A R A L S V Q V G I G V A L V A L L C A A G L A F Y L T - - - - P L S F W L C V A A VG C V V N D L W D - R D I D P Q V E R T K - V R P L A S K A L T V K V G I I T A I A F F L C A A G L A F Y F N P W T N P L S F W L C V A A VG C V V N D L W D - R D I D P E V E R T R - D R P L A A R T L S I K V G I A V G I I A L F C A A I L A Y Y L N - - - - S L S F W L S V A A VG C V V N D L W D - R D I D P E V E R T R - D R P L A S R A L S V K V G I V V A I V S L A C A A I L A F Y L N - - - - P L S F W L C V A A VG C V V N D L W D - R D I D S E V E R T R - D R P L T S R A L T I Q T G I I I V I I A F A C A G V I A L Y L N - - - - P L S F W L S V A A VG C I V N D C W D - R N I D P H V Q R T Q - N R P L A S R R L S L G V A L G L M V I A L L C A W G L T F Y L T - - - - P L G Y W L A V A A VG C I A N D L W D - R R F D G R V E R T K - Q R P L A R G A I R P T S A L V L L I V L L S L S L A V V L S L D E A S R Q L C L L L S I G A LG C V A N D I W D - K R I D Q K V L R T K - N R P L A A N K I S T K T A Y L I L I F L I I C S F F L T L S L P E N G R L L S L S L A F F A LG C I A N D L W D - R R I D R Q V E R T R - E R P L A K G S I R I C T A V G L L I V L L L L S L L V V A S L P T P S Q R L C L A L A I L A LG C I A N D L W D - R K I D S K V K R T K - N R P L A N G S L S I V E A F S L L I F L L I I S L K V I L L L P T P S Q N L S L K L A A M A LG C V I N D L W D - R D I D P L V E R T R - A R P L A S R E L A V P V A V V T F A V S L L C A W G L T F F L N - - - - P F T F W L C V A A VG C T W N D I T D - R D L D D K V E R T R - S R P I P A G Q V T A K Q A A V F M V L L C L I G L V V L L Q F N - - - - S F A I A T G I S S LG C T F N D I V D - R D F D A Q V A R T A - A R P I P S G A V S R K K A V V F L G A Q L L V G L A V L L S L N - - - - G F S I W L G V A S LG C V V N D Y A D - R K F D G H V K R T A - N R P L P S G A V T E K E A R A L F V V L V L I S F L L V L T L N - - - - T M T I L L S I A A LT N F I N E I Y D F R K G A D T A E R L G P T R T V A A G I I T E Q T M I R V S I V L G V S V F V L G L Y L V A I G G W P I L L I G V L S LG C V . N D L W D F R D I D V E R T . P R P L A . L . . A . . . . I . . L L C . . . L . L . L N L S . W L V A A L


150 160 170 180 190 200 210

P V I V A Y P G A K R V F P V P Q L V L S I A W G F A V L I S W S A V T G D L T - - D A T W V L W G A T V F W T L G F D T V Y A - - M A D RP V I I A Y P L A K R F F P V P Q L V L A I A W G F A V L I S W S A V T Q N L T - - A A T W L L W G A T V F W T L G F D T I Y A - - L S D RP V I L L Y P G A K R V F P V P Q L V L S I A W G F A V L I S W S A V T Q N I S - - Q A T W L L W G A T L L W T L G F D T V Y A - - M S D RP V I V L Y P G A K R V F P V P Q L V L S I A W G F G V L I S W S A V T Q T I T - - Q P T W L L W G A T V L W T L G F D T V Y A - - M S D KP V I I C Y P L A K R F F P V P Q L V L S I A W G F A V L I S W S A A V A K L D - - S A T W I L W G A T I V W T L G F D T V Y A - - M S D RP V I L L Y P L A K R V L P I P Q L V L A V A W G F A V L I P W A A V Q G R L T - - L T T A G L W A A V V M W T L A F D T V Y A - - M P D RP A I L L Y P S A K R W F A Y P Q A V L A F C W G F A V L I P W A A A E R S L T L Q P A L I G C W L A T L L W T F G F D T V Y A - - M A D RP L I L I Y P S A K R W F K Y P Q F I L S I C W G F A V V I P W A A N E G N I N - S I V L L F C W L A T I F W T F G F D T V Y A - - L A D KP P I L L Y P S A K R W F A Y P Q A V L A L C W G F A V L I P W A A S Q A N L K G G W P L L G C W L A T L M W T F G F D T V Y A - - M A D RV P I L I Y P S S K R W F R Y P Q V L L A F C W G F A V L I P W A A S E H S L N G G F P L L T C W L A T M V W T F G F D T V Y A - - M A D KP V I A L Y P G A K R V F P V P Q L V L S L A W G F A A L I S W S A V T G G L E - - T P A W W L W G A T V L W T L G F D T V Y A - - L S D RI I V A A Y P F M K R I T Y W P Q F V L G L A F S W G A L M G F A G T F E R L D - - L V A F A L Y A G S I A W V I G Y D T V Y A - - H Q D AI L V F G Y P F M K R I T Y W P Q A W L G L T F T Y G A L M G W A A V R G S L D - - P A P L L L Y A A C F F W T L H Y D T I Y A - - H Q D KA L A W V Y P F M K R Y T H L P Q V V L G A A F G W S I P M A F A A V S E S V P - - L S C W L M F L A N I L W A V A Y D T Q Y A - - M V D RL F A W A Y T G G P F P I A Y S G L G D V F V F I F F G L V A V G G T Y Y V Q A L S L P M E V L V A A A A P G A F S V C I L L V N N I R D IP . I . . Y P . A K R F . P Q L V L . . A W G F A V L I W A A V L . . . L W . A T . . W T L G F D T V Y A N N M D R


220 230 240 250 260 270 280

E D D R R I G V N S S A L F F G Q Y V G E A V G I F F A L T I G C L F Y L G M I L M L N P L Y W - L S L A I A I V G W V I Q Y I Q L S A P TD D D L K I G I N S S A I F F G K F A P E A V G I F F V L T A T C L A I L G K T L N I N L L I Y G I A W A I A V G L W F K Q Y L K I R K P NE D D Q R I G V N S S A L F F G D Y A P L A I G I F F L G T I I C L T W L G V A I H L H L A F W - I T L G I A S V G W I W Q V L R L R Q P QE D D R R I G V N S S A L F F G N Y A P V A I G I F F A G T I L L L A W L G L L I N L H F A F W - I S L A V A T I G W V W Q S L R L R Q R DE D D Q R L G I N S S A L F F G K Y A N N A V G I C F F L T T I F L G L L G I K M E L H T G F W - I T L G I A T V F W F I Q Y K K L G Q E DP D D R R L G V N S S A L F F G A Y A P L A I A L F Y A L T V V F L M M V G V R A G L T W R F W - L A L A V T T Y F W L R Q S L Q I Y T H ER D D A L I G L N S S A L S L G N R A V V T V R A C Y V L T V V A L G V A A A S A G V H P L F W I F W L G A S L L M Q I S C Q S L N - H R NK Y D I Q I G V N S S A V N L A Y N T K K T I Q I C Y F L T S S F L A I C A F I N Q L N F V F W P I W L I T G F L M Q K D I L K I F P E S KR D D A N L A L K S S A L S L G S H A L K T V A F S Y A L A C T F L A S S A V S A G V G W A F W P F W L I A S I G M Q K E T W T L R - G S ND D D K K L G L Q S S A L S L G Q K A L K S V F I C Y A L T S T L I A F S A L T K D I G I I F W P F W L I A S I G M Q K E V F S L K - E L EE D D L K I G I N S S A I F F G R F T P E A I A A F F A G A A L C L A V V G Y Q L E L G S I F Y - V C L I V A T L F W W R E Y R S L V T G DE D D L M V G I K S T A L L F G E N T K L A L V L L F G V A V S L I G V A L K L A D A G W F A W - I G L A A F A A H L V Q Q I V R V D I H DE D D L L V G V K S S A L A L G S N T R P A L V V F S T L M M A L V A A A G W Q A G L S W P F W - A L L G L P A A H L A W Q I W R V D I H DD D D V K I G I K S T A I L F G Q Y D K L I I G I L Q I G V L A L M A I I G E L N G L G W G Y Y W S I L V A G A L F V Y Q Q K L I A N R E RD T D R K V G K M T L P A R I G - - A P A A R A L Y V A L V V L A Y L V P F Y M I S T G Y S L W C L L S L L S I P L A I G M V R T L Y A S E. D D . . I G . N S S A L F G . A A . . I . . . L T . . L A . . G . L . . F W . L . . . . . . Q . .


290 300 310 320 330 340 350

P - - - - - - - - E P K L Y G Q I F G Q N V I I G F V L L A G M L L G W LL - - - - - - - - P R K A Y N R L F E E N V T I G F I L L S G M I S S F I FL - - - - - - - - P N P A Y G E M F R Q N V W I G F I V L A G M I F G S LL - - - - - - - - P N S V Y G K M F R Q N V W I G F I L L A G M I A G S F- - - - - - - - - I L K P Y A Q I F R Q N V W I G F I L L A G M I T G L I FR R D T Q T T T L P I Q T Y G R Y F N E N V W L G F L L W V G M W C P R PA - - - - - - - - T M A S F G L H F R R Q V Q L G S L L L L G L I V S R G L S GQ - - - - - - - - S I K K I G D H F K N Q S I Y G G L I L L G F I I A SQ - - - - - - - - P I T T Y G Q H F Q H Q V L L G A M L L L G L I L G R I SA - - - - - - - - N T N K F G K H F R N Q V F L G G L I L F G L I L G N F- - - - - - - - - D P A R F N R I F N E N V T V G F V I L T G L I G G T L IS - - - - - - - - - - P L C L K L F K S N R D A G L L L F G G L V A D A V S R S M AS - - - - - - - - - - P V C L M I F K S N R F V G W L L L A A I V A G R A P G G VE - - - - - - - - - - - A C F K A F M N N N Y V G L V L F L G L A M S Y W H FG Q - - - - - - A L N A V L A G T G K V L T V H G L L F S L G L V I P N I I S I F R P

D T Q T T T . . G F . N V . G . . L L . G . I . G . P

The multiple alignment was generated by BLOSUM30 using Clustal W. S6803, Synechocystis sp. PCC 6803; S7002, Synechococcus sp. PCC 7002; A7120, Nostoc sp. PCC 7120; Npun, Nostoc punctiforme; Tery, Trichodesmium erythraeum; Telon, Thermosynechococcus elongatus BP-1; S8120, Synechococcus sp. WH8102; PmarMED4, Prochlorococcus marinus MED4; PmarMIT9313, P. marinus MIT9313; PmarSS122, P. marinus SS120; Gvio, Gloeobacter violaceus PCC7421; Rpal, Rhodopseudomonas palustris; Rrub, Rhodospirillum rubrum; Ecol, E. coli; CT1511, Chlorobium tepidum.

297

A.1.5. An amino acid sequence alignment of UbiD homologs in cyanobacteria and other

organisms

S6803S7002A7120NpuncTelonS8102PmarMIT9313PmarSS122GvioRpalRspheEcolBH3930

10 20 30 40 50 60 70 80

M A R D L R G F I Q L L E T R G Q L R R I T A E V D P D L E V A E I S N R M L Q A G G P G L L F E N V K - - - - - G S P F P V A V N L M G T V EM A R D L R A F I K L V E E R G Q L R R I K A L V D S D L E I A E I A N R M L Q V G G P A L L F E N V R - - - - - G T S V P V V V N L L G T V EM A R D L R G F I K I L E E R G Q L Q R I S A L V D P D L E I A E I S N R M L Q K G G P G L L F E N V K - - - - - G A S F P V A V N L M G T V EM A R D L R G F I K I L E E K G Q L R R I S A L V D P E L E I A E I S N R M L Q K G G P G L L F E N V K - - - - - G A S F P V A V N L M G T V EM T K D L R H Y L Q L L E Q R Q Q L R R I T V P V D P D L E M A E I C N R L L A A G G P A L L F E N V I - - - - - G S P Y P V A I N L L G T L E

M A L F R S G P A T R D L R G F L Q L L D Q R G Q L K R I T A A V D P D L E L A A I A D R V L S Q G G P A L L F E N V I - - - - - G S S M P V A V N T L G T V EM P L L R P G P A S Q D M R D F L A L L E Q R G Q L R R I T A P V E P D L E L A A I T D R V L G L G G P A L L F E N V I - - - - - G S S M P V A V N L M G T L EM N V F Q N G V G S Q D M R D F L S L L E K K G Q L R R I S K P V D P D L E L A A I S D R V L S M G G P A L L F E N V I - - - - - G S S M P V A I N L L G T I E

M G R D L R T F L Q L L E S R G Q L R R I S A P V E R D L E V A E I A N R L L A A G G P A L L F E N V K - - - - - G S P F P V A V N L L G T V EM L S R V K P P F P D L R A F S G Y L E S R G Q L H R I R K P V S V V H D L T E I H R R V L H A G G P A L L I E N P I K A D G T P S E M P I L V N L F G T V E

M R S L P I F P D L R A F L D W C A R K G D L S R I A E P V S L R H E T T A V A S K I L R G G G P V L R F E S - - - L R D T S A T M P L V A N L F G T R EM D A M K Y N D L R D F L T L L E Q Q G E L K R I T L P V D P H L E I T E I A D R T L R A G G P A L L F E N - - - - P - K G Y S M P V L C N L F G T P K

M Y Q N L Q E C I N D L E K H G H L I R I R E E V D P Y L E M A A I H L K V Y E A G G P A L L F E N V K - - - - - G S N Y Q A V S N L F G T M EM L R P M . D L R . F L L L E R G Q L R R I . A P V D P D L E . A E I R . L . G G P A L L F E N V K T G S S P V A V N L G T V E


