Phosphatidylglycerol depletion affects photosystem II activity in Synechococcus sp. PCC 7942 cells

REGULAR PAPER

Phosphatidylglycerol depletion affects photosystem II activityin Synechococcus sp. PCC 7942 cells

Balazs Bogos Æ Bettina Ughy Æ Ildiko Domonkos Æ Hajnalka Laczko-Dobos ÆJosef Komenda Æ Leyla Abasova Æ Krisztian Cser Æ Imre Vass ÆAnna Sallai Æ Hajime Wada Æ Zoltan Gombos

Received: 19 March 2009 / Accepted: 3 September 2009 / Published online: 18 September 2009

� Springer Science+Business Media B.V. 2009

Abstract The role of phosphatidylglycerol (PG) in pho-

tosynthetic membranes of cyanobacteria was analyzed in a

Synechococcus sp. PCC 7942 mutant produced by inacti-

vating its cdsA gene presumably encoding cytidine

50-diphosphate-diacylglycerol synthase, a key enzyme in

PG synthesis. In a medium supplemented with PG the

Synechococcus sp. PCC 7942/DcdsA cells grew photoau-

totrophically. Depletion of PG in the medium resulted (a)

in an arrest of cell growth and division, (b) in a suppression

of O2 evolving activity, and (c) in a modification of Chl

fluorescence induction curves. Two-dimensional PAGE

showed that in the absence of PG (a) the amount of the PSI

monomers increased at the expense of the PSI trimers and

(b) PSII dimers were decomposed into monomers.

[35S]methionine labeling confirmed that PG depletion did

not block the de novo synthesis of PSII proteins but slowed

down the assembly of the newly synthesized D1 protein

into PSII core complexes. Retailoring of PG was observed

during PG depletion: the exogenously added artificial

dioleoyl PG was transformed into photosynthetically more

essential PG derivatives. Concomitantly with a decrease in

PG content, SQDG content increased, but it could not

restore photosynthetic activity.

Keywords Oxygen-evolving activity �Phosphatidylglycerol � PS II acceptor side � Synechococcus

Abbreviations

2D-BN Two-dimensional blue native gel electrophoresis

Chl Chlorophyll

CP43 43 kDa chlorophyll-binding protein

DCMU 3-(3, 4-dichlorophenyl)-1, 10-dimethylurea

DGDG Digalactosyldiacylglycerol

OD Optical density

pBQ 1, 4-p-benzoquinone

PG Phosphatidylglycerol

PQ Plastoquinone

QA Primary quinone electron acceptor of PS II

QB Secondary quinone electron acceptor of PS II

RC Reaction center

SQDG Sulfoquinovosyldiacylglycerol

WT Wild type

Introduction

The lipid composition of photosynthetic membranes is

highly conserved among cyanobacterial strains and higher

plant chloroplasts (Wada and Murata 1998). One of the lipid

classes is phosphatidylglycerol (PG), which is an indis-

pensable component of photosynthetic membranes. In

cyanobacterial cells PG is the only representative of the

B. Bogos � B. Ughy � I. Domonkos � H. Laczko-Dobos �L. Abasova � K. Cser � I. Vass � A. Sallai � Z. Gombos (&)

Institute of Plant Biology, Biological Research Center,

Hungarian Academy of Sciences, P. O. Box 521, 6701 Szeged,

Hungary

e-mail: [email protected]

J. Komenda

Institute of Microbiology, Academy of Sciences, Opatovicky

mlyn, 37981 Trebon, Czech Republic

J. Komenda

Institute of Physical Biology, University of South Bohemia,

Zamek 136, 37333 Nove Hrady, Czech Republic

H. Wada

Department of Life Sciences, Graduate School of Arts

and Sciences, University of Tokyo, Komaba, Tokyo 153-8902,

Japan

123

Photosynth Res (2010) 103:19–30

DOI 10.1007/s11120-009-9497-0

glycerophospholipids (Wada and Murata 1998). PG is an

anionic lipid with a negatively charged phosphoglyceryl

headgroup attached to the diacyl-glycerol backbone. This

negative charge plays an important role in establishing pro-

tein–lipid interactions in photosynthetic membranes,

resulting in the formation of protein oligomers (Domonkos

et al. 2008). Although PG is a minor component of photo-

synthetic membranes, it plays important roles, in addition to

the formation of the photosynthetic apparatus, in a number of

various photosynthetic functions as presented below.

The importance of PG was studied in chloroplasts of

eukaryotic photosynthetic organisms, such as Chlamydo-

monas reinhardtii (Sato et al. 1995), and in higher plants

(Ariizumi et al. 2002; Hagio et al. 2002; Xu et al. 2002). It

has been demonstrated by using isolated PSII particles

treated with phospholipase A2 that PG molecules partici-

pate in PSII dimer formation (Kruse et al. 2000). The

availability of the complete genomic sequence of Syn-

echocystis sp. PCC 6803 (Kaneko et al. 1996) and the

unpublished, draft genomic sequence of Synechococcus sp.

PCC 7942 opened the way for using molecular genetic

approaches to study the structural and functional roles of

PG in vivo. Studies on Synechocystis sp. PCC 6803 iden-

tified PG-phosphate synthase (PgsA) as a key enzyme

involved in PG biosynthesis (Hagio et al. 2000). Experi-

ments with the Synechocystis sp. PCC 6803/DpgsA mutant

revealed the important role of PG in the structural orga-

nization and functioning of the photosynthetic apparatus.

