Cyanobacterial hydrogenases: diversity, regulation and applications

R E V I E W A R T I C L E

Cyanobacterial hydrogenases:diversity, regulationandapplicationsPaula Tamagnini1,2, Elsa Leitao1, Paulo Oliveira3, Daniela Ferreira1,2, Filipe Pinto1, David James Harris4,5,Thorsten Heidorn3 & Peter Lindblad3

1IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal; 2Departmento de Botanica, Faculdade de Ciencias,

Universidade do Porto, Porto, Portugal; 3Department of Photochemistry and Molecular Science, The Angstrom Laboratories, Uppsala University,

Uppsala, Sweden; 4CIBIO/UP, Centro de Investigacao em Biodiversidade e Recursos Geneticos, Universidade do Porto, Vairao, Portugal; and5Departamento de Zoologia e Antropologia, Faculdade de Ciencias da Universidade do Porto, Porto, Portugal

Correspondence: Paula Tamagnini, IBMC –

Instituto de Biologia Molecular e Celular, Rua

do Campo Alegre, 823. 4150-180 Porto,

Portugal. Tel.: 1351 2260 74900; fax: 1351

2260 99157; e-mail: [email protected]

Received 5 January 2007; revised 12 July 2007;

accepted 9 August 2007.

First published online October 2007.

DOI:10.1111/j.1574-6976.2007.00085.x

Editor: Annick Wilmotte

Keywords

cyanobacteria; hydrogenase; hup ; hox ; hyp ;

transcriptional regulator.

Abstract

Cyanobacteria may possess two distinct nickel-iron (NiFe)-hydrogenases: an

uptake enzyme found in N2-fixing strains, and a bidirectional one present in both

non-N2-fixing and N2-fixing strains. The uptake hydrogenase (encoded by hupSL)

catalyzes the consumption of the H2 produced during N2 fixation, while the

bidirectional enzyme (hoxEFUYH) probably plays a role in fermentation and/or

acts as an electron valve during photosynthesis. hupSL constitute a transcriptional

unit, and are essentially transcribed under N2-fixing conditions. The bidirectional

hydrogenase consists of a hydrogenase and a diaphorase part, and the correspond-

ing five hox genes are not always clustered or cotranscribed. The biosynthesis/

maturation of NiFe-hydrogenases is highly complex, requiring several core

proteins. In cyanobacteria, the genes that are thought to affect hydrogenases

pleiotropically (hyp), as well as the genes presumably encoding the hydrogenase-

specific endopeptidases (hupW and hoxW) have been identified and characterized.

Furthermore, NtcA and LexA have been implicated in the transcriptional regula-

tion of the uptake and the bidirectional enzyme respectively. Recently, the

phylogenetic origin of cyanobacterial and algal hydrogenases was analyzed, and it

was proposed that the current distribution in cyanobacteria reflects a differential

loss of genes according to their ecological needs or constraints. In addition, the

possibilities and challenges of cyanobacterial-based H2 production are addressed.

Introduction

Cyanobacteria, one of the largest and most important

groups of bacteria on Earth, are able to perform oxygenic

photosynthesis using water as an electron donor and may be

found in almost any ecological niche from fresh to salt water,

terrestrial and extreme environments (Whitton & Potts,

2000). The knowledge on such a diverse group of prokar-

yotic organisms has greatly increased since cyanobacterial

genomes became available. In 1996, the entire sequence of

Synechocystis sp. PCC 6803 was published (Kaneko et al.,

1996; Nakamura et al., 1998), and since then, many other

cyanobacterial genome projects have been completed and

released, including that of Nostoc punctiforme ATCC 29133/

PCC 73102, one of the largest microbial genomes sequenced

so far (Meeks et al., 2001; Anderson et al., 2006).

Fossil traces of cyanobacteria are claimed to have been

found from around 3.5 billion years ago (Schopf, 2000), and

ancestors of cyanobacteria most probably played a key role

in the formation of atmospheric oxygen, and are thought

to have evolved into present-day chloroplasts of algae and

green plants (Miyagishima, 2005; Mulkidjanian et al., 2006).

Cyanobacteria display a relatively wide range of morpholo-

gical diversity, including unicellular, filamentous and colo-

nial forms. Some filamentous strains form differentiated

cells specialized in nitrogen fixation – heterocysts, and

spore-like resting cells – akinetes. A number of nonhetero-

cystous strains are also able to perform N2 fixation under

certain conditions. The fact that several cyanobacteria

are able to reduce nitrogen and carbon under aerobic

conditions may be responsible for their evolutionary and

ecological success. In cyanobacteria, as in any diazotrophic

FEMS Microbiol Rev 31 (2007) 692–720c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

bacteria, the reduction of N2 to NH3 is accompanied by the

formation of molecular hydrogen (Berman-Frank et al.,

2003). The H2 produced by the nitrogenase is rapidly

consumed by an uptake hydrogenase, an enzyme that has

been found in almost all the N2-fixing cyanobacteria exam-

ined so far, with one reported exception – Synechococcus sp.

BG 043511 (Ludwig et al., 2006). Additionally, these strains

may contain a bidirectional hydrogenase, an enzyme that is

generally present in the non nitrogen-fixing cyanobacteria

(Tamagnini et al., 2002, 2005), but absent in Gloeobacter

violaceus PCC 7421, a cyanobacterium that possesses a

number of unique characteristics such as the absence of

thylakoids (Nakamura et al., 2003; Ludwig et al., 2006). The

distribution of genes related to hydrogenases among repre-

sentative cyanobacterial strains is displayed in Table 1. Both

cyanobacterial hydrogenases are NiFe enzymes, which are

the most common hydrogenases found in bacteria and

Archaea. The core enzyme consists of an ab heterodimer

with the large/a subunit hosting the bimetallic active site,

and the small/b-subunit containing the FeS clusters, which

function as electron transfer domains between the electron

acceptors/donors and the catalytic center of the enzyme

(Fig. 1). In general, the NiFe hydrogenases are divided into

four groups, with the cyanobacterial uptake hydrogenases

clustering together with the cytoplasmic H2 sensors of group

2, and the bidirectional enzymes belonging to group 3

comprising the bidirectional heteromultimeric cytoplasmic

hydrogenases (for reviews on this subject, see Vignais et al.,

2001; Vignais & Colbeau, 2004).

In the present review, recent advances on cyanobacterial

hydrogenases, have been summarized focusing on achieve-

ments on the diversity and molecular regulation of both the

uptake and the bidirectional enzyme.

Photobiological production of H2 by microorganisms is

of great public interest because it promises a renewable

energy carrier from nature’s most plentiful resources: solar

energy and water. Cyanobacteria and green algae are the

only organisms known so far that are capable of both

oxygenic photosynthesis and hydrogen production. In a

separate section, the possibilities and challenges in cyano-

bacterial-based hydrogen production are outlined.

Uptake hydrogenase

The cyanobacterial uptake hydrogenase, found exclusively in

N2-fixing strains and encoded by the hup – hydrogen uptake

– genes, is at least a heterodimeric enzyme with a large

subunit of about 60 kDa containing the active site (HupL)

and a small subunit of c. 35 kDa playing a role in electron

transfer (HupS) (Fig. 1). Because the physiological and

biochemical data point to a membrane-bound enzyme

(Houchins & Burris, 1981b; Houchins, 1984; Lindblad &

Sellstedt, 1990; Rai et al., 1992), and the hydropathy profiles

of the HupL and the HupS proteins do not indicate any

transmembrane domains (Tamagnini et al., 2005), the

existence of a polypeptide that anchors the HupSL hetero-

dimer to the membrane seems likely. In fact, analysis of the

available genomes revealed the presence of ORFs whose

products could potentially fulfill this anchoring role (Lind-

berg, 2003). However, to date no definitive proof was

obtained, and the existence of both a soluble and a mem-

brane-bound form of the enzyme cannot be excluded (see

for e.g. Houchins & Burris, 1981b).