90 100 110 120 130 140 150 160

R I C W A M N M D H P L E L E D L G K K L A L L Q Q P K P P K K I S Q A I D F G K V L F D V L K A K P G - R N F F P P C Q E V V I D G E N L D L N Q I P L I R PR I C W A M K M E K P E E L E D L G R K L A M L Q Q P K P P K K I S Q A I D F G K V L F D V L K A K P G - G S F F P P C Q Q V V V E Q E E L D L G L I P M L R PR I C W A M N M Q H P Q E L E T L G K K L S M L Q Q P K P P K K I S Q A I D F G K V L F D V L K A K P G - R D F F P A C Q Q V V V Q G D D V D L N T L P L I R PR I C W A M N M Q H P E E L E T L G K K L S M L Q Q P K P P K K I S Q A I D F G K V L F D V V K A K P G - R D F F P A C Q Q V V L Q G N D L D L N K L P L I R PR V C W A M N M D D P L E L E T L G E K L G K L Q Q P K P P K T L S Q A L D F G K I L F D V V R A K P S - R D L L P P C Q Q V V I T A P D L D L R Q L P L I R PR V V W S M G L E R A E Q L E D L G S R L A L L Q Q P R P P K G L S E T K Q F A R V F W D L V K A K P D - R D L T P P C R Q Q I F K G D A V N L Y N I P L I R PR V V W S M G L D N A Q Q L E D L G T R L A L L Q Q P R P P K G L Q E T R Q F A S V F W D L I K A R P D - L D L T P P C H Q Q V L R G D A L N L D N L P L I R PR V V W S M G L S S Q D E L E G L G K K L S Y L Q Q P E P P K G I K K T I E F G G I L W D L L Q A R P D - L D L T P P C H Q R L L K G E D V N L D K L P L I R PR V V W S M G L E E Q Q Q L E A L G E K L G K L Q K P R P P R S L Q D A L E F A P L L F D V I K A R P G R V L F A P E C Q Q V V L E G K A V D L D R I P M I R PR V A W G L G - I L P E N L S R L G E A L A E M R E P A P P Q S L T D A L S K L P M A K A A L A M R P K - L A K S A P V Q E V V L T G D A V D L G R L P V Q I PR V A A G L G - L G L E Q I P E L G A F L A A L R A P A P V A G M R D A L S R W P Q L Q A A L N T R A K - I V R S A E A Q E V V H E G A A V D L G M I P V P T CR V A M G M G Q E D V S A L R E V G K L L A F L K E P E P P K G F R D L F D K L P Q F K Q V L N M P T K - R L R G A P C Q Q K I V S G D D V D L N R I P I M T CR S K F I F R - - - - - Q T W Q S A E N V V A L R N - D P M S A L K H P F A H A R T A L A A S K A L P L K K S R L P A G F E E I T I S D - - - - - - L P L I Q HR V . W . M G . L E L G K L A L Q Q P . P P K . . A . . F . . . L . D V L K A . P . P P C Q Q V V . G . V D L L P L I R P


170 180 190 200 210 220 230 240

Y P G D A G K I I T L G L V I T K D C - - - - - E T G T P N V G V Y R L Q L Q S - - - - - K T T M T V H W L S V R G G A R H L R K A A E Q G - - K K L E V A I AY P K D A G K N I T L G L V I T K D C - - - - - E T G V P N V G V Y R L Q L Q S - - - - - K N T M T V H W L S V R G G A R H L R K A A E N G - - K K L E V A I AY P S D A G K I I T L G L V I T K D C - - - - - E T G T P N V G V Y R L Q L Q S - - - - - K N T M T V H W L S V R G G A R H L R K A A E R G - - K K L E I A I AY V G D A G K I I T L G L V I T K D C - - - - - E T G T P N V G V Y R L Q L Q S - - - - - K N T M T V H W L S V R G G A R H L R K A A E R G - - K K L E V A I AY P K D A G K I M T L G L V I T K D C - - - - - E T G I P N V G I Y R L Q L Q S - - - - - P T T M T V H W L S V R G G A R H L R K A A A Q G - - K K L E V A V AW P G D A G G V I T L G L V I T K D P - - - - - E T G V P N V G V Y R L Q R Q S - - - - - V N T M T V H W L S V R G G A R H L R K A A A M G - - K K L E V A V AW P G D A S G V I T L G L V I T K D P - - - - - E T G V P N V G V Y R L Q R Q S - - - - - P K T M T V H W L S V R G G A R H L R K A A A M G - - Q K L E V A I AW P G D A G K I I T F G L V I T K D P - - - - - E T N T P N V G V Y R L Q Q Q S - - - - - I N T M T V H W L S V R G G A R H L R K A S A M G - - K K L E V A I AY A G D A G R I L T F G L V I T H D P - - - - - E D G T P N V G I Y R L Q Q Q S - - - - - R D T M T V H W L S V R G G A R H L R K A A E R G - - Q K L P V A I AW P G E P A P L I T W G L V F T K P P - - - P G A H G T D N V G V Y R M Q V L G - - - - - K D R L I M R W L A H R G G A K H H H Q W K A D K - - R E M P V A I VW P G D A G P L V T W P V V L T R P H G T S A E E T L H Y N A G V Y R A Q V I G - - - - - R D R L I M R W L A H R G G A G H C R S W M R A G - - E P M P V A L AW P E D A A P L I T W G L T V T R G P H K - - - E - - R Q N L G I Y R Q Q L I G - - - - - K N K L I M R W L S H R G G A L D Y Q E W C A A H P G E R F P V S V AW P D D G G A F I T L P Q V Y S E D P D K - - P G I M N A N L G M Y R V Q L T G N E Y E L D Q E V G L H Y Q I H R G I G V H Q T K A N Q K G - - E P L K V S I FW P G D A G . . I T L G L V I T K D P K S E T G P N V G V Y R L Q L Q S N E Y E L . . T M T V H W L S V R G G A R H L R K A A G P G K K L E V A I A


250 260 270 280 290 300 310 320

L G V D P L I I M A A A T P I P V D L S E W L F A G L Y G G S G V A L A K C K T V D L E V P A D S E F V L E G T I T P G E M L P D G P F G D H M G Y Y G G V E DV G I D P I L I M A A A T P I P V D L S E W L F A G L Y G G S G V A L T K C K T V D L E V P A D A E F I L E G T I T P G E M L P D G P F G D H M G Y Y G G V E DL G V D P L I I M A A A T P I P V D L S E W L F A G L Y G G S G V Q L A K C K T V D L E V P A D S E F V L E G T I T P G E I L P D G P F G D H M G Y Y G G V E DL G V D P L I I M A A A T P I P V D L S E W L F A G L Y G G S G V Q L A K C K T V D L E V P A D S E F V L E G T I T P G E V L P D G P F G D H M G Y Y G G V E DV G V H P L I I M A A A T P I P V D L S E W L F A G L Y G G G G I H L A K C K T L D L E V P A Q S E F V L E G T I T P G E V L P D G P C G D H M G Y Y G G V E DI G V H P L L V M A A A T P I P V Q L S E W L F A G I Y A G E G V R L T P C K T L D L Q V P S H S E V V L E G T I T P G E V L P D G P F G D H M G F Y G G V E DI G V H P L L I M A A A T P I P V Q L S E W L F A G L Y A G E G V R L T G C K T L D L K V P S H S E V V L E G T I T P G E E L E D G P F G D H M G F Y G G V E SI G V H P L I L M A A A T P I P V T L S E W L F A G L Y A G K G V R L S N C K T V N L Q V P S H S E I V L E G T I A P G E E M N D G P F G D H M G F Y G G I E KV G V D P L L V M A A A T P I P V E L S E W V F A G L Y A G E G V Q L A Q C R T S D L L V P A H S E F V L E G T I T P G E V L P D G P F G D H M G Y Y G G V E DI G A D P S M I L S A V L P L P E T V S E I K F A G L L G G E R P S L T P C Q T I P I S V P A D A E I V L E G F V S P T E T A P E G P Y G D H T G Y Y N A V E EL G C D P A L L L A A A L P L P E Q V S E L T F S G V L R G A R T P L V A G R T V P L M V P A T A E I V V E G W V H P G D M A P E G P F G D H T G Y Y N S V E DL G A D P A T I L G A V T P V P D T L S E Y A F A G L L R G T K T E V V K C I S N D L E V P A S A E I V L E G Y I E Q G E T A P E G P Y G D H T G Y Y N E V D SV G G P P A H S L S A V M P L P E G L S E M T F A G L L S G R - - R F R Y S Y V D G Y C I S H D A D F V I T G E I P P G D T K P E G P F G D H L G Y Y S L I H D. G V D P L . I M A A A T P I P V L S E W L F A G L Y . G G V L . C K T . D L V P A S E F V L E G T I T P G E L P D G P F G D H M G Y Y G G V E D


330 340 350 360 370 380 390 400

S P L V R F Q C L T H R K N P V Y L T T F S G R P P K E E A M M A I A L N R I Y T P I L R Q Q V S E I T D F F L P M E A L S Y K A A I I S I D K A Y P G Q A K RS P L V R F H C L T H R K D P I Y L A T F S G R P P K E E A M M A I A L N R I Y T P I L R Q Q V S E I V D F F L P M E A L S Y K A A I I S I E K A Y P G Q A R RS P L I R F Q C M T H R K D P I Y L T T F S G R P P K E E A M M A I A L N R I Y T P I L R Q Q V S E I V D F F L P M E A L S Y K A A I I S I D K A Y P G Q A R RS P L I R F E C M T H R K N P I Y L T T F S G R P P K E E A M M A I A L N R I Y T P I L R Q Q V S E I V D F F L P M E A L S Y K A A I I S I D K A Y P G Q A R RS P V I H F H C L T H R R N P I Y L T T F S G R P P K E E A M I A L A L N R I Y T P I L R Q Q V P E I V D F F L P M E A L S Y K A A I I S I D K A Y P G Q A R RS P L V R F H C M T Q R R D P V F L T T F S G R P P K E E A M L A I A L N R I Y T P I L R Q Q I P E I T D F F L P M E A L S Y K L A V I S I D K A Y P G Q A K RS P L V R F H C V T Q R R D P I F L T T F S G R P P K E E A M L A I A L N R I Y T P I L R Q Q V S E I V D F F L P M E A L S Y K L A V I A I D K S Y P G Q A K RS P L I R F H C I T S R K D P I Y L T T F S G R P P K E E A M L A I A L N R I Y T P I L R R Q I N E I T D F F L P M E A L S Y K L A I I S I D K S Y P G Q A R RS P L V R I H T V T H R H D P I Y H T T F S G R P P K E E A M I A I A L N R I Y T P L L R A Q V P E V R D F F L P M D A L S Y K L A V L S I K K A Y P G Q A R RF P V M R I T A I T M R R H P I Y L S T Y T G R P P D E P S R L G E A F N D V F L P V A R R Q F P E I V D L W L P P E A C S Y R I A V A S I K K R Y P G Q A R RF P V L R V S A I T H R K D P L Y L T T H T G R P P D E P S V I G E V F N D L A M P V F R Q Q I P E V K D L Y L P P A A C S Y R I A I V S I D K R Y P G Q A R RF P V F T V T H I T Q R E D A I Y H S T Y T G R P P D E P A V L G V A L N E V F V P I L Q K Q F P E I V D F Y L P P E G C S Y R L A V V T I K K Q Y A G H A K RF P V M K V H K V Y A K Q G A I W P F T V V G R P P Q E D T S F G A L I H E L T G D A V K L E I P G V K E V H A V D A A G V H P L L F A I G S E R Y T P Y Q K VS P L . R F H C . T H R . D P I Y L T T F S G R P P K E E A M . A I A L N R I Y T P I L R Q Q V P E I V D F F L P M E A L S Y K . A I I S I D K A Y P G Q A R R


410 420 430 440 450 460 470 480

A A L A F W S A L P - - - - - - - Q F T Y T K F V I V V D K S I N I R D P R - - - Q V V W A I S S K V D P V R D V F I L P E T P F D S L D F A S E K I G L G G RA A L A F W S A L P - - - - - - - Q F T Y T K F V I V V D K D V N I R D P R - - - Q V V W A I S S K V D P V R D V F I L P D T P F D T L D F A S E K I G M G G RA A L A F W S A L P - - - - - - - Q F T Y T K F V I V V D K D I N I R D P R - - - Q V V W A I S S K V D P T R D V F I L P N T P F D T L D F A S E K I G L G G RA A L A F W S A L P - - - - - - - Q F T Y T K F V I V V D K D I N I R D P R - - - Q V V W A I S S K V D P V R D V F I L P N T P F D T L D F A S E K I G L G G RA A L A F W S A L P - - - - - - - Q F T Y T K F V I V V D K E I N I R D P R - - - Q V V W A I S S K V D P S R D V F I L G E T P F D S L D F A S E K I G L G G RA A M A F W S A L P - - - - - - - Q F T Y T K F V V V V D K H I N V R D P R - - - Q V V W A I A A Q V D P Q R D L F T L A D T P F D S L D F A S E Q L G L G G RA A M A F W S A L P - - - - - - - Q F T Y T K F V V V V D A N I N V R D P R - - - Q V V W A I A A Q V D P Q R D L F V L E N T P F D S L D F A S E H L G L G G RA A M A F W S A L P - - - - - - - Q F T Y T K F V V V V D S T I N V R D P R - - - Q V V W A I S S L V D P Q R D L L V M E N T P F D S L D F A S E N I G L G G RA A M A F W T A L A - - - - - - - Q F T Y T K F V I V V D E D I D I R D P S - - - Q V V W A I A S K V D P E R D L F V I P R T P F D T L D F A C E R I G L G A RL M M G L W S M L P - - - - - - - Q F S Y T K L L I I V D D D V D V R D W A - - - D V M W A V S T R C D T S R D M V S I S D T P I D Y L D F A S P K S G L G G KV M M A L W G M L A - - - - - - - Q F S Y T K M V I V V D E D I N P R D W D - - - D V A W A M A T R M D P S R D V V L L E K T P M D Y L D F A S P E P G L A G KV M M G V W S F L R - - - - - - - Q F M Y T K F V I V C D D D V N A R D W N - - - D V I W A I T T R M D P A R D T V L V E N T P I D Y L D F A S P V S G L G S KK Q P A E L L T I A N R I L G T G Q L S L A K Y L F I T A E Q D K P L D T H K E E E F L T Y L L E R I D L H R D I H F Q T N T T I D T L D Y S G T G L N T G S KA A M A F W S A L P N R I L G T G Q F T Y T K F V I V V D D I N . R D P R K E E Q V V W A I S S . V D P R D . F . L T P F D . L D F A S E . I G L G G R


490 500 510 520 530 540 550 560

M G I D A T T K I P P E T D H E W G E V L E - - - - - S D P A M A E Q V S Q R W A E Y G L G D I N L T E V N P N L F G Y D VM G I D A T T K I P P E T D H E W G E V L E - - - - - S D P K I A E R C D R R W A E Y G L A D I D L T A V D P N K F G Y E M NM G I D A T T K I P P E T D H E W G A P L E - - - - - S D A D V A A M V E R R W A E Y G L A D L Q L G E V D P N L F G Y D M KM G I D A T T K I P P E T D H E W G S P L E - - - - - S D P D V A A M V E R R W A E Y G L A E L Q L G E V D P N L F G Y D M KM G I D A T T K I P P E T D H P W G D P L T - - - - - S D P E V A R R V T E R W Q E Y G L G D I D L T A V D A T R F G Y E L D P A F R W RL A I D A T T K V G P E K N H D W G E P L S - - - - - R P A D L E E R V S A R W S E L G L D G L G Q D E P D P S L F G Y A L D R L I Q G L K T S PM A I D A T T K I G P E K R H E W G E P L S - - - - - R D A D L E S R V D A R W Q E L G L E D L G S E E P D P S L F G Y V M E S L C R Q A M V K K TL A I D A T T K V G P E K N H E W G N P L I - - - - - H S S N L S K K I D E R W N E L G L S D L K P N H A D P S L F G Y V I D E I I K F N Q I T K SL G I D A T T K I P P E T D H A W G E P L R - - - - - A D P A T A E Q V S K R W A E Y G L G D I P L G G V D P R A W G Y S G KL G I D A T N K I G T E T E R E W G K V L E - - - - - M D K D V I A R V D A M W T S L G L S P E H Q P A A G Q R R L I RI G I D A T N K I G P E T H R E W G E V M T - - - - - Q S P E A E A F A D R L I A K L R I G AM G L D A T N K W P G E T Q R E W G R P I K - - - - - K D P D V V A H I D A I W D E L A I F N N G K S AV V I A A Y G D K K R E L C Q E V P E L F K G L Q T F Q N P R L V M P G V V A L E G P S F T S Y E R A Q E E F R A F S E T I T Q N G E L P S C P M IM G I D A T T K I P P E T . H E W G E P L G L Q T F D P . . . . V . R W E G L . D . V D P L F G Y

298 The multiple was generated by BLOSUM30 using Clustal W. S6803, Synechocystis sp. PCC 6803; S7002, Synechococcus sp. PCC 7002; A7120, Nostoc sp. PCC 7120; Npun, N. punctiforme; Telon, T. elongatus BP-1; S8120, Synechococcus sp. WH8102; PmarMIT9313, P. marinus MIT9313; PmarSS122, P. marinus SS120; Gvio, G. violaceus PCC7421; Rpal, R. palustris; Rsphe, Rhodobacter sphaeroides; Ecol, E. coli; BH3930, Bacillus halodulans.