Maintenance of this mutant, defective in PG synthesis

required exogenously added PG to the medium. When

cultured in the absence of PG the PG content of the isolated

thylakoid membranes decreased gradually leading to lethal

impairments in a number of photosynthetic processes.

The effect of PG on individual steps of the electron

transport chain was also studied in this mutant, and a

perturbation in the vicinity of the secondary quinone

acceptor (QB) was observed (Gombos et al. 2002). We

found that depletion of PG resulted in decreased photo-

synthetic oxygen-evolving activity. Similar results were

obtained with a cytidine 50-diphosphate (CDP)-diacyl-

glycerol synthase (cdsA) mutant. This mutant harbors an

upstream mutation in PG biosynthesis in the same strain as

described by Sato et al. (2000).

X-ray crystallographic data showed the presence of PG

molecules in PSI and its absence in PSII reaction centers

(RCs) (Jordan et al. 2001; Zouni et al. 2001). Even 3.8 A

resolution measurements did not allow the detection of lipid

molecules in PSII (Zouni et al. 2001). The structure of PSII

was published with better resolution, suggesting the presence

of a PG molecule in the vicinity of D1 and the 43 kDa

chlorophyll-binding protein (CP43) (Loll et al. 2005).

However, recent X-ray crystallographic data with 2.9 A

resolution identified the presence of two PG molecules per

monomer of PSII. The previously known (Loll et al. 2005)

and the newly identified PG molecule are located on the

cytoplasmic side, close to the QB site (Guskov et al. 2009).

Both of the PG molecules are situated on the cytoplasmic

face of a bilayer domain formed by 3 MGDG, 2 DGDG, an

SQDG and the mentioned two PG molecules. This bilayer

island is encircled by protein subunits D1, CP43, PsbE, PsbF,

PsbJ, and PsbK that form the PQ-PQH2 exchange cavity. In

the case of the PSI RCs X-ray crystallographic measure-

ments also indicated the presence of three PG molecules

(Jordan et al. 2001), but PSI activity was not significantly

affected by short-term PG depletion (Hagio et al. 2000).

Nevertheless, PG depletion affected PSII activity earlier than

it did PSI processes. This finding suggested that the strength

of PG binding by PSII might be much weaker than that by the

PSI RCs. Recent reports confirmed the deleterious effect of

PG deprivation not only on photosynthetic function but also

on the structure of photosynthetic RCs in cyanobacteria

(Domonkos et al. 2004; Sakurai et al. 2003). Since PG is

required for functional binding of extrinsic proteins to the

PSII core (Sakurai et al. 2007) we concluded that PG affects

the functionally important proteins involved in photosyn-

thetic electron transport.

More detailed investigation of the effects of PG deple-

tion revealed the importance of PG in the maintenance of

primary quinone acceptor (QA) to QB electron transfer

within the PSII RC (Gombos et al. 2002). The character-

ization of a phycobilisomeless mutant of Synechocystis sp.

PCC 6803, PAL/DcdsA, that was defective also in PG

synthesis has strengthened the view that active PSII RC

needs PG (Laczko-Dobos et al. 2008). The results

emphasized that PG is active on the acceptor side of PSII

rather than on its donor side.

In addition to Synechocystis sp. PCC 6803, Synecho-

coccus sp. PCC 7942 would be another experimental object

to study the effect of PG depletion on photosynthetic and

other cellular processes. The unicellular Synechococcus sp.

PCC 7942 is a non-diazotrophic, obligatory photoautotro-

phic cyanobacterial strain.

In Synechocystis sp. PCC 6803 both PG and sulfoquino-

vosyldiacylglycerol (SQDG) are essential lipids (Aoki et al.

2004; Hagio et al. 2000) for the organism to maintain growth

and photosynthetic activity. However, in Synechococcus sp.

PCC 7942 SQDG is not essential, as it has been demonstrated

by Guler and co-workers (1996). Loss of the sulfolipid in

SQDG-deficient cells was compensated for by an increase in

cellular content of PG. To investigate the role of PG

in photosynthetic processes of Synechococcus sp. PCC 7942,

we inactivated the cdsA gene of this organism.

In the present article we demonstrate, by using fluores-

cence decay kinetics and two-dimensional blue native

SDS-PAGE (2D-BN/SDS-PAGE), that in the mutant Syn-

echococcus sp. PCC 7942/DcdsA PG depletion suppresses

20 Photosynth Res (2010) 103:19–30

123

electron transport around QB and affects the oligomeric

state of PSI and PSII, respectively. These results demon-

strate that PG is an indispensable lipid in both Synecho-

cystis sp. PCC 6803 (Hagio et al. 2000) and Synechococcus

sp. PCC 7942 cells unlike SQDG which is dispensable for

the maintenance of cellular functions in Synechococcus sp.

PCC 7942 (Sato 2004).

Materials and methods

Organisms and growth conditions

Synechococcus sp. PCC 7942/DcdsA cells were selected

and grown on BG11 agar plates supplemented with

500 lM sodium acetate. Synechococcus sp. PCC 7942 WT

and mutant cells were cultured in unbuffered BG11 liquid

medium supplemented with 500 lM sodium acetate. The

cultures were irradiated at 30�C with 30–35 lmol pho-

tons m-2 s-1 of continuous white light. Aeration was

performed without additional CO2, using a gyratory shaker

at 100 rpm. 50 ml liquid cultures were maintained in

150 ml Erlenmeyer flasks in the presence of 20 lM diol-

eoyl-PG (18:1/18:1 PG; P-9664; Sigma, St. Louis),

50 lg ml-1 kanamycin or 8 lg ml-1 chloramphenicol. PG

depletion was carried out by washing the cells twice with

PG-free medium, and culturing them thereafter in PG-free

medium.