Immunolocalization studies, using antibodies produced

against hydrogenases from other bacteria, showed that the

hydrogenase antigens are present in both the vegetative cells

and heterocysts of N. punctiforme, and several symbiotic

Nostoc strains (Lindblad & Sellstedt, 1990; Rai et al., 1992;

Tamagnini et al., 1995). However, these studies do not

clarify whether the enzyme is in its active form in both

cell types. In Anabaena/Nostoc sp. PCC 7120, the uptake

hydrogenase activity was essentially associated with the

particulate fraction of the heterocysts (Houchins & Burris,

1981b); however, one must bear in mind that in this strain

the hupL gene undergoes a rearrangement, allowing its

expression in the heterocysts only, and that this process does

not occur in N. punctiforme (Oxelfelt et al., 1998). Moreover,

the presence/levels of the cyanobacterial uptake hydrogenase

are certainly dependent on the growth conditions. In

heterocystous cyanobacteria grown in air and without

combined nitrogen, the uptake hydrogenase activity is

mainly confined to heterocysts, where it is protected from

oxygen inactivation; however, the exact location of the

enzyme in cyanobacteria should be further investigated in

both heterocystous and nonheterocystous strains.

A strong correlation between the nitrogen-fixation pro-

cess and the uptake hydrogenase activity has been demon-

strated for cyanobacteria (Lambert & Smith, 1981;

Houchins, 1984; Wolk et al., 1994; Oxelfelt et al., 1995;

Schutz et al., 2004), and this indicates that the main

physiological function of the uptake hydrogenase is to

reutilize and regain the H2/electrons produced by the H2

evolution through the nitrogenase. This recycling has been

suggested to have at least three beneficial functions to the

organism: (1) it provides ATP via the oxyhydrogen reaction,

minimizing the loss of energy; (2) it removes the oxygen

from nitrogenase, thereby protecting it from inactivation;

and (3) it supplies reducing equivalents (electrons) to

various cell functions (Bothe et al., 1977, 1991; Howarth &

Codd, 1985; Weisshaar & Boger, 1985; Smith, 1990).

Physical organization of hup genes and thecorresponding proteins

The physical arrangement of the structural genes encoding

the uptake hydrogenase is very similar in all the

FEMS Microbiol Rev 31 (2007) 692–720 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

693Cyanobacterial hydrogenases

Tab

le1.

Dis

trib

ution

of

gen

esre

late

dto

hyd

rogen

ases

inre

pre

senta

tive

cyan

obac

terial

stra

ins

Org

anis

ms

Bid

irec

tional

hyd

rogen

ase

Upta

ke

hyd

rogen

ase

hupL

reco

mbin

ase

Bid

irec

tional

spec

ific

endopep

tidas

e

Upta

kesp

ecifi

c

endopep

tidas

e

Oth

er

mat

ura

tion

gen

es

Gen

Ban

k

acce

ssio

n

num

ber

/

Ref

eren

ces

hoxF

UY

HhoxE

hupSL

Xis

C�

hoxW

hupW

hyp

FCD

EAB

Unic

ellu

lar

non-N

2-fi

xing

G.vi

ola

ceus

PCC

7421

��

��

��

NC

_005125

Nak

amura

etal

.

(2003)

Synec

hocy

stis

sp.

PCC

6803

1 Appel

&Sc

hulz

(1996)

1�

1�

1 Scat

tere

d

NC

_000911

Kan

eko

etal

.

(1996)

Unic

ellu

lar

N2-fi

xing

C.w

atso

nii

WH

8501

��

1�

11 Sc

atte

red

NZ_

AD

V00000000

Fila

men

tous

nonhet

erocy

stousL.

maj

usc

ula

CC

AP

1446/4

1N

D1

ND

11 O

per

on

Leitao

etal

.(2

005,

2006)

N2-fi

xing

T.er

ythra

eum

IMS

101

��

1�

11

NC

_008312

Fila

men

tous

het

erocy

stous

N2-fi

xing

A.va

riab

ilis

ATC

C29413

1 Schm

itz

etal

.

(1995)

11 H

appe

etal

.(2

000)

�1

11

NC

_007413

Nost

oc

sp.

PCC

7120

11

1 Car

rasc

oet

al.(1

995)

11

11 G

ubili

&Bort

hak

ur

(1996,1998)

NC

_003272

Kan

eko

etal

.(2

001)

N.punct

iform

e

PCC

73102

��

1 Oxe

lfel

tet

al.(1

998)

��

11 O

per

on

Han

sele

tal

.(2

001)

NZ_

AA

AY

00000000

� Rea

rrag

emen

tocc

urr

ing

during

the

diffe

rentiat

ion

of

ave

get

ativ

ece

llin

toa

het

erocy

st.

ND

,not

det

erm

ined

.

FEMS Microbiol Rev 31 (2007) 692–720c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

694 P. Tamagnini et al.

cyanobacteria studied so far: hupS and hupL are contiguous,

with the gene encoding the smaller subunit located up-

stream from the gene encoding the larger one (Carrasco

et al., 1995; Oxelfelt et al., 1998; Happe et al., 2000; Lindberg

et al., 2000; Oliveira et al., 2004; Leitao et al., 2005) (Fig. 2).

Transcriptional start sites have been identified upstream of

the hupS start codon (Happe et al., 2000; Lindberg et al.,

2000; Oliveira et al., 2004; Leitao et al., 2005), and a putative

transcriptional terminator, located immediately down-

stream of hupL, has been found in N. punctiforme (Lindberg

et al., 2000). In agreement, reverse transcriptase (RT)-PCR

experiments, and the sizes of transcripts determined by

Northern blot, indicate that hupSL constitute a transcrip-

tional unit in Anabaena variabilis ATCC 29413, N. puncti-

forme and Lyngbya majuscula CCAP 1446/4 (Happe et al.,

2000; Lindberg et al., 2000; Leitao et al., 2005). In the

unicellular Gloeothece sp. ATCC 27152 and in the filamen-

tous Trichodesmium erythraeum IMS 101 hupW – the gene

encoding for the putative uptake hydrogenase-specific endo-

peptidase – is the ORF located immediately downstream of

hupL, and was shown to be cotranscribed with hupSL in

Gloeothece sp. ATCC 27152 (Oliveira et al., 2004). In other

strains, the position of hupW related to the hupSL varies

considerably, and in the strains examined they are tran-

scribed independently (Wunschiers et al., 2003) (Fig. 2).

Analysis of the predicted proteins encoded by the hupSL

operon demonstrated that whereas HupS has the same

number of amino acid residues in all the cyanobacteria

investigated [320 amino acids (aa)], HupL generally has

531 aa with the exception of the filamentous nonheterocys-

tous L. aestuarii CCY 9616 (six extra), L. majuscula (six extra),

and T. erythraeum (three extra). To date, the physiological

significance (if any) of these extra residues is still unknown.

In the NiFe hydrogenases, the large subunit harbors the

active center that is deeply buried inside the protein, close to

the large interface between the two subunits, and the small

subunit contains the FeS clusters that conduct electrons

between the active center and the physiological electron

acceptor/donor (Vignais et al., 2001; Vignais & Colbeau,

2004). In concordance, the cyanobacterial HupL sequences

contain the four conserved cysteine residues that are in-

volved in the coordination of the bimetallic NiFe center of

the active site, and HupS contains eight cysteine residues

clearly corresponding to those involved in the formation of

the FeS clusters, and a ninth cysteine slightly shifted

compared with other bacteria (Tamagnini et al., 2002). In

addition, HupL contains the C-terminal region that is

presumably cleaved off, by a specific endopeptidase, as the

last step of the maturation of the large subunit. In contrast

with other organisms, HupS lacks both the twin-arginine

HupLHupL

NifKNifK NifDNifD

NifDNifD

NifHNifH

NifHNifH NifKNifK

Dinitrogenasereductase Dinitrogenase

Uptake hydrogenaseUptake hydrogenase

NitrogenaseNitrogenase

NH

N2+ H+

HH22

2H++ 2e−

2H++ 2e−

e−

HupSHupS

HupCHupC??

Hydrogenase

HoxHHoxH

HoxYHoxY HoxFHoxF

HoxEHoxE

Diaphorase

BiBi--directional hydrogenasedirectional hydrogenase

HH22 HoxUHoxU

NAD+

NADH

Hox(EFUYH)2

HupLHupL

NifKNifK NifDNifD

NifDNifD

NifHNifH

NifHNifH NifKNifK

NifKNifK NifDNifD

NifDNifD

NifHNifH

NifHNifH NifKNifK

β α

α β

Uptake hydrogenaseUptake hydrogenase

NitrogenaseNitrogenase

NH

HH22

HupSHupS

HupCHupC??

HupCHupC??