299

A.1.6. An amino acid sequence alignment of UbiH homologs in cyanobacteria and other

organisms

S6803S7002A7120NpuncTelonS8102PmarMED4PmarMI9313PmarSS122GvioRpalRrubEcolMlr2092

10 20 30 40 50 60 70 80

M T F V A A S I T D N Q F D V A I A G G G V V G L V L A A G L R H - - - T G L K IM E K S H L G F D V I I V G G G I V G V T L A A C L K R - - - T N L R I

M A L T Q L T Q T I S P Q P T P P E L R K Y D Y D L V I V G G G I V G L T L A S A L K D - - - S G L N IM A L T Q L E Q P L S P Q P T P P D L R G Y E Y D L V I V G G G I I G L T L A S A L K D - - - S G L S I

M M T S A F A P S E Q S L V A S P Q D L P A V D V A I V G A G I V G L S L A C A L R N - - - S G L A IM A T S S T T F H I L G A G P T G S L A A I A L A S - - - T G C S V

M N N Y L N F K I V G S G P S G L L L A I S L A K - - - L N F N IM V D H L N R F H S A A K G S L P V D I R V K I L G A G P T G T L L A L A L A R - - - L G N S V

M S N L H I K V I G A G P S G S L L A L S L V G - - - N S N A VM A F N N R N T L A A E R G K A A L Q A D C I I I G G G P A G A V L S L L L A R - - - Q G V S V

M P R G H A G D F P P P A L S V R N R P G N S G L V L E T C F A Q L R S G R K D H G A R V R N M T A P R S I V I G G G A F A G L A L A L A L R Q G - - L G P E VM E Y G V S E P L L R G L A A G D P P S A T G P V T G S A D K V A D V L I V G G G L V G G T L A C A L A E - - - K G V S V

M S V I I V G G G M A G A T L A L A I S R L S H G A L P VM Q A Q D D Q Q K G K D M T T A A A G P T E V D V A I I G A G M A G T T L A T L L G N - - - A G R K V

M P R G H A G D F P P P A L S V R N R D . . I . G G G . G . L A . A L S H G . V


90 100 110 120 130 140 150 160

A I I E A L P K E Q A L T K - - - P Q A Y A I S L L S G K I L A G L G V W E N I K D S I G H F E R I Q I S D N D Y R G T V P F A K E D V D E - - - - - L A L G HA I V E A Q P L D V V T Q R - - - Q R A Y A M S L L S Q R L F Q E L G V W E V I A A R S G K Y R N I Q L S D E D F S G V V R F S R Q D L K T - - - - - D F L G FL L I E A K V A S A A V A K - - - G Q A Y A V H L L S A L I Y Q G I G I W D K I L P Q I A K Y R Q V R L S D A D Y P G V V E F E S T D L G T - - - - - P E L G YL L I E A K V A S A A V A K - - - G Q A Y A V H Q L S A L I Y Q G I G V W D K I L P Q I A K Y R R V R L S D A D Y P N V V E F T T D D I H T - - - - - A E L G YA L I E A T P Y R R E N T K - - - G Q A Y A L H Q V S R Y F F E E I G V W D K L M P H V Q P F E V V Q L S D E R F P L T V H F S P A D L G T - - - - - E A L G YV L T D P L T R K E L L S R - - - S R A Y A I T H S S R R L L T D L N L W T S L Q G S L T A F S F L D L R D S A C G G R V V F G L D D L P N S N G R H E A I G WY I T D L L T R E K L I N K - - - D K T Y A I T H S T R K I L S K F N L W S K L K S Y L Y K F D S L S I S D S V T S D F T I L T T S D L D K D I C S L D T I G WT L C D P L T V E E L P A R - - - S R A Y A L T H S S R R L L Q S L D L W A S L I P H L A P F T R L R L D D Q E L N R H T Y F E V D D L S S Q N Q L H G A V G WS I Y E A K S K E Q I L L R - - - D R T Y A I N H S S R R L L Q R I G I W D D L N E F M I P F D S L S L E D Y S V N Q R L V L Y N K D L V N L N S S Y K E V G WI L F E A Q K D F D R D F R - - - - - G D S L H A V V M E L M D D L R L T E R L L E H K H S K L K T L T M V S A V G A A R V A D F S R L P T - - - - H H N Y M TP V I V A D P A L A L R P S - R D P R A T A I V A A C R R L F E A L G V W D E V A P T A Q P I I D M V V T D S H L E D A T R P V F L T F A G E V Q P G E P F A HV V I D G E D P E A L L A A G Y D G R C S A I A L A C Q R L L D T I G L W D L L G G E S Q P I L D I R V V D G G S P L F L H Y A Q A E A Q G - - - - - - P M G YH L I E A T A P E S H A H P G F D G R A I A L A A G T C Q Q L A R I G V W Q S L A D C A T A I T T V H V S D R G H A G F V T L A A E D Y Q L - - - - - A A L G QA L I D P H R V H H D E F R - - - - - A E K I G A D Q M Q L F E K L G L D K M V M P L V T P F T D I D V F R L G - - - - - Q F F A R E - - K - - - - - - K W E Y. L I E A . . . G D R A Y A . . . S . L . G . W . L . . . . . . D . V F D L N . G .


170 180 190 200 210 220 230 240

V A E H P V I L Q A L E N C V E Q C P R I A W F R P A E L I S F T A G E N H K Q V T L Q Q E G R - - - - E I T L Q T K L L V A A D G A R S H T R S L A G I Q T KS G Q H Q V V L E T L Q A S L R G Y G N I R W F C P A Q V T A I H Y E S E Q A I V E L S S E T Q G - - - Q I K I Q G A L V V G A D G P R S L V R Q G A S I K T RV A E H Q A L L E P L Q E F V Q N C P N V T Y L C P A E V V S T N Y Q P N E V V I A V K I A D Q - - - - L N T I R S K L L V A A D G S R S P I R Q A A G I K T HV A E H Q A L L Q P L Q E F V Q D C P N V T Y L C P A E V V N T Q Y Q K D I V A V D I K V A D Q - - - - L Y T V R S K L L I A A D G A R S P I R Q A A G I K T SV G E H S A I A E T L Q S V L Q S A G N V T F Y C P W R V V T N E V H G D R A Q L T L I S G D P G E P K V A H L A A R L V V A A D G G K S P L R Q Q M G I E P KI L D H Q P L M K L L M D R L H Q H H R V E L A L G - C T A P T P G I D D - - - - - L - - - - - - - - - - - - - - - - - I V A A D G P R S T T R S Q W D I G C WV V K H S D L M N V F F D E V D N V D N I F F K S S L D L S F N D I K F D - - - - - - - - - - - - - - - - - - - - - - F E F V S T G A N P N H K K N R N F I D II L D H R P L M E L L I D R L Q T S P K V L L N L G R F D D H H L D G H D - - - - - L - - - - - - - - - - - - - - - - - V V A A D G P R S L T R Q A W G I N T WT L D H K L L M E Y L F Y K I S K S S N I N I K F S S K D N D A N A L Y D - - - - - - - - - - - - - - - - - - - - - - Y T F A A D G T N S R Y R D L W K F K S YM V A Q K D F L N F M V A E A K R Y A N F R V F M A T S V Q R L I E E E G E I R G V V F N G P D G - - - P Q E A R C A L T V G A D G R S S R T R Q M A G I D L VM V E N R Y L V E A L A K R A E A E G V E L R A T P - - V T S Y D A R T E A I G V T L G D G S - - - - - - - V I D A S L L V A A D G A K S K L R Q R A G I A T YM V E N R L L R Q A I L T R L G R L P A A T L L A P A R M T A L R R D L D G V S A T L S D G Q - - - - - - - T V R A R L V V G A D G R R S Q V R E S A G I G I RV V E L H N V G Q R L F A L L R K A P G V T L H C P D R V A N V A R T Q S H V E V T L E S G E - - - - - - - T L T G R V L V A A D G T H S A L A T A C G V D W QA F S Y G A L I N G L R D A L P P Q V P I T I G K V A E V S T G P N R Q R - - - L V L A D G - - - - - - - A V I D A R L L V V A T G Y S E L V R R A I G V E R I. . . H . L . L . . P V . . . L G G E P K . L . V A A D G . S . R . . G I


250 260 270 280 290 300 310 320

G W K Y W Q S C V A F T I Q H Q A P D N T T A F E R F C D T G P - - - - - - - - M G I L P L P G D R A Q I V W T M - - - P H H K A H T L V N L P E A D F I T E LG W K Y W Q S C V T C V V E H D A P R N D V A F E R F W G T G P - - - - - - - - M G V L P L T E N R C Q I V W T N - - - P H A E A Q R L K E L P P E V F L E Y LG W K Y W Q S C I V A F V K P E K P H N D T A Y E R F W P S G P - - - - - - - - F A I L P L P E N R C R I V W T A - - - P H D E A K A L C A L N D E E F L R E LG W K Y W Q S C I V A F V K P E K P H N N T A Y E R F W S S G P - - - - - - - - F A I L P L P E N R C R I V W T A - - - P H E E A K A L C A L D D E Q F L A E LG W Q Y P Q S C V V A T L D V A H P Q P V I A Y E R F W P T G P - - - - - - - - M G V L P L P R N R Y R V V W T L - - - P H P E A E A V A A L D D R S F L A T LK F R Y R Q G C L T S K I A L R G V K P G M A Y E L F R P E G P - - - - - - - - F A I L P L G D G I F Q V V W S A - - - P W T Q C Q R R A D L P T P E F L D E LR K S Y N Q S C L T F E V L V R G N V P R R A Y E I F R K E G P - - - - - - - - L A L L P L D K N K Y Q I I W T A - - - T T S K S M D R L N S S K S F L L D N LN H P Y Q N G C L T A K V L L R G A D P L M A Y E L F R A E G P - - - - - - - - L A V L P M G G E V F Q M V W S G - - - P L R R C Q E R A G L S P S C F L D H LR F K Y N Q E C I T F K A L L R T P F T H R A Y E I F R K D G P - - - - - - - - M A I L P M A N D L Y Q I V L S M - - - P P L K S D Y L L N L T T S H F L D T VK F S Q - A V D V L W F R L P R Y P H E P E G V L Y R V G Q G A - - - - - - - - V M G Q A D R S D V W Q I S Y I I R K G T Y Q K L R A Q G I E G L R R S V T T LG W D Y D Q S G I V V T V E H E R D H N G C A E E H F L P A G P - - - - - - - - F A I L P L T G R R S S L V W S E - - - S R R D A E R I V A L P E K E F Q R E LT L G Y G Q T A I V L T V E H E R S H R G C A V E H F L P A G P - - - - - - - - F A I L P M P G N R S S L V W T E - - - R S D L V P G L L A L P A E H F Q A E LQ E P Y E Q L A V I A N V A T S V A H E G R A F E R F T Q H G P - - - - - - - - L A M L P M S D G R C S L V W C H - - - P L E R R E E V L S W S D E K F C R E LE L S K A H S L S M G F D L A I A P R - D C A Y R A V T C Y G K R A S D R V A Y L T V F P I G D K M R A N M F V Y R T V A E Q W T R D F R A D P Q K - M L C E L

. Y Q S C . . . V P . A Y E . F G P R A S D R V A Y A . L P L R . V W . R P . . . . L F L E L


330 340 350 360 370 380 390 400

R Q R I G D R L G E F H L I N A R R L F P V Q L M Q S D C Y V - Q P R L A L V G D A A H C C H P V G G Q G L N L G I R D G A A L A Q V I A T A H - S Q G E D W GE Q H T G P Q L G R L K L L G D R Q V F P V Q L M Q G D R Y V - D H R L A L V G D A A H C C H P V G G Q G L N L G I R D A A A L A Q T L I E A Q - Q R G Q D L GS Q R F G Q Q M G K L E L L G D R F V F Q V Q L M Q S D R Y V - L P R L A L V G D A A H N C H P V G G Q G L N L G I R D V A A L A Q V I Q K A H - Q A G E D I GT R R Y G D Q M G K L E L L G D R F V F Q V Q L M Q S D R Y V - L P R L A L I G D A A H N C H P V G G Q G L N L G I R D A A A L A Q V I Q T A H - Q A G K D I GQ P Y L D P Q M A E I T A V G D R F V F P A Q F M Q V N S Y A - A D R F V V I G D A A H R C H P V G G Q G L N L G L R D V W N L A Q Q I L A S - - - P L E E I GA A V L P A G I E P D L L L D T P R A F S Q Q W S M A R R L S - R G R G V L L G E A G H R C H P V G G Q G L N L C W R D V A S L R N L A E H G G - - - - - - - TS T I L P D N F K L D Q I N S D I N I F P V S L S F R L P I F N F K K V V F V G D S F H T F H P V G G Q G L N T C W R D V N V I Y D I F N R N - - F P V S N R AA A V L P D G L Q P D V L L D R P A A F P L Q L A F A F R L Q - R G R G V L V G E S G H R C H P V G G Q G L N L C W R D V S T L M N L V K N V D E - - - G K L PA T Y L P I G M E I D T L I N K P Q K F S L S L N V P I K L Q - D Y N R F L I G E S L H S L H P V G G Q G L N L S I R D I D D I M N L L T S S N - - - - - - - -V P E L A D R V G L L K D W S Q A S L L S V E S G Y V R R W Y - R P G L L L I G D A A H V M S P F G G V G I S Y A I Q D A V A A A N V L A G P L R - - - S G R VE K R F G L R L G D I K P L D K P K S F P L G Y F V A Q S F I - A P R L A L I G D A A H V I H P I A G Q G L N M G L R D V A A L A E V V V D A A R - L G I D P GE R R F G D H L G W V R P V G P R F S Y R L T L Q A A N R Y V - D H R L A L V G D A A H G M H P V A G Q G M N Y G L R D V A V L A E R L V A A Q R - L G L D P GQ S A F G W R L G K I T H A G K R S A Y P L A L T H A A R S I - T H R T V L V G N A A Q T L H P I A G Q G F N L G M R D V M S L A E T L T Q A Q - E R G E D M GM P E I A T Q C G N F E V A S P V E V R Q V N L T T T Q G H R - R D G V V F I G D A F V T T C P T P G V G I G R V M V D V D Q L H S V H I P R - - - - - - - W L