Synechocystis sp. PCC 6803/DpgsA mutant cells (Hagio

et al. 2000) were grown photoautotrophically in BG11

medium supplemented with 5 mM HEPES–NaOH (pH

7.5), and 20 lg ml-1 kanamycin. Growth conditions, PG

supplementation and PG depletion were similar to those

used for Synechococcus sp. PCC 7942 cells.

Mutant generation, transformation of cells

Standard molecular biological methods were used during

the targeted insertional mutagenesis of Synechococcus sp.

PCC 7942. Escherichia coli DH5a and XL1-Blue cells

(grown in Luria broth at 37�C) were transformed using

standard methods (Sambrook et al. 1989). RCU1 50-CTC

GAGCAACGCTTGCTTAT-30 and RCD1: 50-AATTCGC

ATTGCCGCTGAGG-30 primer pair was used to amplify

the cdsA locus of Synechococcus sp. PCC 7942/DcdsA.

Primers were designed on the basis of the draft genome

sequence of Synechococcus sp. PCC 7942 available at

http://genome.ornl.gov/microbial/syn_PCC7942/. The cdsA

gene was identified by homology search using the published

sequence of the Synechocystis sp. PCC 6803 cdsA gene (http://

bacteria.kazusa.or.jp/cyanobase/). The amplified 1,596 bp

cdsA PCR fragments were cloned into pMPM-A2 recipient

vector (Mayer 1995) and verified by automatic sequencing.

The 209 bp MunI/HincII fragment of the cdsA gene was

replaced by a kanamycin resistance cassette or a chlor-

amphenicol resistance cassette from pZE21 and pZA3

vectors (Lutz and Bujard 1997), respectively. The resulting

plasmids carrying chloramphenicol resistance, pRC5C1,

and kanamycin resistance, pRC5K1 were used for targeted

insertional mutagenesis of the cdsA gene. Transformation

of WT Synechococcus sp. PCC 7942 cells was done

according to an optimized method (Golden and Sherman

1984). The segregation state of the transformants was

checked by PCR amplification of the cdsA locus from the

mutant chromosome.

Lipid analysis

Lipids were extracted from intact cells according to

standard lipid analytic methods (Bligh and Dyer 1959).

Lipid classes were separated by thin layer chromatogra-

phy, and fatty acids were analyzed on Supelco SP2330

capillary columns in a Hewlett Packard HP6890 gas

chromatography equipment as described earlier (Wada

and Murata 1989).

Spectroscopic measurements of cell density, proteins,

and pigments

Aliquots of cyanobacterial cells were collected and OD

was measured at 750 nm using a Shimadzu UV-3000

Spectrophotometer (Columbia, MD). Cells were pelleted

and extracted with acetone:methanol (7:2) (Ihlenfeldt and

Gibson 1975). The concentration of Chl was calculated

from the absorbance at 663 nm of the extracted samples

using an extinction coefficient of 82 mM-1 cm-1. The

protein content of extracted and pelleted cells was mea-

sured by the Lowry method (Yeang et al. 1998). For

measurements of protein concentration Bovine Serum

Albumin solution was used as a standard.

Absorption spectra were taken using protein normalized

cultures in 3 ml quartz cuvettes using a Shimadzu UV-

1601 spectrophotometer. The cultures were adjusted to

200 lg ml-1 protein content. The baseline was corrected

using two blank cuvettes filled with BG11 medium turned

with the opaque side toward the light path. The initial OD

at 750 nm was adjusted to zero and spectra were taken

using normal scanning speed against a blank sample. Each

of the spectra contains average data of three independent

PG-supplemented and PG-depleted samples in a given time

point. The resulting spectra were further normalized to

each other using the absorption maximum of the phyco-

biliproteins (625 nm) (Shibata 1958).

Photosynth Res (2010) 103:19–30 21

123

Measurements of photosynthetic oxygen-evolving

activity

Photosynthetic oxygen evolution in intact cells was mea-

sured with a Clark-type oxygen electrode (Hansatech

Instruments, Kings Lynn, UK) (Gombos et al. 1991).

Whole electron transport chain was measured from H2O to

CO2 and PSII oxygen-evolving activity from H2O to

exogenously added artificial quinone molecules (500 lM

pBQ). Light from an incandescent lamp at a saturating light

intensity of 500 lmol photons m-2 s-1, equipped with a

red optical filter was used in all oxygen evolution mea-

surements. The Chl concentration of the samples was

adjusted to 5 lg ml-1.

Flash-induced fluorescence relaxation kinetics

The kinetics of the decay of flash-induced variable Chl

fluorescence were measured in the 150 ls to 100 s time

range by a double modulation fluorometer (PS Instruments,

Brno) (Trtilek et al. 1997). Samples containing 200 lg

protein were used for the measurements. In order to show

the changes induced by PG depletion in the shape of the

flash fluorescence transients, the following normalization

was used: the basic level of fluorescence intensities (F0),

measured before the saturating flash, were shifted to the

same level, which was set as 0. Then the initial fluores-

cence amplitudes measured from the 0 level were nor-

malized to the same value, which was set to 1. After this

normalization the initial fluorescence rise in each samples

occurred from the 0 level to 1, and the shape of the decay

kinetics could be compared.