HoxHHoxH

HoxYHoxY HoxFHoxF

HoxEHoxE

BiBi--directional hydrogenasedirectional hydrogenase

HH22 HoxUHoxU

Hox(EFUYH)2

HoxHHoxH

HoxYHoxY HoxFHoxF

HoxEHoxE

BiBi--directional hydrogenasedirectional hydrogenase

HH22 HoxUHoxU

Hox(EFUYH)2

Fig. 1. Enzymes directly involved in hydrogen metabolism in cyanobacteria. While the uptake hydrogenase is present in most of the nitrogen-fixing

strains tested (with only one exception reported so far; see text and Table 1), the bidirectional enzyme seems to be present in non-N2-fixing and N2-fixing

strains but is not a universal enzyme. The existence of a third subunit (HupC) anchoring the uptake hydrogenase to the membrane is yet to be

confirmed, and the molecular weight of the native bidirectional hydrogenase indicates a dimeric assembly of the enzyme complex Hox(EFUYH)2.

FEMS Microbiol Rev 31 (2007) 692–720 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

695Cyanobacterial hydrogenases

1000 bpGloeothece sp. ATCC 27152

S L

hup

W

Trichodesmium erythraeum IMS101

S L

hup

W

Tery0800Tery0790Tery0799Tery0801

FDE CAB

hyp

~ 589 kb

Nostoc punctiforme ATCC 29133 / PCC 73102

FDEB A C

hyp

S L

hup

W

NpR0370NpR0363NpR0364

NpR0366

NpF0371

NpR0365NpF0372

NpF0373

NpR0367

Lyngbya majuscula CCAP 1446/4

FDEB A C

hyp

S L

hup

W

ORF1 ORF3ORF2

ORF4 ORF5 ORF6 ORF7ORF8

ORF9ORF10

ORF11

Nostoc sp. PCC 7120

FDE

alr0691

SB

alr0692 all0675

A

asr0689

C

asr0690

~ 880 kb

alr0693

L

hyp hup

W

hup

asr0697

(a)

Fig. 2. Organization of the loci containing the genes encoding (a) the uptake hydrogenase (hup) and (b) the bidirectional hydrogenase (hox) in selected

cyanobacterial strains (black ORFs). The accessory genes (hyp, hupW and hoxW), encoding proteins involved in the maturation of the hydrogenases are

also depicted, as gray ORFs, as well as some additional ORFs (identified, when available, with the corresponding ORF-number in respective annotated

genomes, and shown as white ORFs). Gloeothece sp. ATCC 27152 (Oliveira et al., 2004 – GenBank accession no. AY260103), Trichodesmium

erythraeum IMS101 (http://genome.jgi-psf.org/finished_microbes/trier/trier.home.html), Lyngbya majuscula CCAP 1446/4 (Leitao et al., 2005 –

GenBank accession no. AF368526), Nostoc punctiforme ATCC 29133/PCC 73102 (http://genome.jgi-psf.org/draft_microbes/nospu/nospu.home.html),

Nostoc sp. PCC 7120 (Kaneko et al., 2001), Synechocystis sp. PCC 6803 (Kaneko et al., 1996), Synechococcus elongatus PCC 7942 (http://genome.

jgi-psf.org/finished_microbes/synel/synel.home.html), Arthrospira platensis FACHB341 (Zhang et al., 2005a, b – GenBank accession nos. DQ309870

and AY345594) and Anabaena variabilis ATCC 29413 (http://genome.jgi-psf.org/finished_microbes/anava/anava.home.html).

FEMS Microbiol Rev 31 (2007) 692–720c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

696 P. Tamagnini et al.

signal peptide at the N-terminal, and the hydrophobic motif

at the C-terminal proposed to be involved in translocation

and anchorage to the membrane, respectively. As mentioned

previously, these general features of the cyanobacterial

hydrogenases cluster them together with the soluble H2-

sensing enzymes (Vignais et al., 2001; Vignais & Colbeau,

2004). However, the construction of hup� mutants proved

that the cyanobacterial uptake hydrogenase is indeed a

Synechocystis sp. PCC 6803

hox

E F U Y H WF DE A2B2C

hyp

B1A1

sll1222 ssl2420

sll1225

~ 192 kb ~743 kb~ 236 kb~ 445 kb~ 79 kb~ 150 kb~ 47 kb~ 477 kb

hyp

1000 bp

Synechococcus elongatus PCC 7942

hox

EDEF

hyphox

U HY W

hyp

AB F Cbk 685 ~bk 433 ~ ~ 172 kb

Arthrospira platensis FACHB 341

unknown

hox

E F U Y H

Anabaena variabilis ATCC 29413

F D E BAC

hyp

Ava4652

~ 52 kb

hox

E F U Y H W

Ava4655

Ava4656

Ava4658

Ava4660

Ava4662 Ava4664

Ava4663

Ava4605

Nostoc sp. PCC 7120

~ 8.8 kb

hox

E F U Y H W~ 65 kb

F D E BAC

hyp

9670lla7670lla5670rla3670rla0570rla

all0768asl0749

asr0697

(b)

Fig. 2. Continued

FEMS Microbiol Rev 31 (2007) 692–720 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

697Cyanobacterial hydrogenases

physiological functional enzyme rather than a regulatory

one (Happe et al., 2000; Lindberg et al., 2002; Lindblad et al.,

2002; Masukawa et al., 2002).

hupL rearrangement in heterocystous strains

Programmed DNA rearrangements have been described in

eukaryotes and prokaryotes but are relatively uncommon

events. In cyanobacteria, developmentally regulated DNA

rearrangements have been reported to occur in heterocys-

tous strains (for a review, see Golden, 1997). Generally, the

ORF is interrupted in the vegetative cells by a 10–60-kb

DNA element, which is excised during the differentiation of

a photosynthetic vegetative cell into a N2-fixing heterocyst,

restoring the structure of the gene/operon and allowing its

expression in heterocysts only.

The rearrangement within hupL (large subunit of the

uptake hydrogenase) was first described for Nostoc sp. PCC

7120 (Carrasco et al., 1995). In the vegetative cells of this

cyanobacterium, hupL is interrupted by a 9.5-kb element

that is excised late during the heterocyst differentiation

process by a site-specific recombination between the 16-bp

direct repeats that flank the element (Fig. 3). The hupL

element contains, in one of its borders, the gene that encodes

the recombinase necessary for the excision – xisC (Carrasco

et al., 1995, 1998, 2005). Site-directed mutagenesis revealed

that the XisC protein has a functional similarity to the phage

integrase family of recombinases. Recently, it has been

unequivocally demonstrated that the inactivation of xisC

blocks the hupL rearrangement and that XisC alone is

sufficient to catalyze the hupL element site-specific recom-

bination in Nostoc sp. PCC 7120 (Carrasco et al., 2005). It

was also shown that the xisC-mutant forms heterocysts

without any obvious developmental defects and that the

mutant grown under N2-fixing conditions (BG110) was not

only defective for hydrogen uptake activity but evolves

H2 (Lindblad et al., 2002; Carrasco et al., 2005). Moreover,

Lindblad et al. (2002) showed that, in a competitive growth

environment with increased light intensity, the wild-type

strain has an advantage over the xisC-mutant, probably

because these specific conditions induced higher rates of

H2 evolution that only the wild type has the capacity of

reutilizing through the oxyhydrogen reaction. These find-

ings support the hypothesis that the uptake hydrogenase

plays a role in minimizing the loss of energy caused by the

nitrogenase-dependent H2 formation.

Despite the hupL element being absent from the two other

heterocystous strains for which genome sequences are avail-

able, A. variabilis and N. punctiforme (see also Oxelfelt et al.,

1998; Happe et al., 2000), DNA hybridization studies

showed that sequences similar to xisC were present in about

half of the heterocystous strains tested (Tamagnini et al.,

2000). These authors also showed that the presence of the

bidirectional hydrogenase is not ubiquitous among hetero-

cystous cyanobacteria, although they could not establish a

correlation between the presence/absence of the bidirec-

tional enzyme and hupL rearrangement.

hupSL intergenic region

The regions between hupS and hupL in cyanobacteria are

longer than in other microorganisms, differ considerably in

Vegetative cell Heterocyst

hupS hupL

9.5 kb ?

enzyme

?

9.5 kb elementcontaining xisC

5´ 3´hupLhupS

Uptake hydrogenase enzyme

hupS hupL

9.5 kb ?