. . . G . . . . F . . L . R . . N R . . L . G D A A H . H P V G G Q G L N L G . R D V . . L A . . . . R G D G


410 420 430 440 450 460 470 480

S L A V L K R Y E H W R K P E N - W L I L G F T D L L D R F F S S H W L P A I A L R R F G L E V L R L V P P A K K L A L R L M T G L L G R K P - - - Q L A T G QS L A V L K R Y E R W R K R E N - L V I L G F T D F L N R L F S N R I W P V V W V R R L G L I V M A K F N P L R L F A L K L M T G Q K G R Q P - - - R I L AN I R V L K R Y E S W R Q K E N - L A I L G F T D M L D R M F S N Q F P P L V F L R R L G L W F L R S V P V L K T F M L K L M I G L K G K T P - - - E L A K RD I Q I L K G Y E R W R K L E N - L T I L G F T D L L D R M F S N N F L P V V V V R R L G L W F L Q R V P L L K V F M L K L M I G L K G R T P - - - E L A K RD R R H L M H F H R Q R W W Q N - V L T L A F T D F L N R L F S N T G W P L V A V R R C G L G L L C L L P P L K R L L L R F M A G L L L P L P G D R E F P R G RS Q R L A R R Y G R S R W A D L - L M V G L A T D L L V R L F S N Q Q P W L L P F R R I G L K A M A R F S W L R R I S L L A M T D G P T Q L L - - K P L P DL N I F K Y K Y Y F I R C L D I - I S T I V V T D S L I Q F F A N N K F F L L P F R K F S F L L L N K F I F M R R I I L N H M T K S L I Y S S I KV E K L P Q V Y A R K R I F D L - V L V G L F T D L I V R F F S S R N I L L L M V R L P L M F L L A R L S L L R Q L L L K A M T D G P I T V I - - R L L P E- - - - L L Q Y K I C R Y I D I - Y T T S F L T H M L I V I F S S N N I I L K V F K L S L F T I L R K S I L F R K I V L S L M T D G I P H I F KG V G H L A A V Q R R R E L P T R L S Q Y Y Q A M I Q D Q L L A T A A N E G I P P K P T L L R R L L R L P G L R E L P T R L L A F G L W P A R L R I K N S I P PQ V D V L E R Y Q R W R R F D T - V A M G V A T N G L N F L F S N N S P L L R G I R D I G L G L V D R L P P L K G A F I R Q A A G L T G E I P - - - R L L K G EA P A L L A E Y E A L R R P D N - L L M L A I T D A L V R L F S N D I A P V A L A R R L G I G A V E R M G P L K R L F M R H A M G T L K L G P E P P R L M R G VD Y G V L C R Y Q Q R R Q S D R - E A T I G V T D S L V H L F A N R W A P L V V G R N I G L M T M E L F T P A R D V L A Q R T L G W V A RE T P G M A A D K I G A F Y D D P V K V A A D K A G M R V S F Y A K Q I T T E T G L E W R V R R L R N N A A R Q L M I I G R K M R H L G P R R - - - E P G M A

. L . Y . R D . . . . T D . L R . F S N L . . R . G L . L . L . . . L . . M G . . . P L


490 500 510 520 530 540 550 560

S L V S Q

N I H RA LP L

L Q

300 The multiple alignment was generated by BLOSUM30 using Clustal W. S6803, Synechocystis sp. PCC 6803; S7002, Synechococcus sp. PCC 7002; A7120, Nostoc sp. PCC 7120; Npun, N. punctiforme; Telon, T. elongatus BP-1; S8120, Synechococcus sp. WH8102; PmarMED4, P. marinus MIT9313; PmarMIT9313, P. marinus MIT9313; PmarSS122, P. marinus SS120; Gvio, G. violaceus PCC7421; Rpal, R. palustris; Rrub, R. rubrum; Ecol, E. coli; Mlr2092, Mesorhizobium loti.

301

A.1.7. An amino acid sequence alignment of UbiX homologs in cyanobacteria and other

organisms

S6803S7002A7120NpunTelonS8102PmarMIT9313PmarSS122GvioEcolRpalRspheApe1647

10 20 30 40 50 60

M A Q P L I L G V S G A S G L I Y A V R A I K H L L A A D - Y T I E L VM D R P L T V G I S G A S G L I Y A V R T L K Y L L E A N - L T V D V V

M S Q H N K P L I I G V S G A S G L I Y A V R A L K Y L L T A D - Y E I E L VM S Q K S K P L I L G V S G A S G L I Y A V R A I K F L L E A D - Y S I E L V

M I A V V T S S S L P I V L G V T G A S G L I Y A V R A I K F L L Q A N - Y P I Q L VM H P Y V L A V T G A S A Q P L A E R T L Q L L L Q A G - R S V H L VM D P Y V L A V S G A S A Q P L A E R S L Q L L L E N N - R D V H L IM S S I V L A I T G A S A Q I L A E R S I D L L L R C N - Q K L D V I

M Q T Q A P S S R P I V L G V A G A S G L I Y A V R T V R F L L A A G - H A V D L VM K R L I V G I S G A S G A I Y G V R L L Q V L R D V T D I E T H L V

M S T Q H R I V V G I S G A S G A A V G L R V V E L L A S V D - C E V H L VM G R G H D A V A R G R S L R R P A D R E A E D R S M R V V L G V S G A S G A A L A A A C A R H L S A L G - A E I D L V

M S C R P S S V S I A V T G S S G V R V A L R L L E A L K G E V - E I R G I IM G R G H D A V A R G R S L R R P . P . V L G V S G A S G . I Y A V R . . . L L A D . . L V


70 80 90 100 110 120

A S R A S Y Q V W Q A E Q N I Q M P G E P S A Q A E F W R S Q A G V E K G G K L I C H R W G D V G A T I A S G S Y R C AA S K A S F M V W Q A E N G I T M P A D P D H Q A K F W R D Q A G V P E K G I L R C H R W G D V G A G I A S G S Y R T KA S K S T Y M V W Q A E Q E T R M P S E P K Q Q E Q F W R E Q A G V A L S G K L R C H P W S D V G A N I A S G S F R T LA S K S T Y M V W Q S E Q E I R M P P E P T Q Q E Q F W R E Q A G V A L S G K L R C H P W G D V G A G I A S G S F A T LA S K A A H Q V W R A E Y G L T L P T Q P D K Q E L F W R E Q A Q V W - E G C L T C H R P T D V A A A I A S G S F R T LL S R G A Y A V F Q A E Q G V Q V P V N P E R Q A S F W R E R L N C S - E G E L F C H R W N D Q S V G I A S G S F R T SF S H G A L Q V W K A E R G I E V P V D P S D Q E K F W R D H L Q V K - N G N L T C H R W N D Q S A C I A S G S F R T RI S K G A Y E V W K S E M N V N I P A E G A K Q A T F W R N R L K N N - Q G E I I C H K W N D N S A T I A S G S F R T KA S R A S F M V W R E E M G T A M P V E P A E Q E R F W R E Q S G E S - G G K L R C H A F A N L A A S I A S G S Y R T RM S Q A A R Q T L S L E T - D F S L R E V Q A L A - - - - - - - - - - - - - - D V T H D A R D I A A S I S S G S F Q T LV S K A A R R T I A Y E V G P N A L D H A A D L V - - - - - - - - - - - - - - H R F H K I E D V G A C I A S G S F R T SI S K A G E R T L V E E I G L E A A D A L R A Q A - - - - - - - - - - - - - - T R V H A I M N V G A A I A S G S A P V AL T R G A E E V A R Y E E G I E P E D L R R L L R - - - - - - - - - - - - S Y A P L Y M E N D M S S P L A S S S N Q P D. S K A A . V W . E G . P . P Q . F W R E Q A G V G L C H . W D V . A I A S G S F R T


130 140 150 160 170 180

G M V V L P C S M S T V A K L A V G M S S D L L E R A A D V Q I K E G K P L V V V P R E T P L S L I H L R N L T S L A EG M L V I P C S M S T V A K I A N G L S S D L L E R A A D V S I K E G R K V V V V P R E T P F S L I H L R N L T Q L A EG M I V I P C S M S T V A K L A G G L S S D L L E R A A D V H L K E G R K L V I V P R E T P F S L I H L R N L T T L A EG M I I I P C S M S T V A K L A V G L S S D L L E R A A D V Q L K E G R K L V I V P R E T P F S L I H L R N L T S L A EG M V I L P C S M S T V A K L A A G L S S D L L E R V A D V H L K E H R P L V L V P R E T P F S L I H L Q N L T Q L A MG M L I V P C S M G T A G R I Q A G V A M D L I E R C A D V H L K E G R P L L I A P R E M P F N L I H L R N L T A L A EG M A I V P C S M G T I G R I N A G V S I D L I E R C A D V H L K E G R P L V I A P R D M P W S I I H L R N L T S L A EG M V I V P C S M G T I G R I A S G S S I N L I E R C A D V H L K E G R P L I I S P R E S P F N L I H L R N M T T L C EG M L V I P C S M S T V G K L A A G L S S D L L E R A A D V Q L K E G R P L V L V P R E T P F S L I H L R N L T A L A EG M V I L P C S I K T L S G I V H S Y T D G L L T R A A D V V L K E R R P L V L C V R E T P L H L G H L R L M T Q A A EG M I V A P C S I R T L S A I A H G D L D N L L V R A A D V Q L K E R R R L V L M L R E T P L H L G H I R S M A Q A T EG M I V A P C S M R S L A A I A H G L D D N L L T R A A S V Q L K E R R R L V L L A R E S P L T L A H L R N M T A V T EA M A I V P A S M K T V G L I A R G I P S S L P A R A A L A V L R L G R R L V V A P R E T P L G V V E L E N M L A I A RG M . I . P C S M T V . . I A . G . S S D L L E R A A D V L K E G R P L V . . P R E T P F S L I H L R N L T L A E


190 200 210 220 230 240

A G V R I V P A I P A W Y H Q P Q S V E D L V D F V V A R A L D Q L A I D C V P L Q R W Q G G M E G EV G V K I V P A I P A W Y H Q P Q T I H D L V D F V V A R A L D Q F E I D C V P L K R W K E E A A S Q K S E NT G V R I V P A I P A W Y H N P Q T I E D L V D F V V A R T L D Q L D I D C V P L K R W E G G K H N SV G V R I V P A I P A W Y H N P Q T I E D L V D F V V A R A L D Q L D I D C I P I Q R W E G R RA G A R I V P A I P A W Y H R P Q T I E D L V D F V I A R A L D Q L G L D C V P L Q R W Q G P L SA G A R I A A P I P A W Y T Q P R T L E E M V D F I V I R L L D G F E D D L A P L Q R W T G P I KA G A K I A P P I P A W Y T K P N T I E E M V D F L V V R L F D L F D E R L A P I N R W N G P S QA G A K I I P C I P A W Y S K P K D L E E V I D F M V V R L Y D L F D L N M K D I N R W K G NA G A R I V P A I P A W Y Q Q P K T I E D L V D F V I G R A L D Q L D I D H S L F E R W H P D TI G A V I M P P V P A F Y H R P Q S L D D V I N Q T V N R V L D Q F A I T L P E D L F A R W Q G AI G A I V A P L S P A F Y Q R P R S I S E M V D H M A L R A I G L L G L N D L E L S A P E W S D D T P P T C P K PM G G I V A P P V P A F Y L R P E S L A A A V E Q I A A R A V D L L G L G R P Q A Q A W EM G G I V V P L T L S F Y I K P S S V E D L V D F A A G K V L D A L G V K V D V Y R R W R G P E E G D. G A . I V P . I P A W Y . P . T I E D L V D F . V . R A L D Q L . . D . P . . R W G K P

The multiple alignment was generated by BLOSUM30 using Clustal W. S6803, Synechocystis sp. PCC 6803; S7002, Synechococcus sp. PCC 7002; A7120, Nostoc sp. PCC 7120; Npun, N. punctiforme; Telon, T. elongatus BP-1; S8120, Synechococcus sp. WH8102; PmarMIT9313, P. marinus MIT9313; PmarSS122, P. marinus SS120; Gvio, G. violaceus PCC7421; Rpal, R. palustris; Rsphe, R. sphaeroides; Ecol, E. coli; Ape1647, Aeropyrum pernix K1.