Protein analysis

Cells containing 75 lg of Chl were radioactively labeled at

29�C and at 60 lmol photons m-2 s-1 with [35S]Met (at

[1,000 Ci mmol-1, Isotope Institute Ltd, Hungary) for

20 min and then used for the isolation of membranes

(Komenda et al. 2004). Cyanobacterial membranes were

prepared by breaking the cells with glass beads (150–

200 lm diameter) at 4�C followed by differential centri-

fugation (Komenda and Barber 1995).

The isolated membranes were solubilized with dodecyl-

b-D-maltoside (dodecylmaltoside/Chl ratio of 40:1, w/w)

and analyzed by BN/SDS-PAGE at 4�C in a 5–14%

polyacrylamide gel (Schagger and von Jagow 1991). The

whole lane from the gel was excised, incubated for 30 min

in 25 mM Tris–HCl buffer (pH 7.5) containing 1% SDS

and then layered onto the top of a denaturing gel. The

protein composition of the complexes was then assayed by

the second dimension SDS-PAGE in a denaturing 12–20%

linear gradient polyacrylamide gel containing 7 M urea

(Komenda et al. 2004). The protein bands were stained

with Coomassie blue.

Results

Inactivation of the cdsA gene of Synechococcus sp.

PCC 7942

The cdsA gene encoding CDP-diacylglycerol synthase,

which is a crucial enzyme in PG synthesis, was identified in

the draft genome sequence of Synechococcus sp. PCC 7942

by homology search from the genomic data of Synecho-

cystis sp. PCC 6803. Following a targeted disruption of the

chromosomal cdsA gene by insertion of a chloramphenicol

(CmR) or a kanamycin (KmR) resistance cassette (Fig. 1a),

the transformants were segregated and verified by PCR

analysis (Fig. 1b). The kanamycin and chloramphenicol

resistance cassettes-containing Synechococcus sp. PCC

7942/DcdsA mutants did not show any phenotypical dif-

ferences compared to each other.

Effect of PG depletion on the growth and pigment

composition of Synechococcus sp. PCC 7942/DcdsA

cells

The growing of Synechococcus sp. PCC 7942/DcdsA

mutant cultures on solid or in liquid media requires exog-

enously added PG. The segregated transformants were able

to grow on PG-supplemented agar plates (Fig. 2a) and

were unable to grow on agar without added PG (Fig. 2b).

We also investigated the growth of Synechococcus sp.

PCC 7942/DcdsA in liquid medium under photoautotrophic

conditions in the presence and in the absence of PG. In the

medium containing PG the growth of Synechococcus sp.

PCC 7942/DcdsA cells was continuous even after 13 days

Fig. 1 Inactivation of chromosomal cdsA. a Physical map of the

Synechococcus sp. PCC 7942 genomic region containing the cdsAgene. In the inactivated strain the KmR or CmR gene cassette replaces

a 209 bp-long MunI/HincII gene fragment. Small black arrowsindicate positions of PCR primers used to amplify the region and to

verify the replacement. b PCR analysis of the WT (lane 1) and of

DcdsA kanamycin- (lane 2) and chloramphenicol- (lane 3) resistant

transformant genomic fragments

22 Photosynth Res (2010) 103:19–30

123

(Fig. 3a). However, when PG-supplemented inoculates

were transferred to a medium without PG, the growth of the

mutant slowed down after 3 days of PG depletion and

reached a plateau after 5 days of culturing. Following a 5-

day culturing period, the optical density at 750 nm (OD750)

increased from an initial value of 0.25 to 1.65. The OD750

of the culture remained at 1.65 for the next 5–6 days and

after 11 days of culturing it decreased to 1.35 (Fig. 3a).

The colony-forming capability of Synechococcus sp. PCC

7942/DcdsA cultures decreased significantly after 9 days of

PG starvation suggesting a reduced survival of the PG-

depleted cells (Fig. 3b).

The growth of Synechococystis PCC 6803/DpgsA under

photoautotrophic conditions in the presence and in the

absence of PG (Fig. 4a) was compared to the growth rate of

Synechococcus sp. PCC 7942/DcdsA grown under the same

conditions (Fig. 3a). The growth of the Synechocystis sp.

PCC 6803 mutant was continuous even after 21 days in the

medium containing PG. The growth of the mutant cells

slowed down after 15 days of culturing and stopped after

17 days in the absence of PG. Following a 17-day culturing

period, the optical density increased from an initial value of

0.20 to 2.70. The OD750 remained at 2.70 for 6 days and

after 23 days of culturing it decreased to 2.55 (Fig. 4a).

The survival of PG-depleted cells was demonstrated by the

recovery of the culture upon readdition of PG to the

medium. On the sixth day of recovery the OD750 of the

culture depleted of PG for 17 days increased from 0.25 to

1.0, that for 19 days increased from 0.25 to 0.90, and that

for 21 days increased from 0.25 to 0.80 (Fig. 4b). The

growth curve of the culture depleted of PG for 23 days was

slower and increased only up to 0.30.

Whereas Synechococcus sp. PCC 7942/DcdsA cells

could recover only after 11 days of PG depletion, Syne-

chococystis PCC 6803/DpgsA cells could recover as late as

23 days of PG depletion.