Uptake hydrogenaseenzyme

?

hupS hupLhupS hupLhupS hupL

5´ 3´hupLhupS

Fig. 3. Schematic representation of the hupL rearrangement occurring in Nostoc sp. PCC 7120 and other heterocystous cyanobacteria (adapted from

Carrasco et al., 2005). In the vegetative cells, hupL is interrupted by a DNA element that is excised late during the heterocyst differentiation process by a

site-specific recombination. Subsequently, the structure of the hupL gene is restored, allowing its expression in the heterocysts only. The destiny of the

9.5-kb excised element is currently unknown. In aerobically grown filaments of Nostoc sp. PCC 7120, most of the uptake hydrogenase activity is

recovered in the membrane fraction of heterocysts (Houchins & Burris, 1981b). The question marks represent events that have not been elucidated so

far: the fate of the excised DNA element, and the attachment of the uptake hydrogenase to a cell membrane.

FEMS Microbiol Rev 31 (2007) 692–720c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

698 P. Tamagnini et al.

size (ranging from 43 to 689 bp; see Table 2) and are not

particularly conserved (except for Nostoc sp. PCC 7120 and

A. variabilis). A prominent feature within the hupSL inter-

genic region of heterocystous strains is the presence of Short

Tandemly Repeated Repetive (STRR) sequences (with the

exception of the relatively short 43-bp region of Nostoc sp.

Mitsui 38901). STRR sequences have previously been shown

to be frequent in heterocyst-forming cyanobacteria and

relatively less frequent in unicellular strains (Asayama et al.,

1996). Indeed, no STRR sequences could be discerned in the

hupSL intergenic region from nonheterocystous cyanobac-

teria. However, in the filamentous nonheterocystous L.

majuscula only about 10% of the intergenic region consists

of nonrepetitive nucleotides, with two distinct sets of Long

Repeated Repetitive (LRR) sequences clearly identified (for

details see Leitao et al., 2005). Because the repetitive

sequences within the hupSL intergenic region are highly

variable or even absent (Table 2), it is unlikely that these

repeats play a direct role in the regulation of gene expres-

sion. However, in all strains, a putative stem-loop structure,

derived via 2D-computer modeling, might occur in the

transcribed RNA (Lindberg et al., 2000; Tamagnini et al.,

2002, 2005). The value of free energy (DG) was determined

for each secondary structure and it was negative in all cases

(ranging from � 136.32 to � 6.9 kcal mol�1), meaning that

the formation of the hairpin is favored. It has been hypothe-

sized that the occurrence of the hairpin may increase the

stability of the transcript, and/or confer a translational

coupling between hupS and hupL by sequestering the ribo-

some-binding site of hupL and thereby preventing the

initiation of translation of this gene (Lindberg et al., 2000).

However, although the sequestration of the hupL RBS may

be effective in N. punctiforme in which the hairpin folds the

entire hupSL intergenic region (Lindberg et al., 2000), it

does not occur in all hupSL intergenic hairpin structures

predicted. Only the construction of specific mutants will

help to clarify the function of these intergenic regions.

hup promoter regions and transcriptionalregulators

As mentioned previously, in all cyanobacteria studied so far

the uptake hydrogenase structural genes are arranged in a

contiguous manner with the gene encoding the smaller

subunit located upstream of the gene of the larger one. The

transcriptional start sites of the hup operons are localized

238, 59, 103 and 259 bp upstream from the hupS start codon

for the unicellular Gloeothece sp. ATCC 27152, the filamen-

tous L. majuscula and the filamentous heterocystous A.

variabilis and N. punctiforme, respectively (Happe et al.,

2000; Lindberg et al., 2000; Oliveira et al., 2004; Leitao et al.,

2005) (Fig. 4). The analysis of the regions upstream the

transcriptional start point (tsp) revealed the presence

of a � 10 and a � 35 box in both L. majuscula and

N. punctiforme, while in Gloeothece sp. ATCC 27152 and A.

variabilis only a � 10 box could be clearly discerned. A putative

Table 2. Size and occurrence of repetitive sequences within the region between of hupS and hupL in cyanobacteria

Organism Size (bp)

Repetitive

sequences

GenBank accession

number/Reference

Unicellular

Crocosphaera watsonii WH 8501 67 No NZ_AADV02000237

Cyanothece sp. ATCC 51142 126 No DQ650318

Gloeothece sp. ATCC 27152 259 No AY260103 Oliveira et al. (2004)

Filamentous nonheterocystous

Lyngbya aestuarii CCY 9616 118 No DQ375444

Lyngbya majuscula CCAP 1446/4 643 LRR AF368526 Leitao et al. (2005)

Trichodesmium erythraeum IMS 101 689 No NZ_AABK04000005

Filamentous heterocystous

Anabaena siamensis TISTR8012 195 STRR AY152844

Anabaena variabilis ATCC 29413 75 STRR Y13216; NC_007413

Happe et al. (2000).

Nostoc HCC 1048 (Mitsui 38901) 43 No AF455566

Nostoc HCC 1061 (Mitsui 56111) 118 STRR AF455567

Nostoc HCC 1075 (Mitsui 91911) 97 STRR AF455568

Nostoc sp. PCC 7120 68 STRR U08013; NC_003272

Carrasco et al. (1995), Kaneko et al. (2001)

Nostoc sp. PCC 7422 144 STRR AB237640

Nostoc muscorum CCAP 1453/12 68 STRR AF455565 Oxelfelt (1998)

Nostoc punctiforme PCC 73102 192 STRR AF030525; NZ_AAAY02000001

Oxelfelt et al. (1998)

LRR, long repeated repetitive; STRR, short tandemly repeated repetitive.

FEMS Microbiol Rev 31 (2007) 692–720 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

699Cyanobacterial hydrogenases

binding site for NtcA (a protein that operates global nitro-

gen control in cyanobacteria) could be found in Gloeothece

sp. ATCC 27152, L. majuscula and N. punctiforme, although

its relative position to the tsp varied depending on the

strain. Moreover, in L. majuscula and N. punctiforme a

possible binding site for the integration host factor (IHF) –

WATCAAN4TTR (Craig & Nash, 1984; Goodrich et al.,

1990; Goodman et al., 1999) – could be recognized in the

region between the NtcA motif and the tsp (Fig. 4). It has

been postulated that the possible binding of the IHF to the

promoter could bend the DNA (Friedman, 1988), and

consequently allow the contact of the NtcA with the RNA

polymerase complex, activating the hupSL transcription. In

the unicellular Gloeothece sp. ATCC 27152, the potential

NtcA-binding site is centered at � 41.5 bp with respect to

the tsp in place of the � 35 box, like in the canonical NtcA-

activated promoters with the consensus sequence signature

GTAN8TAC (Herrero et al., 2001), a structure similar to that

of class II bacterial promoters activated by catabolite acti-

vator protein (CAP). In L. majuscula and N. punctiforme, the

NtcA-binding sites were found to be centered at positions

� 233.5 and � 258.5, respectively, resembling class I CAP-

dependent promoters (Busby & Ebright, 1999; Herrero

et al., 2001, 2004). These data indicate that the type of the

NtcA-activated promoter (class I vs II) is not correlated to

the strategies used by heterocystous and nonheterocystous

cyanobacteria to separate N2 fixation and photosynthesis. In

the filamentous heterocystous A. variabilis, half of a se-

quence motif identical to the consensus Fnr-binding se-

quence was identified 144-bp upstream of the tsp (Happe

et al., 2000) (Fig. 4). Fnr is a regulator of a fumarate nitrate

reductase, which has been found to be involved in the

regulation of the hyp operon in Escherichia coli (Lutz et al.,

1991), and it is responsible for the induction of several

operons in E. coli grown under anaerobic conditions (Spiro

& Guest, 1990). In A. variabilis, although there is no

rearrangement of the hupL gene, hupSL are expressed in

heterocysts only. These differentiated cells have very low

intracellular O2 pressures which led Happe et al. (2000) to

suggest that the hupSL operon in A. variabilis could be

regulated in a manner similar to that of the anaerobically

induced operons in E. coli.