302

A.1.8. An amino acid sequence alignment of UbiE homologs in Synechocystis sp. PCC

6803

Sll1653Sll0418Sll0829Sll0487Slr1039UbiE

10 20 30 40 50 60

M S N S L L T Q P T Q E S V Q Q I F A R I A P - - - - - - Q Y D D L N T F LM P E Y L L L P A G L I S L S L A I A A G L Y L L T A R G Y Q S S D S V A N A Y D Q W T E D G I L E Y Y W G D H I H L G

M A T I F R T W S Y Q Y P W V Y A L V S - - - - - - - R L A T L N - - -M L S N S D Q H R A V Q A L Y N T Y P F P P E - - - - - - P L L Q E P P - -M L E R I L E P E V M D T E A E A M D Y D A M - - - - - - D F Q A V N - - -

M V D K S Q E T T H F G F Q T V A K E Q K A D M V A H V F H S V A S - - - - - - K Y D V M N D L MM P E Y L L L P A G L M L . . . . . . D G I L E Y . N L


70 80 90 100 110 120

S F G Q H - - - H I W K A M A V K - - - - - - - W S G V S - - - P G D R L L D V C C G S G D L A F Q G A K V V G T R G KH Y G D P P V A K D F I Q S K I D N V H A M A Q W G G L D T L P P G T T V L D V G C G I G G - - - S S R I L A K D Y G N- V G G E - - - E R F H Q L P L E - - - - - - - N L A I S - - - P G Q K V L D L C C G G G - - - - Q A T V Y L A Q S G A- P G Y N - - - W R W Q W T A A H N - - - - - F C L G R R P A N Q K V R I L D A G C G T G V G T - E Y L V H L N P E A E- L A - - - - - - - F A Q R A C Q - - - - - - - - L G P S - - - - K A T V L D A G T G T A R I P - I L L A Q L R P A W QS F G I H - - - R L W K R F T I D - - - - - - - C S G V R - - - R G Q T V L D L A G G T G D L T A K F S R L V G E T G KS G P V A . . N V H A M A . G . P G V L D . . C G . G . . . G


130 140 150 160 170 180

V V G L D F C A E L L A I A A G K H K S K - Y A H L P M Q W L Q G D A L A L P F S D N E F D G A T M G Y G L R N V G - -N V T G - I T I S P Q Q V K R A T E L T P - P - D V T A K N A V D D A M A L S N P D G S F D V V W S V E A G P H M P - -T V V G - L D A S P K A L G R A K I N - - - - - V P Q A T Y V Q G L A E D L P F G E G E F D L V H T S V A L H E M T P AV H A V D I S E G A L A V A Q T R L Q K S G V V C D R V H F H H L S L E N L A H L P G Q F D Y I N S V G V L H H L P - -I T A I D F A R S M L A I A K E N V I A A - G C E T Q I C L E F V D A K Q L P Y A N G S F D G V I A N S L C H H L P - -V V L A D I N E S M P K M G R E K L R N I - G V I G N V E Y V Q A N A E A L P F P D N T F D C I T I S F G L R N V T - -. V . . D . S A . . . . . G . . . . A L P . . G F D . . . L . . P A


190 200 210 220 230 240

N I P Q A L T E L Q R V L K P G K K V A I L D F H Q - - - - - - - - - - - - - P G N - - A L A A N F Q R W Y L A - - - -D K A V N A K E L L R V V K P G G I L V V A D W N Q R D D R Q V P L N F W E K P V M - - R Q L L D Q W S H P A N - - - -Q L Q S I I S G V H R V L K P G G I F A L V D L H R - - - - - - - - - - - - - P - - - - - - - S N W L F W P P L - - - -D P V A G I Q A V A E K L A P G G L F H I F V Y A E I G R W E I Q L M Q K A I A I L Q G K K R G D Y Q D G V A V G R E IR P L D F L R E V K R V L K P H G F L L I R D L I R - - - - - - - - - - - - - P E S P - E K L A D F V T A I G S - - - -D K D K A L R S M Y R V L K P G G R L L V L E F S K - - - - - - - - - - - - - P I I - - E P L S K A Y D A Y S F - - - -

. . . R V L K P G G . . . . D . L P . G L . . . G R E I


250 260 270 280 290 300

- - - - - - - - N V V V P M A K Q W R - - - - - - - - L T E E Y A Y L Q P S L D R F P T G P K Q V Q F A L E V G F - - A- - - - - - - - A S I E G N A E N L E A - - - - - - - T G L V E G Q V T T A D W T V P T L P A W L D T I W Q G I I R P Q- - - - - - - - A I F M G L F E T E T - - - - - - - - - - - A W Q L I N T D L G S L L D Q A G - F T V V R K H L Y - - AF A S L P E Y N R L V K R E K E R W S L E N H R D E S F A D M Y V H P Q E T D Y N I D T L F E L I D S A G L E F L G F S- - - - - - - - D Y N D Q Q R K L F A D S L H - - - - A S L T I A E I Q D Y L Q H L G W V G D D G P N L E L H L Y Q - S- - - - - - - - H V L P R I G S L V A N - - - - - - - D A D S Y R Y L A E S I R M H P D Q D T L K A M M Q D A G F - - EF A S L P E Y N . . H R D E S . . . . . . . . . . .


310 320 330 340 350 360

K A V H Y P I A - - - - - - - - - - A G L M G V L V A E KG W L Q Y G I R - - - - - - - - - - G N I K S V R E V P T I L L M R L A N G - - V G L C R F G M F K A V R K N A T Q A NG G S L Q V I Q - - - - - - - - - - A R A N K T V NN P D Y W Q L D R L L G K A P D L M E R A K D L S E K E R Y R L I E L L D P E I T H Y E F F L A K P P I Q I S D W T D DS D R H W T L E - - - - - - - - - - K K F Q S S T I V Q RS V D Y Y N L T - - - - - - - - - - A G V V A L H R G Y K F

. . R L L G K A P D L M . . . . . L L E I F


370 380 390 400 410 420

Q T L L K A L P N L H P C L T G W P S R S L F D Y N Y Q P L E L T E E E F K F L Q A V E A Q T E I Q A N V E K V L T D L

Q T L L K A L P N L H P C L T G W P S R S L F D Y N Y Q P L E L T E E E F K F L Q A V E A Q T E I Q A N V E K V L T D L


430 440 450 460 470 480

G D R S I D L A L V R S L Q T R Q L L T L G N S

G D R S I D L A L V R S L Q T R Q L L T L G N S

The multiple alignment was generated by BLOSUM30 using Clustal W. Sequences with “Sll” and “Slr” prefixes indicate the UbiE homologs of Synechocystis sp. PCC 6803. UbiE indicate the E. coli sequence.

303

A.1.9. An amino acid sequence alignment of Sll1653 homologs in cyanobacteria Sll165389-c70244Alr5252Npun3758Tery3321Tll2373Synw1674Pmm0431Pmt0276Pro0427Gll0127UbiE

10 20 30 40 50 60

M S N S L L T Q P - T Q E S V Q Q I F A R I A P Q Y D D L N T F L S F G Q H H I W K A M A V K W S GM S K P N A A T E I Q G I F N R I A P Q Y D A L N E W I S L G Q H R I W K K M A V K W S GM T N - - - - - K I R A I F D R I A P V Y D Q L N D W L S L G Q H R I W K E M A I K W T GM T N - - - - - E V Q S I F N R I A P V Y D Q L N D W L S L G Q H R I W K E M A V K W S AM T D D - - S I E V K A I F N K I A P V Y D Q L N D L L S L G I H K V W K Q M A V R W S EM T N - - - - - - I Q A L F E R I A P L Y D R L N D Q L S F G L H H V W K Q M A V D W L EM K P G - D P A A V E Q L F D S V A S R Y D Q L N D L L S L G L H R Q W K R Q L Q C L L RM R Y K - K T N E V K S I F N N I S C S Y D F L N S L L S L G L H K Y W K K T L V D L L K

M K P Q - D P I A I K E L F D S V S K N Y D F L N D L F S L G F H R I W K R Q L L T W L KM Q G S - - - Q E I Q G I F N A I A P V Y D Q I N D R M S F G L H R V W K K M A I R W T D

M V D K S Q E T T H F G F Q T V A K E Q K A D M V A H V F H S V A S K Y D V M N D L M S F G I H R L W K R F T I D C S GM V D K S Q E T T H M V I F I A P Y D L N D L S L G H R . W K M A V W .

Sll165389-c70244Alr5252Npun3758Tery3321Tll2373Synw1674Pmm0431Pmt0276Pro0427Gll0127UbiE

70 80 90 100 110 120

V S P G D R L L D V C C G S G D L A F Q G A K V V G T R G K V V G L D F C A E L L A I A A G K H K S K Y A H L P - M Q WV T G G D L A L D V C C G S G D L T L L L A K T V G V Y G K T V G I D F A S Q Q L A I A A Q K Q N R L C P H L D - I Q WA K P G D T C L D L C C G S G D L A L R L A R R V G S T G Q V S G V D F S A N L L E T A K Q R A Q S Q Y P Q P N - I S WA K S G N T A L D L C C G S G D L A L R L A R R V G A T G Y V Y G V D F S C N L L E T A K E R S Q K Q Y P Q P A - I A WP D Y G S T C L D L C C G S G D L A Y M L A K H V G N T G H V F G V D F S R E Q L A I A R N R E P P L L N K I S P I H WL P Q G A T A L D L C C G T G D L T R L L A R R V G R Q G R V V G L D F A A A P L A I A R Q R S D - - - - H Y P Q I E WP A A G E T W L D L C C G T G D L A L E L A R R V R P G G S V L G L D A A A A P L E L A R H R Q S R Q - - P W L N V V FP I N G E N W A D L C C G T G D L S F L I F K K V R P N G S V T G I D S A K E I L N I A K E K S K L I - - E N K F I S W

M A R R R A A V E - - P W L P L S WP A S G E K W I D L C C G T G D L S L P L A R L V R P F G S V I G V D F S R A Q I N C A H K R S L K E - - P W L P I S WC K P G D T V L D L C C G T G D L A F L L A R R V G V A G T V W G V D F A A A Q L A Q A R R R D - - - - - R E G R V R WV R R G Q T V L D L A G G T G D L T A K F S R L V G E T G K V V L A D I N E S M P K M G R E K L R N I G - V I G N V E Y

G . T L D L C C G T G D L A L A R . V G G V G . D F L A . R P I W


130 140 150 160 170 180

L Q G D A L A L P F S D N E F D G A T M G Y G L R N V G N I P Q A L T E L Q R V L K P G K K V A I L D F H Q P G - N A LQ E G D A L A L P Y P D N H F D G A T M G Y G L R N V T D I P Q A L R E L Q R V L K P G K K V A I L D F H R P D - N P GV E A N V L D L P F K D N Q F D A A T M G Y G L R N V T D I P R S L Q E L H R V L K P N A K A A I L D F H R P N - N Q QV E A D V L N L P F D D N Q F D A A T M G Y G L R N V K D I P R S L Q E L H R V L K P G A K A A I L D F H R P S - N P QV E A N A L S L P F P H N Y F D C A T M G Y G L R N V S N I P L C L Q E L Y R V L K P N A K A A I L D M H C P S - S S TL Q G D A L A V P F A P Q T F Q G I T I G Y G L R N V V D I P Q A L R E M F R L L V P G G R A A I L D F S H P Q - T S AQ Q G D A L S T G L P D A S A D G I V M A Y G L R N L A D P V V G L K E M A R L L K P G R L A G V L D F N R L T T A G PE M Q D I L E I D E D L K N Y D G I C M S Y G L R N L V N V E E G L K K V F N L L S D T G R A G F L D F N H S K F N S IL Q G D A L D T G L P S H R F D G A V M A Y G L R N L A D P G A G L I E L R R L L R P G A R A G V L D F N R M G E G S LL N E D V L K A G L P S S T F D G V V M A Y G L R N L S S P E A G L K E I H R L L K P G A R A G V L D F N H T I E G S KL E A D A L D L P F A D D S F D A V T Q G F G L R N V V D I P A C F A Q L R R V L K P G G R A V I L D L H A P A - D A GV Q A N A E A L P F P D N T F D C I T I S F G L R N V T D K D K A L R S M Y R V L K P G G R L L V L E F S K P I I E P L. . D A L L P F D . F D G . T M G Y G L R N V D I P . L E L R V L K P G . . A . I L D F . P .


190 200 210 220 230 240

A A N F Q R W Y L A N V V V P M A K Q W R L T E E Y A Y L Q P S L D R F P T G P K Q V Q F A L E V G F A K A V H Y P I AK Q M F Q Q W Y L D R F V V P L A E R F Q L T P E Y A Y I Q P S I E R F P T G K T Q E K L A K E A G F R N A I H Y G I VF R T F Q Q W Y L D S I V V P L A D R L G V K E E Y A Y I S P S L D R F P I G K E Q V E I A L K V G F T S A T H Y P I AL R A F Q Q L Y L N S F V V P V A N Y L G L K E E Y A Y I S P S L D R F P I G K E Q I E L A R Q V G F A V A T H Y P I AI R W F Q K W Y L D T V V V P V A S N L G L T K E Y A Y I S P S L T K F L T G K D Q V A L A Y K L G F V N A K H Y P I IL E Q F Q Q W Y L Q Q W V V P T A R H Y G L A A E Y D Y L W P S I Q A F P T P P T L C A L I Q Q A G F E R V K H Y P L LA A D F Q R F Y L R R L V V P V A S A A G L K E E Y A Y L E E S L K R F P D G T A Q E R L A L E A G F A E A R H H T L VA D L F Q K I Y L R F V V V P I S K I F R L S K E Y S Y I E K S I K N F P K G N E L M L I A K G V G F K K V E Y R T I FA A R F Q R F Y L R R L V V T V A A Q V G L R E H Y A Y L E A S L Q Q F P K G E V Q E R L A R D A G F A A A S H R P L AN S F F Q K F Y L R N F V V P I A S K M G L K E E Y A Y L E E S L K K F P V G L I Q E Q I A I N V G F Q E A N Y K T L AW R A F Q R W Y L E G W V T A L G R A Q G L E A E Y A Y I A P S L E R F P T G D E Q V R L A Q Q A G F S Q A S H Y P L IS K A Y D A Y S F H V L P R I G S L V A N D A D S Y R Y L A E S I R M H P D Q D T L K A M M Q D A G F E S V D Y Y N L T. F Q . . Y L . V V P . A G L . E Y A Y . P S L . F P G Q L A . G F A H Y P . .


250 260 270 280 290 300

A G L M G V L V A E KG D M M G V L V A T K NN G M M G V L I I S KN G M M G V L V V S K FG G M M G I L V I T KG G L M A I T V A Q KA G Q M G M L L L K RF N Q M G I L I L E KA G Q M G A L L L T AG G Q M G A L L L R AG G T V G V L V A QA G V V A L H R G Y K F. G M G . L . . K F

The multiple alignment was generated by BLOSUM30 using Clustal W. Locus tags are used to indicate each organism (see also Table 6.5): Sll1653, Synechocystis sp. PCC 6803; Alr5252, Nostoc sp. PCC 7120; Npun3758, N. punctiforme; Tery3321, T. erythraeum; Tll2373, T. elongatus BP-1; Synw1674, Synechococcus sp. WH8102; Pmm0431, P. marinus MED4; Pmt0276, P. marinus MIT9313; Pro0427, P. marinus SS120; Gll0127, G. violaceus PCC7421. 89-c70244 indicates a tentative locus in Synechococcus sp. PCC 7002.