PG depletion-induced changes in Chl and protein

content

We also evaluated changes in the pigment composition

during PG depletion of Synechococcus sp. PCC 7942/DcdsA

cells. Initially, the changes were roughly estimated by

measuring the absorption spectra of cells. During the 6-day

depletion period the maximum of the absorption peak of Chl

at about 680 nm considerably decreased in comparison with

the peak of phycobiliproteins at 625 nm (Fig. 5). Hence, the

absorption spectrum of the PG-depleted mutant cells showed

a significant decrease in Chl content after 6 days of PG

depletion while no significant decrease in phycobiliprotein

content was detected during this time.

In order to get a more detailed picture about the changes

in the Chl content of the mutant cells, total protein and total

Chl contents of the cultures were measured. The total

protein content of cells grown in the presence and in the

absence of PG was used to estimate doubling times

(Fig. 6a). The average doubling time of the mutant cells

supplemented with PG was between 36 and 38 h and that

of PG-depleted cells was the same during the first 4 days.

Protein synthesis slowed down between the fourth and the

eighth day of culturing and stopped at a protein content of

340 lg ml-1 after 2–3 cell divisions. After 9 days of PG

starvation the protein content of the cells decreased to

230 lg ml-1.

Fig. 2 The growth of WT (1), Synechococcus sp. PCC 7942/DcdsAkanamycin (2), and Synechococcus sp. PCC 7942/DcdsA chloram-

phenicol resistant (3) cells on agar in the presence (a) and absence (b)

of exogenously added PG

Fig. 3 a Growth curve of

Synechococcus sp. PCC 7942/

DcdsA cells grown in the

presence (filled diamond) and in

the absence (filled square) of

PG, and b the colony forming

capability of PG-depleted cells

of the same mutant plated on

PG-containing agar plates after

9, 10, 11, 12, and 13 days of PG

depletion

Photosynth Res (2010) 103:19–30 23

123

The Chl content of the mutant increased gradually in

cultures supplemented with PG. During the first 3 days of

PG depletion the Chl content of the mutant cells increased

from the initial 2 lg ml-1 to 6.5 lg ml-1, similar as in the

PG-supplemented cells. After 3 days of PG depletion Chl

synthesis stopped reaching a level of 10 lg ml-1. Between

the third and the tenth day of PG starvation the Chl content

stayed at this concentration and during the following

3 days of culturing the Chl content of the mutant cultures

decreased to 1.3 lg ml-1 (Fig. 6b).

In order to determine precisely the Chl content of the

cells, the Chl to protein ratio was calculated. In the cells

grown in the presence of PG this ratio did not change for

13 days of culturing. However, upon PG depletion, the

ratio of Chl to total protein decreased to one-tenth of its

original value, 0.06–0.0057 suggesting a decrease in cel-

lular Chl content (Fig. 6c).

Fig. 4 a Growth curve of Synechocystis sp. PCC 6803/DpgsA cells

cultivated in the presence (dots) and in the absence (line) of PG, and

(b) the growth of the mutant cells in the PG-containing BG 11

medium after they have been cultured in the absence of PG for 17

(filled circle), 19 (filled square), 21 (filled triangle), and 23 (filleddiamond) days. The cells were cultured for 6 days and the growth was

followed by measuring OD750

Fig. 5 Absorption spectra of Synechococcus sp. PCC 7942/DcdsAcells grown in the presence (line) and in the absence of PG for 6

(broken line) and 10 days (dots). The spectra were normalized at

625 nm, the absorption maximum of phycobiliproteins

Fig. 6 Time course of changes in the protein content (a), in Chl

content (b), and in the Chl to protein ratio (c) during growth of PG-

supplemented (open diamond) and PG-depleted (filled square)

Synechococcus sp. PCC 7942/DcdsA cells

24 Photosynth Res (2010) 103:19–30

123

Effect of PG depletion on the lipid content and fatty

acid composition of PG in Synechococcus sp. PCC

7942/DcdsA cells

In order to investigate the influence of PG depletion on the

lipid composition of Synechococcus sp. PCC 7942/DcdsA

cells, lipid analysis was performed on lipid extracts of PG-

supplemented and PG-depleted cells. The relative con-

centration of PG in the extracts of PG-supplemented cells

was 8%. After two cell divisions, the concentration of PG

in the extracted lipids of PG-depleted cells decreased to

near one-fourth (2%) of the original PG content (8%). In

the mutant cells grown in the absence of PG the relative

amount of PG decreased to \2% after 6 days of culturing

(Fig. 7a). Simultaneously with a decrease in PG content,

the content of SQDG increased from 6% to more than 12%

in the PG-depleted cells, suggesting a balance of total

anionic charge that was being kept at about the same level

(Fig. 7a).

Fatty acid analysis of PG-supplemented cells demon-

strated that the cellular PG contained more than 50 mol%

of oleic acid (18:1) that originated from the exogenously

added, artificial dioleoyl-PG. This result suggests that the

cells could take up and incorporate the artificial PG into

their membranes. However, in cells grown in the absence

of PG the fatty acid composition of the isolated PG sig-

nificantly differed from that of the cells grown in the

presence of PG. The amount of oleic acid in the PG frac-

tion of lipids decreased from 50 mol% to\10 mol%, with

a concomitant increase in palmitic acid (16:0) content from

30 mol% to more than 60 mol%. This suggests that the

cells retailored PG in the membranes (Fig. 7b). With this

modification the fatty acid composition of PG became

similar to that of the original PG of the WT, which has

been described previously (Murata et al. 1992).