hyp

NtcA2 NtcA1

hox

Synechocystis sp. PCC 6803

LexA2 LexA1LexA2 LexA1

100 bp

Lyngbya majuscula CCAP 1446/4

hypFNtcA2 NtcA1

LexA

+1

17 bp

+1

ORF

Nostoc punctiforme PCC 73102

21 bp

hoxE

+1

−35 −10−35 −10

−35 −10

−35 −10

−10

−10

−10

168 bp

hup

ATCC 29413

hupS

+1

+1

Fnr103 bp

Gloeothece sp. ATCC 27152

hupS

+1+1

NtcA238 bp

Lyngbya majuscula CCAP 1446/4

hupSNtcA IHF

+1

59 bp

hupSNtcA IHF

Nosctoc punctiforme PCC 73102

259 bp

Anabaena variabilis

hupS

Fig. 4. Promoter regions upstream of hupS, hoxE and hypF in cyanobacteria. The following regions are highlighted: putative NtcA-, IHF-, Fnr- and LexA-

binding sites, the � 10 and � 35 boxes and the transcriptional start points (11). The following ORFs are not to scale. In Nostoc punctiforme, the ORF

represented here is immediately upstream of hypF and in the same direction. Analysis of the available genomes revealed the presence of homologues of

this ORF, in the same position and direction, in other filamentous cyanobacteria, and the encoded proteins can be assigned to COG0583 that includes

transcriptional regulators from the LysR family (Leitao et al., 2006). In Synechocystis sp. PCC 6803 hox promoter region, the two putative pairs of LexA-

binding motifs were identified by two different groups (Gutekunst et al., 2005; Oliveira & Lindblad, 2005).

FEMS Microbiol Rev 31 (2007) 692–720c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

700 P. Tamagnini et al.

The possible interaction between NtcA and the hupSL(W)

promoter regions in cyanobacteria was assessed by perform-

ing band shift assays. These experiments indicate a specific

binding of NtcA to DNA sequences upstream of hupS in the

three cyanobacterial strains tested (Gloeothece sp. ATCC

27152, L. majuscula and N. punctiforme), suggesting, indeed,

the involvement of NtcA in the transcription regulation of

the uptake hydrogenase gene cluster (Lindberg, 2003;

Oliveira et al., 2004; Leitao et al., 2005). The fact that the

transcription of the uptake hydrogenase structural genes

is under the control of the transcriptional regulator that

operates global nitrogen control in cyanobacteria reinforces

the correlation observed between the activity of the

uptake hydrogenase and N2 fixation, already demonstrated

in several filamentous heterocystous cyanobacteria (Hou-

chins, 1984; Wolk et al., 1994; Oxelfelt et al., 1995; Troshina

et al., 1996).

Transcription and expression patterns ofhup genes

The first transcriptional data on cyanobacterial uptake

hydrogenases arose from RT-PCR experiments on Nostoc

sp. PCC 7120, revealing that hupL is expressed only after a

photosynthetic vegetative cell differentiates into a N2-fixing

heterocyst (see above details about the DNA rearrangement

occurring within this strain, Carrasco et al., 1995, 2005).

Subsequent studies with other filamentous heterocystous

strains have shown that hupSL is a transcriptional unit

(Happe et al., 2000; Lindberg et al., 2000), present in cells

grown under N2-fixing conditions (Axelsson et al., 1999;

Happe et al., 2000; Hansel et al., 2001). Non-N2-fixing

cultures of Nostoc muscorum, a strain without the hupL

rearrangement, exhibit no in vivo H2-uptake activity (Ax-

elsson et al., 1999). However, the transfer of N. muscorum

cells from non-N2-fixing (ammonia) to N2-fixing condi-

tions induced the appearance of a transcript (after c. 24 h),

and the relative amounts of transcript increased in parallel

with the H2-uptake activity (Axelsson et al., 1999). A similar

pattern of transcription was observed for A. variabilis

and N. punctiforme, two other strains with noninterrupted

hupL genes (Happe et al., 2000; Hansel et al., 2001). These

authors demonstrated that hupSL transcripts were

missing in A. variabilis and in N. punctiforme cells grown

with ammonia (and in A. variabilis cells grown with

nitrate), but were present in both organisms grown under

N2-fixing conditions.

While the heterocyst provides a microaerobic environ-

ment protecting the oxygen-sensitive nitrogenases and up-

take hydrogenases from the atmospheric and intracellulary

generated oxygen, the nonheterocystous cyanobacteria de-

veloped different approaches. The temporal separation

between photosynthesis (light) and nitrogen-fixation/

hydrogen uptake (dark) seems to be the most common

strategy adopted by the later cyanobacteria (Bergman et al.,

1997; Bohme, 1998; Berman-Frank et al., 2003). In fact, in

the nonheterocystous Gloeothece sp. ATCC 27152 (unicellu-

lar) and L. majuscula (filamentous), grown under nitrogen-

fixing conditions and 12 h light/12 h dark cycles, there is an

evident light/dark regulation with the highest levels of

hupSL(W) transcripts detected during the light phase or in

the transition between the light and dark phase, respectively

(Oliveira et al., 2004; Leitao et al., 2005). It has also been

demonstrated that both organisms exhibit higher hydrogen-

uptake activities during the dark period (in agreement with

the nitrogen fixation rates; see Reade et al., 1999; Lundgren

et al., 2003). In L. majuscula, the increase of the HupL

protein levels coincides with the increase of hydrogenase

uptake activity during the dark phase. In the beginning of

the light phase, no hupSL transcription is detectable, and the

levels of both polypeptides and H2 uptake activity begin to

decline (Leitao et al., 2005). These results suggest that in

L. majuscula, a protein turnover occurs, with degradation

taking place during the light period and de novo synthesis

taking place during the dark phase. The time difference

between the hupSL transcription and the hydrogen uptake

activity, both in Gloeothece sp. ATCC 27152 and L. majuscu-

la, might be due to the complexity of the maturation process

of the uptake hydrogenase. Thus, it is possible that the

translation occurs as soon as the transcript is available, while

the enzyme becomes active only after the maturation

process is completed. The temporal separation between

the photosynthesis and nitrogen fixation/hydrogen uptake

activity may also influence the time lag between transcrip-

tion and activity.

In the presence of combined nitrogen, hupSLW transcrip-

tion is totally repressed in Gloeothece sp. ATCC 27152, while

in L. majuscula the levels of hupSL transcription and

expression are significantly reduced but it is possible to

discern a pattern similar to the one observed in cells grown

under N2-fixing conditions (Oliveira et al., 2004; Leitao

et al., 2005, Ferreira et al., 2007). The results obtained for

L. majuscula under non-N2-fixing conditions could be ex-

plained by the mode of growth of this cyanobacterium, in

which the inner cells are probably not in the same conditions

notably in terms of access to the combined nitrogen.

Besides the source of nitrogen, other factors were proven

to influence the transcription/expression of the cyanobac-

terial uptake hydrogenases. Similar to any NiFe hydroge-

nase, the activity of the cyanobacterial uptake enzyme was

shown to be dependent on nickel availability, and the

addition of external nickel to the growth medium (up to a

certain concentration) increased the uptake hydrogenase

activity in several strains (Xiankong et al., 1984; Daday

et al., 1985; Kumar & Polasa, 1991; Oxelfelt et al., 1995;

Axelsson & Lindblad, 2002). Furthermore, the addition of

FEMS Microbiol Rev 31 (2007) 692–720 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

701Cyanobacterial hydrogenases

exogenous hydrogen was shown to induce hupSL transcrip-

tion and hydrogen uptake activity in N. muscorum and N.

punctiforme (Oxelfelt et al., 1995; Axelsson & Lindblad,

2002), as well as hydrogen uptake activity in Nostoc sp.

PCC 7120 (Houchins & Burris, 1981b). Both cyanobacterial

hydrogenases are affected by the oxygen partial pressure.

Nostoc muscorum and N. punctiforme cultures transferred

from aerobic to anaerobic conditions showed an increase in

both the transcription of hupL and hydrogen uptake activity

(Axelsson & Lindblad, 2002). Similarly, the uptake hydro-

genase activity could be elicited by removing oxygen from

the sparging gas of a culture of Nostoc sp. PCC 7120

(Houchins & Burris, 1981b). The addition of organic carbon

to the culture medium can also influence the hydrogen

uptake activity. Cells of N. punctiforme grown either photo-

or chemoheterotrophically reach both higher nitrogenase

and hydrogen uptake activities than photoautotrophically

grown cells (Oxelfelt et al., 1995). However, the effect of

carbon substrates on the cyanobacterial uptake hydrogenase

activity is difficult to assess, and apparently contradictory

results are reported in the literature (Houchins, 1984;

Kumar et al., 1986; Chen et al., 1989; Margheri et al., 1991).