304

A.1.10. An amino acid sequence alignment of Sll0418 homologs in cyanobacteria Sll0418Slr008980a16268All2121Npun3758Tery4209Tll1726Synw2141Pmm1505Pmt1785Pro1661Glr3039UbiE

10 20 30 40 50 60

M P E Y L L L P A G L I S L S L A I A A G L Y L L T A R G Y Q S S D S V A N A Y D Q W T E D G I L E Y Y W G D H I HM V Y H V R P K H A L F L A F Y C Y F S L L T M A S A T I A S A D L Y E K I K N F Y D - - D S S G L W E D V W G E H M H

M N I L L L A L G I S A T I L A I A L I S Y L L T A R R Y Q S A D T V A D S Y D E W T E D G I L E Y Y W G E H I HM S W L F S T L V F F L T L L T A G I A L Y L I T A R R Y Q S S N S V A N S Y D Q W T E D G I L E F Y W G E H I H

M T N E V Q S I F N R I A P V Y D - - - - - - - Q L N D W L S L G QM Y L Y Y A L G F I L L L V A L G I V I Y L I T P R S Y E S S N T V A N S Y D D W T Q D G I L E F Y W G E H I H

M S H L G L I L V I T V V A L V L L G L A L Y L L F P R K Y E S A R S V A E S Y D N W T K D G I L E F Y W G E H I HM L A G L L L L T G A A G A T A L L I W L Q R D R R Y H S S D S V A A A Y D A W T D D Q L L E R L W G D H V H

M S I F L I S S L V I F L T L L F S S L I L W R I N T R K Y I S S R T V A T A Y D S W T Q D K L L E R L W G E H I HM I L A G T T L T S A V V I W T Q R D R R Y K S S A S V A S A Y D A W T N D Q L L E R L W G E H V H

M I A F L L P I V S L L V L S L I F L W L F N D R K Y K S S E S V S S A Y D S W T N D R L L E K L W G E H I HM G E R T V L N E N I R R F Y D - - A S S G L W E E V W G E H M H

M V D K S Q E T T H F G F Q T V A K E Q K A D M V A H V F H S V A S K Y D V M N D L M S F G IM V M L L . . . . . . . . . . L . T R . Y S S . . V A . Y D W T D G . L E W G E H I H

Sll0418Slr008980a16268All2121Npun3758Tery4209Tll1726Synw2141Pmm1505Pmt1785Pro1661Glr3039UbiE

70 80 90 100 110 120

L G H Y G D P P V A K D - F I Q S K I D N V H A M A Q W G G L D T L P P G T T V L D V G C G I G G S S R I L A K D Y G NH G Y Y G P H G T Y R I D R R Q A Q I D L I K E L L A W A V P Q N S A K P R K I L D L G C G I G G S S L Y L A Q Q H Q AL G H Y G N P P R A K N - F L K A K A D F V H E M V R W G G L D Q L P P G T K V L D V G C G I G G S S R I L A R D Y G FL G H Y G S P P Q R K D - F L V A K S D F V H E M V R W G G L D K L P P G T T L L D V G C G I G G S S R I L A R D Y G FH R I W K - - - - - - - - - - - - - - - - - E M A V K W S A A K - - - S G N T A L D L C C G S G D L A L R L A R R V G AL G H Y G S P P R R K D - F L Q A K A D F V H E M V K W G G L D K L P R G T T V L D V G C G I G G S S R I L A K E Y E FL G H Y G L P P R P K D - F R Q A K V D F V H E M V R W A G L D R L P P G T T V L D V G C G I G G S S R I L A R D Y G FL G H Y G N P P G S V D - F R Q A K E A F V H E L V R W S G L D Q L P R G S R V L D V G C G I G G S A R I L A R D Y G LL G F Y P - L N K N I D - F R E A K V Q F V H E L V S W S G L D K L P R G S R I L D V G C G I G G S S R I L A N Y Y G FL G Y Y G K P P S T R D - F R A A K Q D F V H E L V Q W S G L A Q L P R G S R V L D V G C G I G G S A R I L A R D Y N FL G Y Y E N S Y K T K D - F R Q A K I D F V H K L A H W S G L S T L P K G S R I I D I G C G I G G S S R I L A K D Y G FH G H W E V G E A D K D - R R V A Q V D L V V R L L D W A G - - - I D R A E S I V D V G C G I G G S S L F L A E R F G AH R L W K R - - - - - - - - - - - - - - - - - F T I D C S G V R - - - R G Q T V L D L A G G T G D L T A K F S R L V G EL G H Y G P P K D D F R Q A K . D F V H E . V . W . G L D L P . G . . V L D V G C G I G G S S R I L A R D Y G F


130 140 150 160 170 180

N - - V T G I T I S P Q Q V K R A T E L T P P - - - D V T A K N A V D D A M A L S N P D G S F D V V W S V E A G P H M PE - - V M G A S L S P V Q V E R A G E R A R A L G L G S T C Q F Q V A N A L D L P F A S D S F D W V W S L E S G E H M PD - - V T G I T I S P K Q V E R A T Q L T P P - - - G L T A K F A V D D A M N L S F A D G S F D V V W S V E A G P H M PA - - V T G I T I S P Q Q V Q R A Q E L T P Q - - - E L N A Q F L V D D A M A L S F P D N S F D V V W S I E A G P H M PT G Y V Y G V D F S C N L L E T A K E R S Q K Q Y P Q P A I A W V E A D V L N L P F D D N Q F D A A T M G Y G L R N V KE - - V T G V T I S P K Q V Q R A T E L T P Q - - - G V T A K F Q V D D A L A L S F P D N S F D V V W S I E A G P H M PH - - V T G I T I S P E Q V R R A R E L T P A - - - E L N V R F Q L D D A L A L S F P D A S F D V V W S I E A G P H M PD - - V L G V S I S P A Q I R R A T E L T P A - - - G L S C R F E V M D A L N L Q L P D R Q F D A V W T V E A G P H M PN - - V T G I T I S P A Q V K R A K E L T P Y - - - E C K C N F K V M D A L D L K F E E G I F D G V W S V E A G A H M ND - - V L G I T I S P A Q V K R A S Q L T P E - - - G M T C Q F Q V M D A L D L K L A N G S F D A V W S V E A G P H M PD - - V V G I T I S S E Q V K R A N Q L T P K - - - E L K C H F E I M N A L N L K F E D G S F D G V W S V E A G P H I LR - - V E G I T L S P V Q C K R A A E R A R E H H L D G R A H F Q V A D A H R M P F A D G R F D L V W S L E S G E H M AT G K V V L A D I N E S M P K M G R E K L R N I G V I G N V E Y V Q A N A E A L P F P D N T F D C I T I S F G L R N V T

G V G I T I S P Q V . R A E L T P . . . F V D A L L F D . S F D . V W S . E A G P H M P


190 200 210 220 230 240

D K A V N A K E L L R V V K P G G I L V V A D W N Q R - - D D R Q V P L N F W E K P V M R Q L L D Q W S H P A N A S I EN K A Q F L Q E A W R V L K P G G R L I L A T W C H R P I D P G N G P L T A D E R R H L Q A I Y D V Y C L P Y V V S L PD K A I F A Q E L L R V L K P G G K L V V A D W N Q R - - D D R Q I P L N W W E K P V M T Q L L D Q W A H P K F S S I ED K A I F A K E L M R V L K P G G I M V L A D W N Q R - - D D R Q K P L N F W E K P V M Q Q L L D Q W S H P A F S S I ED I P R S L Q E L H R V L K P G A K A A I L D F H R P - - - - - - - - S N P Q L R A F Q Q L Y L N S F V V P - V A N Y LD K V K Y G S E M M R V L K P G G I L V V A D W N Q R - - D D R Q K P L N Y W E K P V M R Q L L D Q W S H P A F S S I ED K Q Q F A K E L L R V L K P G G I L V V A D W N Q R - - D D R Q Q P L N F W E R L I M R Q L L D Q W A H P A F A S I ED K Q R F A D E L L R V L R P G G C L A A A D W N R R - - A P K D G A M N S T E R W V M R Q L L N Q W A H P E F A S I SN K T K F A D Q M L R T L R P G G Y L A L A D W N S R - - D L Q K Q P P S M I E K I I L K Q L L E Q W V H P K F I S I ND K Q R Y A D E L L R V L R P K G V L A V A D W N R R - - D Y E D G E M T S L E R W V M R Q L L D Q W A H P E F A S I KN K Q L F A D E M L R V L R P G G V L A V A D W N R R - - D Y A K K E I G F L N S L V L K Q L L N Q W S H P D F A T I YD K A Q F L R E C H R V L R P G G R F V F V T W C C R - - - - - H G A L D A R D Q K W L G A I Y R I Y H L P Y I L S I ED K D K A L R S M Y R V L K P G G R L L V L E F S K P - - - - - - - - - - - - I I E P L S K A Y D A Y S F H V L P R I GD K F A E L L R V L K P G G L . V A D W N R P I D P L N E . V M . Q L L D Q W H P F . S I


250 260 270 280 290 300

G N A E N L E A T G L V E G Q V T T A D W T V P T L P A W L D T I W Q G I I R P Q G W L Q Y G I R G N I K S V R E V P TD Y E A I A R E C G - - F G E I K T A D W S V A V A P F W D R V I E S A F - D P R V L W A L G Q A G - P K I I N A A L CG F S E L L E E T G L V Q D Q V I N A D W S Q E T L P S W L H T I W V G A I R P W G W L Q Y G L P G F I K S V R E I P TG F S E L L A A T G L V E G E V I T A D W T K Q T L P S W L D S I W Q G I V R P E G L V R F G L S G F I K S L R E V P TG L K E E Y A Y I S - - - - - - P S L D R - - - - - - - - - - - - - - - - - F P - - - - - I G K E Q - I E L A R Q V G FG F S E Q I A E T G L V E G E V A T A D W T Q E T L P S W F E S I W Q G I V R P K G L I K F G F S G F I K S L R E V P TG F A E A L A A T G L V A G E V M T A D W T Q E T L P S W L D S I W Q G I V R P E G L I R F G L P G L V K S L R E V P TG F R A N L E A S P H Q R G L I S T G D W T L A T L P S W F D S I A E G L R R P W A V L G L G P K A V L Q G L R E T P TE F S S I L I N N K N S S G Q V I S S N W N S F T N P S W F D S I F E G M R R P N S I L S L G P G A I I K S I R E I P TG F R R N L L H S P F A C G T V E S D D W T R S I L P S W N D S I L E G F R R P G A V L G L G P A A L V K G F R E I P TG F R N N L S D S I Y S A G R V E T D D W T K Y T I P S W N D S I I E G I R R P N V F F D L G L G S F F K A I R E V P TS Y T Q L L G E T G - - F S G I R T T D W S D R V A R F W S L V I D S A L - E P A V L W K V I A Q G - P T V I K G A L AS L V A N D A D S Y - - - - - - R Y L A E S I R M H P - - - - - - - - - - - D Q - - - - - - - - D T L K A M M Q D A G FG F L . . G L V G V T . D W T T L P S W D S I G . R P . . . . G G . K . R E . P T


310 320 330 340 350 360

I L L M R L A N G V G L C R F G M F K A V R K N A T Q A NL R L M K W G Y E R G L V R F G L L T G I K P L VI L L M R L A F G T G L C R F G M F Q A V R G E G N P V T K N R T T Q Q A I S R NL L L M R L A F G T G L C R F G M F R A L R A D T V R S S A E Q T S A I K V A Q KA V A T H Y P I A N G M M G V L V V S K FM L L M R L G F G A G L C R F G M F R A V K S N S V P V S T E T A T T E V A N AF L L M R I A F G M G L C R F G M F R A V R A E I P A V S L E P A P Q V N CL L L M H W A F A T G L M Q F G V F R L S RI L L M D W A F K K G L M E F G V Y K C R GI L L M R W A F A H G L M Q F G V F R S R DI V L M R W A F H T G L M Q F G V F R S R GM Q L M R R S Y A R G L V R F G V F A A Q K A E GE S V D Y Y N L T A G V V A L H R G Y K F. L L M R . A F . G L . R F G . F . . . V S E

305 The multiple alignment was generated by BLOSUM30 using Clustal W. Locus tags are used to indicate each organism (see also Table 6.5): Sll0418 and Slr0089, Synechocystis sp. PCC 6803; All2121, Nostoc sp. PCC 7120; Npun3758, N. punctiforme; Tery4209, T. erythraeum; Tll1726, T. elongatus BP-1; Synw2141, Synechococcus sp. WH8102; Pmm1505, P. marinus MED4; Pmt1785, P. marinus MIT9313; Pro1661, P. marinus SS120; Gll3039, G. violaceus PCC7421. 80a16268 indicates a tentative locus in Synechococcus sp. PCC 7002.

306

A.1.11. An amino acid sequence alignment of Sll0829 homologs in cyanobacteria

Sll082989a46731All3750Npun0871Tery1223Tll1604Synw1749Pmt1242Gll1483UbiE

10 20 30 40 50 60

M A T I F R - T W S Y Q Y P W V Y A L V S - - - - R L A T L N V G G E E R F H Q L P L E N L A IM A T F L R - T L S Y R Y Q W L Y D T I S - - - - R L A A L T V G G E T R F R N L A L Q G L T GM A T I F R - D L S Y R Y Q W L Y D S I S - - - - R V A A L T V G G E A R F R K L A L Q G L T IM A T I L R - D W S Y R Y Q W L Y D S I S - - - - R L A A L S V G G E A R F R Q L A L Q A L T IM A T I L R - D W S Y R Y Q W L Y D G I S - - - - R L A S L S V G G E T R F R Q L A L E G L I IM A T I L R - T W S Y Q S P W L Y D T I S - - - - A L A A I A V G G S D R L H R L A W Q D L N LM S S F L R - P L A Y R H R W I Y D L V T - - - - A V S S L S V G G V A R L R G L G L E A L G PM T S P L R - T F A Y E H R W F Y D L V T - - - - T I S A L S V G G V K R L R S L G L T A L Q D

M E A P A W N N A L A T I L R - D W S F A C A P L Y E A I T - - - - S G A A V L A G G E R H F R Q A A W Q N V S LM V D K S Q E T T H F G F Q T V A K E Q K A D M V A H V F H S V A S K Y D V M N D L M S F G I H R L W K R F T I D C S GM V D M A T I L R E . S Y W L Y D I S S K Y D . A A L . V G G E R F R L A L . L .