PG depletion-induced changes in the oxygen-evolving

activity of Synechococcus sp. PCC 7942/DcdsA cells

We measured changes in net and PSII oxygen-evolving

activities of PG-depleted Synechococcus sp. PCC 7942/

DcdsA cells. In the first 4 days of PG depletion there were

no detectable changes in oxygen-evolving activities

(Fig. 8) compared to those of PG-supplemented mutant

cells (data not shown). Between the fourth and the eighth day

of culturing the oxygen-evolving activity of the PG-depleted

cells decreased gradually, while this activity in PG-supple-

mented cells remained identical. During this time of PG

depletion the PSII and the H2O to CO2 oxygen-evolving

activities were also impaired compared to those of the PG-

supplemented mutant cultures, suggesting that PG is an

indispensable component of the photosynthetic apparatus

(Fig. 8). After 11 days of culturing, the oxygen-evolving

activity of the PG-depleted cells was severely blocked.

Effect of PG depletion on the synthesis and assembly

of PSII protein subunits

The protein composition of the isolated thylakoid mem-

branes was studied by 2D-BN/SDS-PAGE. Radioactively

labeled and isolated membranes of cells grown in the

presence and in the absence of PG were analyzed. On

Coomassie blue-stained 2D gels the trimers and monomers

of PSI and dimers and monomers of PSII RCs were clearly

separated from the thylakoid membranes of PG-supple-

mented cells (Fig. 9a). After 5 days of PG depletion the

oligomers of both photosystems were clearly destabilized

as evidenced by 2D gel of PG-depleted membrane proteins

in which a large fraction of PSI trimers and all PSII dimers

were missing (Fig. 9b). The corresponding autoradiograms

Fig. 7 Changes in the anionic lipid content of PG-depleted cells. aTime course of changes in PG (filled triangle) and SQDG (filleddiamond) contents. b Time course of changes in 18:1 (times) and 16:0

(filled diamond) fatty acid composition of PG

Photosynth Res (2010) 103:19–30 25

123

reflecting new synthesis of membrane proteins and their

assembly into complexes showed an efficient synthesis of

the D1 protein into monomeric as well as dimeric PSII core

complexes in the PG-supplemented cells. In contrast, in the

PG-depleted cells the majority of the newly synthesized D1

protein was detected in the fraction of unassembled pro-

teins and only \30% became assembled in the PSII

monomer (Fig. 9c, d). Taken together the data obtained by

2D protein analyses showed the importance of PG for

oligomerization of both photosystems and for efficient

assembly of the D1 protein into PSII.

Flash-induced fluorescence relaxation kinetics

In dark-adapted samples, illumination with a single satu-

rating flash forms QA-, the primary plastoquinone (PQ)

electron acceptor of PSII, and this results in a rapid rise of

variable fluorescence. The subsequent relaxation of fluo-

rescence reflects different reoxidation routes of QA-. The

detailed kinetics of this fluorescence relaxation have earlier

been described for WT Synechocystis sp. PCC 6803

(Allahverdiyeva et al. 2004).

Previously it was shown that DGDG deficient Arabid-

opsis thaliana mutants have characteristic changes in flash-

induced fluorescence quantum yield. The dgd1 mutation

causes 90% reduction in the relative DGDG content of the

mutant plants, resulting possible structural changes at the

donor side of PSII complex and increased susceptibility to

high-temperature stress in intact leaves. However, the

acceptor side of the PSII complex was not affected by the

drastically decreased DGDG content, as it was shown by

QA- reoxidation measurements (Reifarth et al. 1997).

For comparison flash-induced fluorescence decay mea-

surements were first done on Synechocystis sp. PCC 6803/

DpgsA mutant samples cultured in the presence and in the

absence of PG. In PG-supplemented cells, the relaxation of

the fluorescence was similar to that in the WT and was

dominated by a fast component (500 ls), related to the

reoxidation of QA- by QB

-. The middle phase (3–5 ms)

arises from QA- reoxidation in the RCs, which have an

empty QB site at the time of the flash and are to bind a

plastoquinone (PQ) molecule from the PQ pool. Finally, the

slow phase (8–10 s) represents QAQB- reoxidation through

equilibrium with QA- QB via a reverse reaction with the S2

state. Cells that were cultured for 7 days in the absence of PG

yielded a clear difference in fluorescence relaxation kinetics

from those of the PG-supplemented cells. Relaxation of the

middle phase fluorescence yield slowed down in the absence

of PG, suggesting a clear inhibition of QA- to QB electron

transfer (Fig. 10a). In the presence of 3-(3, 4-dichloro-

phenyl)-1, 10-dimethylurea (DCMU) there were no signifi-

cant differences in the decay curves between PG-depleted

and PG-supplemented samples, suggesting that there is no

significant donor side inhibition in the DpgsA mutant of

Synechocystis sp. PCC 6803.

The same measurements of variable fluorescence decay

were performed using the Synechococcus sp. PCC 7942/

DcdsA strain. In contrast to Synechocystis sp. PCC 6803/

DpgsA, after 7-day PG depletion the fast phase of the relaxa-

tion did not slow down, and even accelerated in the absence of

PG. In addition, the amplitude of the slow-phase was higher

and decay time was faster in the absence of PG, than in PG-

supplemented cells (Fig. 10b). These data indicate a modifi-

cation of the QB binding site, leading to the accumulation of

QA- due to a decreased equilibrium constant for sharing the

electron between the QA- QB and QAQB

- states.