Bidirectional hydrogenase

The soluble or loosely membrane associated cyanobacterial

bidirectional hydrogenase might be present in both N2- and

non-N2-fixing strains (Tamagnini et al., 2000, 2002). Initi-

ally, the bidirectional hydrogenase was thought to be com-

posed of four subunits (encoded by the hox – hydrogen

oxidation – genes), in which HoxFU constitute the diaphor-

ase part, and HoxYH constitute the hydrogenase part

(Schmitz et al., 1995; Appel & Schulz, 1996; Boison et al.,

1996, 1998; Sheremetieva et al., 2002). However, because

HoxE was shown to copurify with the active bidirectional

enzyme, the cyanobacterial bidirectional hydrogenase is

considered to be a heteropentameric enzyme encoded by

hoxEFUYH, HoxE belonging to the diaphorase part

(Schmitz et al., 2002). Bidirectional hydrogenases with more

than four subunits have also been identified in other

bacteria, such as the photosynthetic purple sulfur bacteria

Thiocapsa roseopersicina and Allochromatium vinosum which

contain heteropentameric cyanobacterial-type bidirectional

hydrogenases (Rakhely et al., 2004; Long et al., 2007), and

Ralstonia eutropha, which possess two HoxI subunits besides

HoxFUYH (Burgdorf et al., 2005). In recent years, the

number of reports showing the presence of a functional

and active bidirectional hydrogenase in cyanobacteria has

increased significantly, ranging from unicellular strains

(Gloeocapsa alpicola CALU 743 – Sheremetieva et al.,

2002; Troshina et al., 2002) to filamentous nonheterocystous

(L. majuscula – Schutz et al., 2004; Leitao et al., 2005;

Arthrospira and Spirulina spp. – Zhang et al., 2005a, b), and

filamentous heterocystous strains (Nostoc spp. – Tamagnini

et al., 2000; Schutz et al., 2004). Furthermore, the increasing

number of cyanobacterial sequenced genomes is contribut-

ing toward a better understanding of both the distribution

and the diversity of this enzyme.

The physiological function of the bidirectional hydroge-

nase in cyanobacteria is not totally clear. It has been

suggested that the enzyme acts as an electron valve during

photosynthesis in Synechocystis sp. PCC 6803. This is based

on the fact that hoxH� mutants are impaired in the oxida-

tion of PSI, have higher fluorescence of PSII and have

different transcript levels of the photosynthetic genes psbA,

psaA and petB when compared with the wild type (Appel

et al., 2000). The enzyme has also been proposed to play a

role in fermentation functioning as a mediator in the release

of excess reducing power under anaerobic conditions (Stal &

Moezelaar, 1997; Troshina et al., 2002). Furthermore, it has

been suggested previously that the bidirectional hydroge-

nase could be part of the respiratory complex I (Appel &

Schulz, 1996; Schmitz & Bothe, 1996), because only 11

subunits out of 14 conserved subunits of the prokaryotic

complex I have been identified in cyanobacteria. Some of

the subunits of the bidirectional hydrogenase indeed show

sequence similarities with the missing subunits of the

respiratory complex I (Schmitz et al., 1995). However, the

bidirectional hydrogenase has been demonstrated to be

absent from several cyanobacterial strains (Tamagnini et al.,

1997, 2000; Schutz et al., 2004; Ludwig et al., 2006). More-

over, N. punctiforme, a strain naturally lacking the bidirec-

tional hydrogenase (Tamagnini et al., 1997), has rates of

respiration comparable to cyanobacteria containing the

bidirectional hydrogenase (Boison et al., 1999). In addition,

mutants of hoxU in Synechococcus sp. PCC 6301 (former

Anacystis nidulans) (Boison et al., 1998) and hoxEF in

Synechocystis sp. PCC 6803 (Howitt & Vermaas, 1999)

showed nonimpaired respiratory O2 uptake while being

affected in H2 evolution. Furthermore, inactivation of hoxH

in Synechocystis sp. PCC 6803 and Nostoc sp. PCC 7120

resulted only in a small decrease in the growth rate com-

pared with the respective wild types (Appel et al.,

2000; Masukawa et al., 2002). Taking into account all the

data, it seems that in general the bidirectional hydrogenase

does not play an essential role for cell survival in the strains

where it is present.

Attempting to shed some light on the physiological

function of the bidirectional hydrogenase, Cournac et al.

(2004) demonstrated that the bidirectional hydrogenase in

Synechocystis sp. PCC 6803 is insensitive to light, reversibly

inactivated by O2 and can be quickly reactivated by NADH

or NADPH. This work also reported H2 evolution by cells

incubated anaerobically in the dark, after an adaptation

period. This dark H2 evolution was enhanced by exogen-

ously added glucose and resulted from the oxidation of

FEMS Microbiol Rev 31 (2007) 692–720c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

702 P. Tamagnini et al.

NAD(P)H produced by fermentation reactions. Upon illu-

mination, a short (o 30 s) burst of H2 output was observed,

followed by rapid H2 uptake, and a concomitant decrease in

CO2 concentration in the cyanobacterial cell suspension,

which were both linked to photosynthetic electron transport

in the thylakoids (Cournac et al., 2004). Moreover, in this

experimental setup, in anoxia (or microaerobiosis) and in

the presence of H2, H2 uptake was of the same magnitude as

photosynthetic activity and could therefore contribute sig-

nificantly to CO2 fixation. Therefore, although the bidirec-

tional hydrogenase in Synechocystis sp. PCC 6803 is

constitutively expressed in the presence of O2 (Appel et al.,

2000), it probably plays a role mainly under anaerobic or

microaerobic conditions, and at the onset of light before the

enzyme is inactivated by photosynthetic O2. In the ndhB

mutant M55, which is defective in the type I NADPH-

dehydrogenase complex (NDH-1) and produces only low

amounts of O2 in the light, H2 uptake was negligible during

dark-to-light transitions, allowing several minutes of con-

tinuous H2 production. It was further shown that two

pathways of electron supply for H2 production operate in

M55, namely photolysis of water at the level of photosystem

II and carbohydrate-mediated reduction of the plastoqui-

none pool. When comparing the features of the Synechocys-

tis sp. PCC 6803 hydrogenase with those of the homologous

NAD1-dependent hydrogenase of R. eutropha, despite se-

quence homologies between the two enzymes, their char-

acteristics are not identical, which might indicate that this

enzyme might have slightly different functions in different

organisms (Cournac et al., 2004).

If the function of the bidirectional hydrogenase is still open

to debate, its subcellular localization is not less controversial.

The bidirectional hydrogenase can be found in both the

heterocysts and the vegetative cells (Hallenbeck & Benemann,

1978; Houchins & Burris, 1981a), and in Nostoc sp. PCC 7120

appears in the soluble fraction after cell disruption, and

consequently has been considered to be a soluble enzyme

(Houchins & Burris, 1981b). Nevertheless, investigations in

other cyanobacteria suggest a weak association of the bidirec-

tional hydrogenase with cell membranes: in A. variabilis and

Synechocystis sp. PCC 6803, an association with the thylakoid

membrane was proposed (Serebriakova et al., 1994; Appel

et al., 2000), while in Synechococcus sp. PCC 6301 immuno-

logical data implied an association with the cytoplasmic

membrane (Kentemich et al., 1989, 1991).

Physical organization of hox genes and thecorresponding proteins

In cyanobacteria, the structural genes encoding the bidirec-

tional hydrogenase are organized in a dissimilar way (see

Fig. 2). In some strains (e.g. Synechocystis sp. PCC 6803 and

A. variabilis), the hox genes are localized in one cluster,

although interspersed with different ORFs at diverse posi-

tions. In other cases, the hox genes are found in two different

clusters separated by several kilobase (c. 333 and 8.8 kb in

Synechococcus sp. PCC 6301 and Nostoc sp. PCC 7120,

respectively). Despite this fact, the similarities at the de-

duced amino acid level of their homologous hydrogenase

proteins range between 55% and 81%.

The bidirectional hydrogenase has been purified from

several cyanobacterial strains: A. cylindrica (Hallenbeck &

Benemann, 1978), Spirulina maxima (Llama et al., 1979),

Microcystis aeruginosa (Asada et al., 1987), Synechococcus sp.