70 80 90 100 110 120

S P - - G Q K V L D L C C G G G Q A T V Y L A Q S G A T - - - V V G L D A S P - - K A L G R A K I N - - - - V P Q A T YD K - - S L K I L D L C C G A G Q T T Q F L T Q Y S D D - - - V T G L D I S P - - L A I E R A K K N - - - - V P Q A N YQ A - - N T S V L D V C C G S G Q A T Q L L V K S S Q N - - - V T G L D A S P - - L S L Q R A R Q N - - - - V P E A N YH S - - D T Q V L D L C C G S G Q T T Q F L V K I S Q N - - - V T G L D A S P - - K S L Q R A R L N - - - - V P E A S YE E - - N T Q I L D L C C G S G Q G T N F L A K Y S Q S - - - V T G L D A S P - - L S I N R A K K N - - - - V P S A K YP R - - T A V V L D L C C A H G I V T Q A L T Q A F D Q - - - V T G L D A A P - - K A I A R A R E R - - - - V P Q A T YH L N P D A A V L D L C C G S G E A A A P W L E A G Y R - - - V T G L D I S P - - R A L A L A A Q R - - - - H P A M T RK I S K G A P V L D L C C G S G E T A A P W I E A G F A - - - V T G L D L S P - - K A L A L A A E R - - - - T P Q L Q CS A - - A S S V L D L C C G P G G A T R Y L A A T G A R - - - V T G L D R S E - - G S L R H A R A R - - - - V P G A H FV R - R G Q T V L D L A G G T G D L T A K F S R L V G E T G K V V L A D I N E S M P K M G R E K L R N I G V I G N V E Y

V L D L C C G . G . T L . T G K V T G L D . S P S M L R A . N I G V V P A Y


130 140 150 160 170 180

V Q G L A E D L P F G E G E F D L V H T S V A L H E M T P A Q L Q S I I S G V H R V L K P G G I F A L V D L H R P S N WV V A P A E K M P L P D Q Q F D L V H T S A A L H E M T P T Q L S Q I F Q E V Y R V L K P G G I F T F I D L H Q P T N PV E A F A E K M P F P D N Q F D I V H T S A A L H E M E P Q Q L R E I I Q E V Y R V L K P G G V F T L V D F H T P T N PV E A F A E E M P F T D N L F D V V H I S V A L H E M Q P Q Q L R K I I D E V Y R V L K P G G I F T L V D F H A P T N PV E G F A E D M P F S S N Q F D L V H T S A A L H E M N Y E Q L R Q I I Q E V Y R V L K P S G I F T F V D F H S P T N PV Q A F A E K M P F A D A T F D L V H T S M A L H E M T A A Q R D A I L A E V W R I L K P G G W F A L I D F H R P Q V PV E G L A E D P P L A D G S F A A I Q L S V A L H E F P R S D R E A V L R S C L R L L Q P G G W L V V V D L H - P A G PI E G M A E K P P L N T S Q F A A I Q I S L A L H E F S S E E R Q Q V L K A C M R L L Q P G G W L V L I D L H - P A G PV Q G R A E A M P F A G A S F D L V H T S V A L H E M E P T Q R R T I I R E V L R V L K P G G T F A L I D Y H R P T N PV Q A N A E A L P F P D N T F D C I T I S F G L R N V T D K - - D K A L R S M Y R V L K P G G R L L V L E F S K P I I EV . A E M P F D F D . V H T S . A L H E M Q L I . E V . R V L K P G G F . L . D . H P . N P


190 200 210 220 230 240

L F W P P L A I F M G L F E T E T A W Q L I N T D L G S L L D Q A G F T V V R - - - - - K H L Y A G G S L Q V I Q A R AF F V P S L Y T F M F L F E T E T A W Q L I K T N L A E K L T T T G F E I H K - - - - Q - E Q Y A G G S L Q M I Q S R KI F W P G L T V F L L L F E T E T A W Q L L K T D L A G L L T E I G F E A G - - - - - Q S I L Y A G G S L Q V I Q A K KI L W P G I S L F L L L F E T E T A W Q L L K T D L A G L L T E T G F D V S - - - - - K P I L Y A G G S L Q V I Q A K KL F W P G F A M F L W L F E T E T S W K F I N T N I L E L L T T I G F N L V D T K L P L P I L Y A G G S L Q V I Q V Q KL L W P G I A L F F W L F E T E T A W Q L L Q T D L A E Q L R L Q G F T V E R - - - - - Q T Y H L G R S L Q V L H G R KW L Q L P Q Q L F C A L F E T D T A T A M L E D D L P A Q L K Q L G F S A V N - - - - - Q E L L A G Q A L Q R I T A T RC L K L P Q Q L F C A L F E T E T A L T M L Q A D L P K Q L R E M G Y S T I E - - - - - Q E L L A G R A L Q R I T A R LL Y W P G L A L F L W V F E T H T A W E L L A T D L P G L L A E C G F S T L E - - - - - Q Q L L A G G S L Q T V Q A R KP L S K A Y D A Y S F H V L P R I G S L V A N D A D S Y R Y L A E S I R M H P D Q D T L K A M M Q D A G F E S V D Y Y N. W P . . . F L F E T E T A W L L T D L . L G F . . L A G G S L Q I Q A . K


250 260 270 280 290 300

N K T V NP G S E P L K N

P C A N DS A A S T SPPL T A G V V A L H R G Y K FP A H R G Y K F

The multiple alignment was generated by BLOSUM30 using Clustal W. Locus tags are used to indicate each organism (see also Table 6.5): Sll0829, Synechocystis sp. PCC 6803; All3750, Nostoc sp. PCC 7120; Npun0871, N. punctiforme; Tery1223, T. erythraeum; Tll1604, T. elongatus BP-1; Synw1749, Synechococcus sp. WH8102; Pmt1242, P. marinus MIT9313; Gll1483, G. violaceus PCC7421. 89a46731 indicates a tentative locus in Synechococcus sp. PCC 7002.

307

A.1.12. An amino acid sequence alignment of Sll0487 homologs in cyanobacteria Sll0487All0012Npun5885Tery3855Tlr1948Synw0487Pmt1474Glr1180UbiE

10 20 30 40 50 60

M L S N - - - S D Q H R A V Q A L Y N T Y P F P P E P L L Q E P P P G Y N W R W Q W T A A H N F CM S D P - - - Q A V S S A V A K L Y D T Y P F P P E P I L D E P P P G Y N W R W N W L A A H S F CM S D S - - - Q T V S A A V A K L Y N T Y P F P P E A L L D E P P P G Y N W R W N W L A A H N F CM K D R - - - I N I S S A V E G L Y D T F P F P P D P L T D E A P P G Y N W R W S W P V A Y S F CM T S A - - - A E I T A A V R Q L Y N T Y P F P P E P L L D E P P P G Y N W R W S W P A A Y S F CM A E P G N R D A A T P V V S A F Y D R F P F P G D P L Q D G P P P G Y N W R W C H Q S V L A A V

M S S V A Q P - - C D E A T P V V S A F Y D R F P Y P G D P L Q D G P P P G Y N W R W S V D M A Y A V CM D I T R A V Q S L Y E R Y P F P P D P L S D K P P P G W N W R W S Y S F A H S F C

M V D K S Q E T T H F G F Q T V A K E Q K A D M V A H V F H S V A S K Y D V M N D L M S F G I H R L W K R F T I D - - CM V D K S Q E T M . . A V L Y . T Y P F P P D P L D E P P P G Y N W R W W . A F C

Sll0487All0012Npun5885Tery3855Tlr1948Synw0487Pmt1474Glr1180UbiE

70 80 90 100 110 120

L G R R P - - - - A N Q K V R I L D A G C G T G V G T E Y L V H L N P E - A E V H A V D I S E G A L A V A Q T R L Q K ST G R K P - - - - A K Q D I R I L D A G C G S G V G T E Y L V H L N P Q - A Q V V G I D L S A G T L A V A K V R C Q R ST G Q K P - - - - Q K Q D I R I L D A G C G T G V S T E Y L V H L N P Q - A S V V G I D L S T G A L D V A K E R C Q R ST G Q K P - - - - K K Q D I R I L D A G C G T G S S T D Y L V H L N P Q - A S V V G I D L S S G A L R V A K E R C S R ST G R Y P - - - - S Q L D V A I L D A G C G T G V G T E Y L A H L N P Q - A K I T A L D L S E A A L A I A C E R C R R SH G A V P - - - A G T V R P R I L D A G C G T G V S T D Y L C H L N P G - A E V L G I D I S E G A L A V A R E R L Q R ST G S V V R K C A G S E P L K I L D A G C G T G V S T D Y L A H L N P G - A E I L A V D I S L G A L E V A R E R L Q R SR G W R P - - - - E P R P I R I L D A G C G T G V S T E Y L A V Q N P E - A Q V T A I D I S E A S L A L A K R R C A G HS G V R R - - - - - - - G Q T V L D L A G G T G D L T A K F S R L V G E T G K V V L A D I N E S M P K M G R E K L R N IT G . P R K C A . R I L D A G C G T G V S T E Y L . H L N P T A V . . I D I S E G A L A V A . E R C Q R S


130 140 150 160 170 180

G V V C D - - R V H F H H L S L E N L A H L P G Q F D Y I N S V G V L H H L P D P V A G I Q A V A E K L A P G G L F H IG A N - - - - R V E F H H L S L Y D V E Q L P G E F D L I N C V G V L H H L P D P I R G I Q A L A K K L A P G G L M H IG A N - - - - R V E F H H L S L F D V E Q L P G E F D L I N C V G V L H H T S D P I R G I Q A L A Q K L A P G G L M H IG A S - - - - R V D F H H L S I Y D V G R L E G E F K F I N C V G V L H H L P D P I R G I Q N L A L K L A P G G I M H IG A T - - - - N V Q F H H L S L E E V A Q L G Q T F Q M I N C V G V L H H L P E P Q R G I Q A L A D V L A P G G I V H IG A A A Q V S Q L R Q E Q R S L L D L E S E - G P F D Y I N S V G V L H H L D Q P E S G L R S L A G R L A P D G L L H LG G N D Q - A H V R I E N Q S L L E L E D E - G Q F D Y I N S V G A L H H L R Q P E A G L K A L A S R L K P G A L L H LA N V - - - - - - Q F A Q M S L T D A G D L E G Q F D L I N C V G V L H H L A D P K A G L A A L A A K L A P G G L L H IG V I G - - - N V E Y V Q A N A E A L P F P D N T F D C I T I S F G L R N V T D K D K A L R S M Y R V L K P G G R L L VG A Q V . V F H H L S L D . L G F D I N C V G V L H H L D P . G I Q A L A K L A P G G L . H I


190 200 210 220 230 240

F V Y A E I G R W E I Q L M Q K A I A I L Q G K K R G D Y Q D G V A V G R E I F A S L P E Y N R L V K R E K E R W S L EF V Y G E L G R R E I Q L M Q K A I A L L Q N E K Q G D Y R D G V Q V G R K I F A S L P E N N R L V K R E K E R W A M EF V Y G E L G R W E I Q L M Q K A I A L L Q G D K K G D Y R D G V Q V G R Q I F A S L P E N N R I V K Y E K Q R W S L EF V Y A E L G R W E I R L M Q Q A I A I L Q G N E R G N Y K D G V K I G R Q L F A S L P E N N R I V K Y E K E R W S W EF V Y G E Y G R W E I K L M Q Q A I A L L Q G S D R H N Y R E G V A I G R Q L F A A L P E N N R L K K R E Q E R W S L EF L Y A D A G R W E I H R T Q Q A L T L L D - - - V G T G R E G L R L G R E L L A S L P E G N R L A R H H C E R W A V DF L Y A E A G R W E I H R I Q R F L S S L G - - - V G T D E Q G L R L A R Q L F E I L P E H N R L R L N H E Q R W A I DF V Y G E L G R W E I R L M Q E A I R L L R - D G R D P L A D G L K V G R A L F E A L P E D N R L K S A D R - R W V L EL E F S K P - - - I I E P L S K A Y D A Y S - - - - - - - - - - - - - - - - - F H V L P R I G S L V A N D A D S Y R Y LF V Y . E . G R W E I . L M Q . A I A L L Q G . G Y . D G V . G R L F A S L P E N R L V K E . E R W . E


250 260 270 280 290 300

N H R D E S F A D M Y V H P Q E T D Y N I D T L F E L I D S A G L E F L G F S N P D Y W Q L D R L L G K A P D L M E R AN Q R D E C F A D M Y V H P Q E V D Y N I D T L F E L I D A S G L E F I G F S N P S F W N L D R L L G K A P E L I E R SN H K D E H F A D M Y V H P Q E I D Y N I E T L F E L I D A S G L E F I G F S N P S F W D L E R L L S K A P E L V E R AN K K D E C F A D M Y V H P Q E I H Y N I D T L F E L I E A S G L E F L G F S N W E Y W D L G R L L G K S P E L I E R AN Q R D E C F A D M Y V H P Q E I D Y T I A S L F E L I R A S G L E F I G F S N P Q V W Q L E R L L G S Q P E L L Q R AC A A D A N F A D M Y L H P Q E T S Y D L Q R L F A F I E A A D L H F A G F S N A E V W D P A R L L N G - - E L L E R AC V A D V N F A D M Y L H P Q E T S Y N L E R L F A F V A S A D L E F V G F S N P K V W D P V R L L Q G - - E L L E M AN H H D T C F V D M Y V Q V Q E I R Y T I P T L F D L I A S S G L G F L G F S N P G F W R L E R L V G K A P W L L E R AA E S - - - - - - I R M H P D Q - - - - - D T L K A M M Q D A G F E S V D Y Y N - - - - - - - - L T A G - - - - - - - VN . D E F A D M Y V H P Q E . Y N I . T L F E L I . A S G L E F . G F S N P . W L . R L L G K A P E L . E R A


310 320 330 340 350 360

K D L S E K E R Y R L I E L L D P E - I T H Y E F F L A K P P I Q I S D W T D D Q T L L K A L P N L H P C L T G W P S RE K L S D R Q R Y R L I E L L D P E - V T H Y E F F L G R P P I K K A D W S N D N A L L Q A I P E L N P C I D G F P S QK E L S D R Q L Y R L I E L L D P E - V T H Y E F F L G R P P L V K T D W S D D K A L L A A I P E L N P C I E G F P S QI D L N D Q E R Y R L I E L L D P Q - I S H Y E F F L G R P P L Q K V D W S H D E L L K A A I P E L S P C I Q G W P S EQ Q L D P I Q Q Y Q L I E C L D P E A M T H F E F F L A K P P L P R H D W A A D R E L L A A I P W R H P C I D G W P G AQ A L P Q R Q Q W L L V E Q L D P N - I S H F E F F L S S Q P V Q P A S W S - D A A L Q A A K G L R Q P C L W G E P - DL A L P Q R Q Q W Q L I E Q L D P D - I S H F E F F L S K G P Q A S L G W T D D Q E L L A A T G K L S I C L W G W P G QA T L S D K E R F R L I E L L D P V - V A H Y E F F L G A P P L E R L E L D D D - R L D A A L P V R S P Y L Y P W P E RV A L H R G - - - - - - - - Y K F

L . . Q Y R L I E L L D P . A . . H Y E F F L . . P P . . . D W . D D L L A A . P L P C . G W P S


370 380 390 400 410 420

S L F D Y N Y Q P L E L T E E E F K F L Q A V E A Q T E - - I Q A N V E K V L T D L G D R S I D L A L V R S L Q T R Q LC I F N Y D Y Q I V N L S A L E F E F L Q K C D G - - - - - - N S T V A E I L V S V Q - - - L S L D E V R S L I Q Q Q LC F F N Y N Y Q I V N L S E P E F E F M Q K C D A - - - - - - N S T V A E I L A D V Q - - - L G L D E V R K L L Q Q Q LN I F D G D Y R L V N L S R P E Y E F L E A C T Q G V S N K S Q S T V G E I L T D V P - - - V G V E T V R S L Q S R L LC V F N C H Y E V I H L N Q A E Q E F L N A A S G - - - - - - K A T V A E L L A Q Q P D - - F N L A E V R S L L A R H LP I L D R N M Q P L Q L S D A E R Q L L R R V D E Q - - - - - P N T P L G A M A E - - - - - - - P A V I R D L A A R Q LS L L D F E M A P L E L S V E Q V E L L K A I E A T - - - - - P D T A L G C L P L G W D S K R I S S L A R D L Q R R Q VQ V F D H E Y E L V E L S E A E V A F L Q R C N G - - - - - - R Q T L G Q L R G D I A Q - - - A R I R E L L E H F L L Q

. F D Y . . L S E E F L . . . N K T V . . L . . . D . V R L R Q L


430 440 450 460 470 480

L T L G N SV M L I P G A K G Y A SI L L A P AI L L S P N S G Y HL L L G V K EL L L K AL L L S P G N V A S TL A P P P G L L S S K M R V

L L L P R V

The multiple alignment was generated by BLOSUM30 using Clustal W. Locus tags are used to indicate each organism (see also Table 6.5): Sll0487, Synechocystis sp. PCC 6803; All0012, Nostoc sp. PCC 7120; Npun5885, N. punctiforme; Tery3855, T. erythraeum; Tlr1948, T. elongatus BP-1; Synw0487, Synechococcus sp. WH8102; P. marinus MIT1474; Glr1180, G. violaceus PCC7421.