In the presence of DCMU the relaxation kinetics of

fluorescence were quite similar (Fig. 10b), both in the

presence and in the absence of PG, suggesting that there

are no detectable instabilities at the donor side of PSII. In

conclusion, the fluorescence decay data obtained with the

PG-depleted Synechococcus sp. PCC 7942/DcdsA cells

provided evidence for a strong disturbance at the acceptor

side of PSII after 7 days of PG depletion but there were no

indication for the impairment of the donor side of PSII.

Fig. 8 Time course of oxygen-evolving activity during PG depletion

of Synechococcus sp. PCC 7942/DcdsA cells. The oxygen-evolving

activity was measured from H2O to CO2 (open square) and from H2O

to an added artificial electron acceptor, pBQ (filled square). The

initial activity of the PG-supplemented cells before the start of PG

depletion (100%) was 124 lmol O2 mg Chl-1 h-1

26 Photosynth Res (2010) 103:19–30

123

Fig. 10 Relaxation of flash-

induced Chl fluorescence yield

in a Synechocystis sp. PCC

6803/DpgsA mutant and bSynechococcus sp. PCC 7942/

DcdsA mutant cells. The

measurements were performed

after single flash excitation in

PG-supplemented cultures in

the presence (filled triangle) or

in the absence (filled circle) of

DCMU and in 7-day-old, PG-

depleted cultures in the presence

(open triangle) and in the

absence (open circle) of DCMU

Fig. 9 2D-BN/SDS-PAGE of radioactively labeled membrane pro-

teins from Synechococcus sp. PCC 7942/DcdsA cells either supple-

mented with PG (PG?) or depleted of PG for 5 days (PG-). The

upper panels (a, b) show Coomassie stained gels, the lower ones (c, d)

the corresponding autoradiograms. Designations: PSI(3) and PSI(1),

PSI trimeric and PSI monomeric core complexes; PSII(2) and PSII(1),

PSII dimeric and monomeric core complexes. 6 lg of Chl was loaded

for each sample

Photosynth Res (2010) 103:19–30 27

123

Discussion

To get a generalized view of the effects of PG on the

functions and structure of photosystems in prokaryotic

photosynthetic organisms, a particular mutant strain, Syn-

echococcus sp. PCC 7942/DcdsA, was constructed, and

subjected to studies as outlined below.

Effect of PG depletion on the growth and pigment

composition of Synechococcus sp. PCC 7942/DcdsA

cells

Inactivation of the cdsA gene gave rise to a Synechococcus

sp. PCC 7942/DcdsA mutant defective in PG synthesis. The

growth of mutant cells was suppressed upon depletion of

PG from the cells. However, similar to other mutants

defective in PG synthesis (Hagio et al. 2000; Laczko-

Dobos et al. 2008), addition of PG to the medium made the

growth rate of mutants indistinguishable from that of the

WT, suggesting that the cells require a continuous supply

of PG for photoautotrophic growth. The pigment compo-

sition of cells was altered by PG depletion. Similar to

Synechocystis sp. PCC 6803/DpgsA, the Chl content

gradually decreased simultaneously with a decrease in PG

content of the cells, although the phycobiliprotein content

did not change significantly. The growth of Synechococcus

sp. PCC 7942/DcdsA slowed down and stopped earlier than

that of Synechocystis sp. PCC 6803/DpgsA. This demon-

strates that PG depletion has a more severe effect on cel-

lular functions in Synechococcus sp. PCC 7942 than in

Synechocystis sp. PCC 6803 cells. While the Synechocystis

sp. PCC 6803 mutant defective in PG synthesis stopped

growing after 17 days of PG depletion, this occurred earlier

(7 days after PG depletion) with Synechococcus sp. PCC

7942/DcdsA cells. The recovery of PG-depleted Synecho-

cystis sp. PCC 6803/DpgsA was superior to that of Syn-

echococcus sp. PCC 7942/DcdsA. Following 23 days of PG

depletion the readdition of PG led to an incomplete

recovery.

While complete recovery occurred even after 21 days of

PG depletion corresponding to four cell divisions of Syn-

echocystis sp. PCC 6803 mutant cells, the Synechococcus

sp. PCC 7942 mutant cells could not recover after 12 days

of PG depletion which corresponds to two cell divisions.

Effect of PG depletion on the lipid content and fatty

acid composition of PG in DcdsA cells

Decrease in PG content of Synechococcus sp. PCC 7942/

DcdsA cells was followed by gas chromatography. Fol-

lowing two cell divisions, the PG content of the cells

decreased to one-fourth of the original value. The decrease

of PG was compensated for by an increase in SQDG

content of Synechococcus sp. PCC 7942/DcdsA cells.

Earlier it has been postulated that in photosynthetic

organisms loss in PG following severe phosphate limitation

can be balanced by an increase in SQDG content (Yu and

Benning 2003). PG can be substituted by SQDG ensuring a

constant sum of anionic charged lipids in the cells. Sulfur-

starvation concomitantly upregulated PG synthesis and this

could compensate for a decrease in SQDG content

(Sugimoto et al. 2008). Our analysis of fatty acid content

showed that the exogenously added dioleoyl-phosphati-

dylglycerol was converted to more essential PG which

contains palmitic acid at position sn-2 (Murata et al. 1992).