PCC 6301 (Schmitz et al., 1995, 2002) and Synechocystis sp.

PCC 6803 (Schmitz et al., 2002), but the data collected by

Schmitz et al. (2002) finally helped to clarify the picture of

the subunit composition and molecular mass of the cyano-

bacterial bidirectional hydrogenase. Thus, it is widely ac-

cepted that the bidirectional hydrogenase is composed of

five subunits, HoxE, HoxF, HoxU, HoxY and HoxH, with

apparent molecular weights of c. 20, 61, 28, 24 and 49 kDa,

respectively. The molecular weight of the native protein

(375 kDa) indicates a dimeric assembly of the enzyme

complex, Hox(EFUYH)2 (Schmitz et al., 2002).

Similar to the uptake hydrogenase, the large subunit of the

hydrogenase dimer (HoxH) harbors the active metal center

containing nickel and iron. The two metal atoms are held in

close proximity by two disulfide bridges provided by two

cysteine residues of the protein. The iron has two cyanide ions

and one carbon monoxide as ligands, whereas the nickel ion

is coordinated by two additional cysteines (Volbeda et al.,

1995). The small subunit of the hydrogenase dimer (HoxY),

and the different components of the diaphorase part of the

bidirectional hydrogenase (HoxF and HoxU) also contain

several conserved cysteine residues putatively involved in the

coordination of FeS clusters (Schmitz et al., 2002; for a review,

see Tamagnini et al., 2002). In addition, in the middle region

of HoxF, typical glycine-rich binding sites for NAD1

(GxGxxGxxxG) and flavin mononucleotide (GxGxxxxGx10

GxxG) can be identified (Schmitz et al., 1995). HoxE may be

involved as a bridging subunit in membrane attachment.

Moreover, a functional role in electron transport directed to

membrane components, as demonstrated experimentally for

the Hox-hydrogenase of Thiocapsa roseopersicina (Rakhely

et al., 2004), could be considered because sequence motifs for

binding of an additional FeS cluster are present in this gene

(Schmitz et al., 2002).

hox promoter regions and transcriptionalregulators

The information about the transcription and regulation of

the hox genes is limited in cyanobacteria, but the under-

standing of these mechanisms is now emerging. Recent

studies showed that the hox genes in Synechocystis

FEMS Microbiol Rev 31 (2007) 692–720 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

703Cyanobacterial hydrogenases

sp. PCC 6803 are transcribed as a single operon (Gutekunst

et al., 2005; Oliveira & Lindblad, 2005; Antal et al., 2006)

with the transcription start point located 168-bp upstream

of the hoxE start codon (Gutekunst et al., 2005; Oliveira &

Lindblad, 2005).

Up to now, only one regulator – LexA – has been proven

to bind and regulate the transcription of the hox genes in

cyanobacteria. Two independent studies (Gutekunst et al.,

2005; Oliveira & Lindblad, 2005) demonstrated an interac-

tion between LexA and the promoter region of the bidirec-

tional hydrogenase in Synechocystis sp. PCC 6803. However,

two distinct regions were analyzed and both were demon-

strated to be targets for this interaction. Oliveira & Lindblad

(2005) showed that LexA binds to a region located between

the nucleotides � 198 and � 338 bp, respective to transla-

tional start point, while Gutekunst et al. (2005) found that

LexA interacts further upstream on the hox promoter, at the

positions � 592 to � 690 bp, in relation to the hoxE ATG

codon (see Fig. 4). Furthermore, a LexA-depleted mutant

showed a reduced hydrogenase activity compared with the

wild-type, suggesting that LexA works as a transcription

activator of the hox genes in Synechocystis sp. PCC 6803

(Gutekunst et al., 2005). Synechocystis sp. PCC 6803 LexA

has been detected in different proteomic studies (Wang

et al., 2000; Gan et al., 2005; Srivastava et al., 2005; Fulda

et al., 2006; Kurian et al., 2006; Slabas et al., 2006), and its

transcript has also been identified in microarray experi-

ments (Hihara et al., 2001; Kamei et al., 2001; Li et al., 2004;

Singh et al., 2004; Tu et al., 2004; Shapiguzov et al., 2005).

Interestingly, in some proteomic studies, LexA has been

identified in association with thylakoid membrane fractions

(Wang et al., 2000; Srivastava et al., 2005), which represents

an unexpected location for a transcription regulator.

Based on the observations that the bidirectional hydro-

genase activity is directly affected by the redox status of the

cell, either in photosynthesis or in fermentation, and that

the regulation of the hox gene expression can be operated by

LexA, hypothesis was recently put forward on the direct

involvement of the transcription regulator LexA as a med-

iator of the redox-responsive regulation of the hox gene

expression in Synechocystis sp. PCC 6803 (Antal et al., 2006).

Interestingly, the expression of the cyanobacterial DEAD-

box RNA helicase, crhR, which is regulated in response to

conditions that elicit reduction of the photosynthetic elec-

tron transport chain, was recently shown as being directly

controlled by LexA in Synechocystis sp. PCC 6803 (Patter-

son-Fortin et al., 2006). Transcript analysis indicated that

lexA and crhR are divergently expressed, with the respective

transcripts accumulating differently under conditions,

which, respectively, oxidize and reduce the electron trans-

port chain, suggesting that LexA works as a repressor of the

crhR transcription (Patterson-Fortin et al., 2006). Although

these results are in agreement with the initial hypothesis, the

signal transduction pathways directly or indirectly involved

in the regulation of LexA, and consequently its downstream

targets, definitely require further investigation.

Transcription and expression patterns of hoxgenes

The number of studies focusing on the transcription and

regulation of the hox genes in cyanobacteria is scarce.

Nevertheless, transcripts of the bidirectional hydrogenase

have been shown to be present in NH41-grown filaments,

and in both vegetative cells and heterocysts under nitrogen-

fixing conditions in A. variabilis (Boison et al., 2000). In

addition, hoxFUYH were shown to be transcribed as a single

unit together with other two ORFs with unknown function.

However, it should be kept in mind that these experiments

were performed using RT-PCR and do not exclude addi-

tional promoters within the operon (Boison et al., 2000). On

the other hand, in the unicellular Synechococcus sp. PCC

6301 and Synechococcus sp. PCC 7942 the hox genes are

located apart and give rise to two different transcripts

(Boison et al., 2000; Schmitz et al., 2001). While hoxEF are

cotranscribed in both strains, the second transcript is

constituted by hoxUYH together with hoxW, hypA and hypB

in Synechococcus sp. PCC 6301 (Boison et al., 2000), and by

hoxUYHW only in Synechococcus sp. PCC 7942 (Schmitz

et al., 2001). For the last strain, using real-time PCR and

reporter gene constructs, it was suggested that a second

promoter might be present between hoxH and hoxW

(Schmitz et al., 2001). Furthermore, it was demonstrated

that the hox genes have a circadian clock expression

(Schmitz et al., 2001), a fact that has also been demonstrated

for hoxE in Synechocystis sp. PCC 6803 (Kucho et al., 2005).

Very few studies focusing on the regulation of hox genes

transcription have been performed in cyanobacteria. Analy-

sis of the transcription of hoxY and hoxH in G. alpicola,

under combined nitrogen-limiting growth conditions, de-

monstrated an increase in the enzyme activity, but no

regulation at the transcript level (Sheremetieva et al., 2002).

In contrast, Northern blot analyses of the hox genes expres-

sion in Synechocystis sp. PCC 6803 under combined nitro-

gen-limiting growth conditions demonstrated an increase in

transcription (Antal et al., 2006), followed by an increase in

enzyme activity (T.K. Antal, P. Oliveira & P. Lindblad,

unpublished data). A similar increase in the hox genes

transcription has also been observed with microarray in

Synechocystis sp. PCC 6803 cells undergoing nitrogen starva-

tion for 4 h (Osanai et al., 2006 – supplementary material).