308

A.1.13. An amino acid sequence alignment of Slr1039 homologs in cyanobacteria Slr103991c179267All3016Tlr1953Synw1148Pmm0662Pmt0792Pro0816Glr3970UbiE

10 20 30 40 50 60

M L E R I L E P E - - - - V M D T E A E A M D Y D A M D F Q A V N L - - A F A Q R A C Q L G P S K - - - - - - - - - -M A L T R I L E P E - - - - V M D D P A E A I A Y D A M D F R A V N Q - - D F A D L V V T T Y S Q E T - - - - - - - - -

M H R Q L E P E - - - - V M D S W E E A S E Y D A M D F T E V N N - - A F A E E A V A C G L S E H - - - - - - - - -M P L P R I L E P E - - - - V M E T P E T A A A Y D A M D F T A V N T - - A F C D D L A A V L P R V D Q P - - - - - - -

M Q R L P E P E - - - - L M V D P A Q V L A Y A A A D F S G G D Q - - R T L D R I E A L L S S A S G R A A - - - - -M E R T V E P E - - - - L M E R E D Q V D S Y A K A D F S E G E N - - N L I S Q I N H Y L I K N N I Y L S - - - - -

M P E P E - - - - L M D D P L Q A R A Y A E A D F S C G D E - - A L I Q R L Q E Y L I S L C K I P S - - - - -M Q R V T E P E - - - - L M L A P D Q V Q A Y A E A D F S S S E A - - S M L S E V D L L I R D R G M H I D - - - - -

M L E R V L E P E - - - - V M D S Q A E A E A Y D A M D H G E V N A - - R F V A D V L A L G P A D G - - - - - - - - -M V D K S Q E T T H F G F Q T V A K E Q K A D M V A H V F H S V A S K Y D V M N D L M S F G I H R L W K R F T I D C S

M M R . . E P E H F G F . M . A A Y A D F . V K Y . . . T I D C S

Slr103991c179267All3016Tlr1953Synw1148Pmm0662Pmt0792Pro0816Glr3970UbiE

70 80 90 100 110 120

- - - - - A T V L D A G T G T A R I P I L L A Q L R P A - W Q I T A I D F A R S M L A I A K E N V I - - - A A G C E T Q- - - - - A F V L D L G T G T A Q I P I L L G Q Q R P Q - W Q I K G T D L A Q S M L A L G Q K N V V - - - A A G L T A Q- - - - - G L V L D A G T G T A R I P V L I C Q K R P Q - W Q L V A I D M A E N M L Q I A T Q H V Q - - - Q S G L Q E H- - - - - L K V L D V G T G N G R I L Q L L H Q R Y P H - W Q L T G I D L S P A M L A I A R H H S - - - - - - - - - P Q- - S D P A V I L D L G C G P G N I S L P L A K R F P E - S Q V I G V D G S R A M L Q V A R D R - - - - - A N Q Q G L S- - E K - E L I V D L G C G P G N I S E K L S T K W P N - A N V I G I D G S K E M I R I A E L N K K N S L N R S R L K N- - P G - S M I V D L G C G P G N I C E R L Q R L W P E - V M V L G I D G A Q A M L D H A L Q R Q K A - - M A A E L K R- - R K - K L I V D L G C G P G N I T Q R L A D Q W P E - A K V L G L D D S P E M L M Y A R K K Q K E - - K I S F L E R- - - - - - P W L D L G T G P A H L P V L L A S E R P D - I R I T A V D L A A P M L A I A R R R V E - - - A A G L A G RG V R R G Q T V L D L A G G T G D L T A K F S R L V G E T G K V V L A D I N E S M P K M G R E K L R - - - N I G V I G NG V . L D L G G G I . L P T . . . G . D . M L . A . . S L . G .


130 140 150 160 170 180

I C L E F V D A K Q L P Y A N G S F D G V I A - - - - N S L C H H L P R P L D F L R E V K R V L K P H G F L L I R D L II E L V Y A D A K N L P W P D Q S F D V I I S - - - - N S L I H H L P D P L P C F Q E M R R L L K P Q G N I I V R D L FI R L E L V D A K R L P Y E D G I F D L V V S - - - - N S L V H H L P D P L P F F A E I K R V C K P Q G G I F I R D L LL N F I E G D A K T L P F A A A S F D V V I S - - - - N S L V H H L A D P Q P A L G E M L R V L R P Q G T L F I R D L CI D L R C S T L Q D L A L E P V D L I V S - - - - - - N S L L H H L H E P G L L W G L T R E L A A P G C R V L H R D L RL R Y I C A D I K S L K S S D I S F E K N I S L L V S N S L I H H I T Y L D D F F N C I K R L S S D L T I N F H K D L KL T Y R C S N L S S L V N Q C V D L K C S A A L V V S N S L L H H L H D P N L L W Q V T K Y L A A P G A F V F H R D L RI T Y K K I N I S Q I A N G D F P F R E C A D L V V S N S L I H H I H D P L I F I K A L K N I S K H G A I H F H R D L RI T L V H G D A K A P A L G A A R F A C V F S - - - - N S L V H H L P Q P A P F W Q A C A R L L A P G G V L F V R D L AV E Y V Q A N A E A L P F P D N T F D C I T I - - - - S F G L R N V T D K D K A L R S M Y R V L K P G G R L L V L E F SI . D A K L . F . . . S L V V S N S L . H H L D P . . . R . . . P . G . F . R D L


190 200 210 220 230 240

R P E S P E K L A D F V T A I G S D Y N D Q - Q R K L F A D S L H A S L T I A E I Q D Y L Q H L G W V G D D G P N L E LR P D S T A A I D Q I V A A A E G P G F D A R Q R Q L F W D S L H A A F T V P E I E A I A T E A G L T K - - - - - - - AR P E D E A T M N A L V A S I G N E Y D D Y - Q K K L F R D S L H A A L T L D E V N Q L I I T A G L T G - - - - - - - VR P Q T L A E L R A L V E Q Y A G Q D T P Q - Q Q K L F A D S L H A A L T L R E M R H L L R T L P Q S P Q - - - - - R WR P A A - L A E V H R L Q Q L H C F D A P A V L I Q D F C A S L V A A F T P E E V Q Q Q L A L A E L D G - - - - - - - LR P I D - E Q S A L Y L K E K C G E K Y N E I L T N D Y Y A S L K A S Y T S K E L K D F I F E N K L S S - - - - - - - LR P L S - T K H A I A L Q Q K H M Q E A P S I L I R D Y L A S L H A A F T L E E V Q S Q L V H A G L D H - - - - - - - LR P S S - L E E A L A I K Q K H L P T A P S V M E K D F I A S L K A A Y T S R E I S Q Y L K D S E L N S - - - - - - - FR P D S L S R R D A L V E Q Y A A G C D D D - Q R R L F A E S L Q A A L I P A E V A S Q A R A A G L T G - - - - - - - AK P I I - E P L S K A Y D A Y S F H V L P R I G S L V A N D A D S Y R Y L A E S I R M H P D Q D T L K A M M Q - - - - DR P . . . . . . F . S L . A A . T . E . . . L P N L .


250 260 270 280 290 300

H L Y Q S S D R H W T L E K K F Q S S T I V Q RQ V Y Q S S E R H W T L I F P Q P NE I Y Q S S D R H W T A K R S W T NQ A I L T S D R H W T V V A Q RT V E M E D D R Y L V V S G L V DE V F E E G D Q Y L V I Y G K VH V I E V E D R Y L D V F G T IK V V E V D D R Y L D V F G V I IQ V A M S S D R H W T L I R R G A VA G F E S V D Y Y N L T A G V V A L H R G Y K F

V . D R . . .

The multiple alignment was generated by BLOSUM30 using Clustal W. Locus tags are used to indicate each organism (see also Table 6.5): Slr1039, Syechocystis sp. PCC 6803; All3016, Nostoc sp. PCC 7120; Tlr1953, T. elongatus BP-1; Synw1148, Synechococcus sp. WH8102; Pmm0662, P. marinus MED4; Pmt0792, P. marinus MIT9313; Pro0816, P. marinus SS120; Glr3970, G. violaceus PCC7421. 91a179267 indicates a tentative locus in Synechococcus sp. PCC 7002.

309

A.2.1. Polypeptide composition of isolated PS I complexes isolated from the wild-type,

menG, menF, rubA, rubA menG, and rubA menB mutants of Synechococcus sp. PCC

7002

M and T indicate monomeric and trimeric PS I complexes isolated from the respective wild type (WT) and mutant strains. SDS-PAGE gel containing 16% acrylamide and 6 M urea were used to resolve the polypeptides (electrophoresis at 80 mV for 12 h at room temperature). Small blue, yellow, and red arrows indicate polypeptide bands that are not detectable in the wild-type PS I trimeric forms. The polypeptide bands were visualized after silver staining.

310

Presence of all the polypeptides was confirmed in the PS I complexes isolated

from menF and menG mutants. Immunoblotting analysis showed that PsaA and PsaB

polypeptides were present in the PS I complexes isolated from the rubA menB and rubA

menB double mutants, while PsaC, PsaD, and PsaE were absent. The absence of the latter

three polypeptides is expected as a result of the inactivation of the rubA gene, as

previously reported [Shen G, Antonkine ML, van der Est A, Vassiliev IR, Brettel K, Bittl

R, Zech SG, Zhao J, Stehlik D, Bryant DA, Golbeck JH (2002) J Biol Chem 277: 20355-

20366]. In the rubA menB mutant, no PsaL was detectable when the isolated PSI

complexes were analyzed, while it was detectable when thylakoid membranes was

analyzed. This suggests that PsaL was lost during the preparation of PS I complexes. The

absence of PsaL is also visible in the SDS-PAGE gel (a band immediately below PsaF is

missing). It has been reported that PsaL is required for the formation of trimeric forms of

PS I complexes [Chitnis VP and Chitnis PR (1993) FEBS Lett 336: 330-334]. Indeed,

after the first sucrose-density gradient centrifugation, most of the chlorophyll (an thus, PS

I complexes) was collected in the PS I monomeric fraction from the rubA menB mutant.

311

A.2.2. Illustration of genetically engineered PS I in cyanobacteria.

Strain names are shown below the respective PS I complexes. WT, wild-type strains of Synechocystis sp. PCC 6803 (containing PhyQ) and Synechococcus sp. PCC 7002 (containing MQ-4); DMQ, demethylmenaquinone-4; DphQ, demethylphylloquinone; DMPBQ, 2,3-dimethyl-6-phytyl-1,4-benzoquinone; PQ, plastoquinone-9; AQ, 9,10-anthraquinone. The PS I core is shown in green, while the PsaC subunit is shown in blue. P700 and A0 are indicated by purple circles, A1 is indicated by red diamonds, and [4Fe-4S] (FX, FA, FB) clusters are indicated by yellow squares. All the PS I complexes shown here have been generated, except for the DMPBQ-containing complexes, which are expected to be produced in a menF cyclase (in α-tocopherol biosynthetic pathway) double mutant that is yet to be constructed. For constructions of the mutants, see Chapter 2 and 3. The rubA menG mutant in Synechococcus sp. PCC 7002 was generated by transforming the rubA mutant with the menG::accC1 construct (see the construction of the rubA menB double mutant described in Chapter 2). The PS I complex devoid of quinone and [4Fe-4S] clusters has been recently generated by treating the PS I complexes isolated from the rubA menB mutant with AQ followed by extensive washing (Y. Sakuragi, B. Zybailov, G. Shen, R. Balasubramanian, B.A. Diner, I. Karygina, Y. Pushkar, D. Stehlik, D.A. Bryant, and J.H. Golbeck, unpublished).

CURRICULUM VITAE

YUMIKO SAKURAGI Date of Birth: April 2, 1973 Nationality: Japanese Place of Birth: Tokyo, Japan

EDUCATION

1996 B. S., Biology, Department of Biology, Tokyo Metropolitan University, Japan

1999 M. S., Biology, Department of Biology, Tokyo Metropolitan University, Japan

2004 Ph.D. in Biochemistry, Microbiology, and Molecular Biology, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, USA

HONORS

1996 Danish Government Scholarship, Denmark

1999 Braddock Graduate Fellowship, The Pennsylvania State University

2000 First place in the competition for poster presentation, American Association for Microbiology, Allegheny Branch Meeting, Pennsylvania, USA

2001 Invited speaker at the 7th Cyanobacterial Workshop, California, USA

2002 Center for Biomolecular Structure & Function Travel Award, The Pennsylvania State University

2003 Second place in the competition for oral presentation in Environmental Engineering, Ecology and Soil Science, 6th Annual Environmental Chemistry Symposium, The Pennsylvania State University

2003 Invited speaker at the 11th International Symposium on Phototrophic Prokaryotes, Tokyo, Japan

2003 Alumni Association Dissertation Award, The Pennsylvania State University

PROFESSIONAL MEMBERSHIPS 2000 American Society of Microbiology

2002-2004

American Association for the Advancement of Science

cyanobacterial quinomics studies of quinones in cyanobacteria

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