The oleic acid esterified to the sn-1 position of the glycerol

backbone was replaced by a stearyl residue that normally

binds to this position (Murata et al. 1992). This retailoring

process was earlier detected in Synechococcus sp. PCC

6301 as a result of nitrate starvation (Gombos et al. 1987).

Effect of PG depletion on the synthesis and assembly

of PSII protein subunits

Protein analysis of Synechococcus sp. PCC 7942/DcdsA by

2D-BN/SDS-PAGE supported our earlier results on the

effect of PG depletion on the protein content of PAL/

DcdsA (Laczko-Dobos et al. 2008). PG depletion leads to

conversion of both the dimeric form of PSII and the tri-

meric form of PSI into their respective monomer forms.

This is a sign of a general destabilization of RC oligomers.

However, Takahashi and coworkers suggested that in intact

cells PSII exists in monomeric form (Takahashi et al.

2009). Formation of PSII RCs was suppressed and it

occurred much earlier in PG-depleted Synechococcus sp.

PCC 7942/DcdsA than in PG-depleted Synechocystis sp.

PCC 6803/DpgsA cells. This suggests that PG is a more

important partner of protein subunits for RC oligomer

formation in Synechococcus sp. PCC 7942 than in Syn-

echocystis sp. PCC 6803. In addition, the finding of the

majority of newly synthesized D1 among the non-assem-

bled thylakoid proteins supports the view that PG is also

necessary for a correct incorporation of membrane proteins

into the complexes.

Effect of PG depletion on photosynthetic activities

PG depletion affects photosynthetic processes in both

cyanobacterial species studied so far. In Synechocystis sp.

PCC 6803/DpgsA defective in PG synthesis the oxygen-

evolving activity may decrease to 60% of its original value

following 9 days of PG depletion (Hagio et al. 2000).

Suppression of photosynthetic activity was even more

extensive in Synechococcus sp. PCC 7942/DcdsA in which

an 11-day-long PG depletion was enough to decrease

photosynthetic activity to about 10% of its original value.

28 Photosynth Res (2010) 103:19–30

123

Whereas the effects of a 21-day-long PG depletion were

reversible in Synechocystis sp. PCC 6803/DpgsA cells, in

Synechococcus sp. PCC 7942/DcdsA cells 14 days of PG

depletion were sufficient to inhibit photosynthetic pro-

cesses irreversibly and readdition of PG to the cells did not

restore photosynthesis. Apparently, SQDG, another anionic

lipid constituent of photosynthetic membranes is not an

obligatory component of the photosynthetic machinery.

The SQDG-deficient mutant of Synechococcus sp. PCC

7942 grew without exogenously added lipid and had active

PSII without SQDG (Aoki et al. 2004; Guler et al. 2000).

Since the increase in SQDG content on the expense of PG

content could not restore photosynthetic activities in Syn-

echococcus sp. PCC 7942/DcdsA cells, we conclude that

PG is indispensably needed for this photosynthetic organ-

ism. The result suggests that maintaining the constant sum

of negatively charged lipids is not enough for the mainte-

nance of active electron transport processes.

We concluded as follows:

i. Synechococcus sp. PCC 7942/DcdsA is a mutant

defective in PG synthesis and is more susceptible to

PG depletion than Synechocystis sp. PCC 6803/DpgsA

or PAL/DcdsA.

ii. PG depletion affects the lipid composition of Syn-

echococcus sp. PCC 7942/DcdsA. The decrease in PG

content is counterbalanced by an increase in SQDG

content in the cells. Added artificial dioleoyl-PG was

transformed to essential PG species originally present

in the WT cells. However, neither the increased level

of SQDG nor the retailoring of fatty acid composition

can compensate for the negative effects of the reduced

PG content.

iii. The oligomerization of PSII and PSI was severely

perturbed by PG depletion in Synechococcus sp. PCC

7942/DcdsA following a short-term PG depletion. The

newly synthesized D1 protein was observed in the

non-assembled protein region of 2D gels showing the

importance of the lipid for the efficient assembly of

proteins into complexes.

iv. PG depletion resulted in a relatively rapid decrease in

PSII oxygen-evolving activity related to the pertur-

bation on the acceptor side of PSII while its donor side

was not primarily inactivated. In contrast to Synecho-

cystis sp. PCC 6803/DpgsA, perturbation of the PSII

acceptor site in Synechococcus sp. PCC 7942/DcdsA

is not caused by the PG-deficiency-induced inhibition

of electron transport between QA and QB, but seems to

be due to a decreased equilibrium constant for sharing

the electron between the QA- QB and QAQB

- states.

v. Synechococcus sp. PCC 7942/DcdsA provided a new

experimental system to support the importance of PG

in the correct formation of photosynthetic RCs and in

the functions of electron transport in an obligatory

photoautotrophic cyanobacterium.

Acknowledgments The authors thank Dr. Ghada Ajlani, CEA Sa-

clay, Gif-sur-Yvette, France, for her advice in preparing the cdsAplasmid construction, as well as for reading and correcting the man-

uscript. We are grateful to Prof. Ferenc Solymosy for reading and

correcting the manuscript. This work was supported by grants from

the Hungarian Science Foundation (OTKA; grant nos. T 60109 and T

68692), by the Ministry of Education, Youth and Sports of the Czech

Republic (project no. MSM6007665808) and by the Czech Academy

of Sciences (Institutional Research Concept no. AV0Z50200510,

Czech–Hungarian bilateral research priority project and project

IAA400200801).

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