Furthermore, a transfer to a low level of oxygen in

A. variabilis induced both the enzyme activity as well as the

relative amount of hoxH (Sheremetieva et al., 2002). It has

long been demonstrated that microaerobic/anaerobic con-

ditions influence hox transcription and bidirectional

FEMS Microbiol Rev 31 (2007) 692–720c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

704 P. Tamagnini et al.

hydrogenase activity in heterocystous cyanobacteria (Hou-

chins & Burris, 1981a; Houchins, 1984; Serebryakova et al.,

1994; Schmitz & Bothe, 1996; Axelsson & Lindblad, 2002;

Sheremetieva et al., 2002). The bidirectional hydrogenase in

Nostoc sp. PCC 7120 is active in both vegetative cells and in

heterocysts in aerobically grown filaments, with heterocysts

having several fold more activity than vegetative cells. When

the filaments were transferred to anaerobic conditions, the

activity of the bidirectional hydrogenase increased by about

two orders of magnitude with approximately the same

activity levels in both types of cells (Houchins & Burris,

1981a). Similar results have been observed in A. variabilis

(Serebryakova et al., 1994). In contrast to the filamentous

cyanobacteria, the activity of the bidirectional hydrogenase

in the unicellular G. alpicola is not directly dependent on

oxygen (Troshina et al., 2002). Higher activity is observed

under nitrogen starvation and low light, and it was sug-

gested that the bidirectional hydrogenase could act as an

alternative electron donor to PSI after inactivation of PSII

due to nitrogen starvation. Under dark anoxic conditions,

the unicellular cyanobacterium G. alpicola produces H2

catalyzed by the bidirectional hydrogenase (Troshina et al.,

2002). In addition, the unicellular strain Chroococcidiopsis

thermalis CALU 758 contains a bidirectional hydrogenase

with some catalytic properties more related to an uptake

hydrogenase, i.e. not inducible under anaerobic conditions

or under nitrate-starving conditions (Serebryakova et al.,

2000).

Because the bidirectional hydrogenase in cyanobacteria is

a metal-dependent enzyme, containing nickel and iron in

its active center and FeS clusters involved in electron

transfer, the availability of these elements in the growing

medium has been a subject of research. Axelsson & Lindblad

(2002) showed that in the heterocystous N. muscorum

CCAP 1453/12, the addition of external nickel to the

growing medium increased the mRNA abundance of hoxH

(monitored by RT-PCR). Making use of reporter gene

constructs, Gutekunst et al. (2006) were able to show that

the transcription of the bidirectional hydrogenase genes in

Synechocystis sp. PCC 6803 increased with lower concentra-

tions of iron, the signal being 10 times higher in cells grown

with 0.22mM iron compared with nonstarved cells. In the

same work, measurements of the hydrogenase activity

revealed a reduction of the enzyme activity alongside the

decrease in the iron concentration. The increase in tran-

scription of the hox genes, when the cells undergo iron

starvation, might be a feedback mechanism to compensate

for the lack of functionally active enzyme (Gutekunst et al.,

2006). The availability of sulfur in the growth medium has

also been shown to influence the bidirectional hydrogenase

activity in Synechocystis sp. PCC 6803 and G. alpicola (Antal

& Lindblad, 2005). Both strains showed an enhanced (more

than fourfold) H2 production capacity during fermentation

via hydrogenase, when grown under sulfur starvation con-

ditions.

Although the understanding of the regulation and the

physiological role of the bidirectional hydrogenase is becom-

ing clearer, intriguing recent results on the hydrogenase

activity from two substrains of Synechocystis sp. PCC 6803

have shown that they do not have comparable values

(Gutekunst et al., 2006). The authors suggested that these

phenotypic differences in the hydrogenase activity might be

due to divergences in their metabolism. In fact, maintenance

of these strains in culture collections, or under various

laboratory conditions, may have led to spontaneous muta-

tions and unintended selective pressures, resulting in the

observable variations in each subculture (Ikeuchi & Tabata,

2001). Therefore, special care must be taken when interpret-

ing results coming from different laboratories and different

cyanobacterial strains, even from the same strain, but

cultured in different laboratories.

Maturation of cyanobacterialhydrogenases

The biosynthesis/maturation of NiFe-hydrogenases is a

highly complex process requiring at least seven core proteins

for the incorporation of the metal ions and CO and CN

ligands in to the active center, the orientation of the FeS

clusters within the small subunit and the cleavage of the C-

terminus as the final step in the maturation of the large

subunit (for a recent review on this subject, see Bock et al.,

2006, and also Casalot & Rousset, 2001; Blokesch et al., 2002;

Mulrooney & Hausinger, 2003; Kuchar & Hausinger 2004;

Vignais & Colbeau, 2004; Theodoratou et al., 2005). The

genes encoding the proteins involved in the maturation of

hydrogenases were firstly characterized for E. coli, and while

most of the Hyp proteins affect hydrogenases pleiotropically,

the large subunit of each hydrogenase is proteolytically

processed by a specific endopeptidase (Lutz et al., 1991;

Jacobi et al., 1992; Menon et al., 1994; Rossmann et al., 1995;

Theodoratou et al., 2005; Bock et al., 2006). Homologues of

the hyp genes are present in all organisms capable of forming

NiFe hydrogenases. Although little is known about the

biosynthesis/maturation of the cyanobacterial hydrogenases,

several genes presumably involved in this process have been

identified clustered or scattered throughout the genomes of

several cyanobacterial strains (Boison et al., 1996; Gubili &

Borthakur, 1996, 1998; Kaneko et al., 1996; Sakamoto et al.,

1998; Hansel et al., 2001; Tamagnini et al., 2002; Wunschiers

et al., 2003; Hoffmann et al., 2006; Leitao et al., 2006). The

presence of a single copy of most of the hyp genes (hypFC-

DEAB) in the genome of cyanobacteria, regardless of

possessing only the uptake hydrogenase (e.g. N. puncti-

forme), the bidirectional hydrogenase (e.g. Synechocystis sp.

PCC 6803) or both enzymes (e.g. Nostoc sp. PCC 7120)

FEMS Microbiol Rev 31 (2007) 692–720 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

705Cyanobacterial hydrogenases

suggests that they might be responsible for the maturation

of both hydrogenases. In contrast, the genes encoding for the

putative hydrogenase C-terminal endopeptidases – hupW

and hoxW – were identified and seem to be specific for the

cyanobacterial uptake and the bidirectional hydrogenase,

respectively, resembling the situation in other organisms

(Wunschiers et al., 2003; Oliveira et al., 2004; Leitao et al.,

2006).

Physical organization of hyp genes and thecorresponding proteins

The hyp genes in cyanobacteria are frequently clustered and

in the vicinity of the structural genes of one of the hydro-

genases (Fig. 2), with a well-known exception – the uni-

cellular non-N2-fixing Synechocystis sp. PCC 6803 – in which

the hypABCDEF genes are scattered throughout the genome.

Still, in this organism the homologs hypA2 and hypB2 are

clustered (Kaneko et al., 1996), but these two do not seem to

play a key role in the maturation of the bidirectional

hydrogenase (Hoffmann et al., 2006). In three Synechococ-

cus, closely related strains (Synechococcus elongatus PCC

6301, Synechococcus elongatus PCC 7942 and Synechococcus

sp. PCC 7002) hypABFC are together and downstream of

hox genes, while hypD and hypE are apart in the two first

organisms (Boison et al., 1996). In the heterocystous strains,

N. punctiforme, Nostoc sp. PCC 7120 and A. variabilis and in

the N2-fixing but nonheterocystous L. majuscula, the hyp

genes are located in a cluster with all genes orientd in the

same direction, and relatively close to the uptake hydro-

genase structural genes, although in the opposite direction

(Gubili & Borthakur, 1998; Hansel et al., 2001; Leitao et al.,

2006). However, this organization does not constitute a

pattern for N2-fixing strains, because it contrasts with the

organization observed for other nonheterocystous strains,

such as the filamentous T. erythraeum, in which hyp genes

are located much further upstream of hupSL (ca. 589 kb),

and the unicellular Crocosphaera watsonii WH 8501, in

which the genes are scattered over the genome resembling

the non-N2-fixing Synechocystis sp. PCC 6803. When the

genes are grouped, the order varies in non-N2-fixing com-

pared with N2-fixing strains being hypABFC and hypFC-

DEAB, respectively. In the former case, ORFs interspersed

with the hyp genes can be found in several organisms.

The putative cyanobacterial Hyp proteins possess con-

served motifs and may fulfill functions similar to the

corresponding proteins in other organisms (Tamagnini

et al., 2002; Vignais & Colbeau, 2004; Hoffmann et al.,

2006; Leitao et al., 2006). It is believed that from the two

metal ions present in the active center of a NiFe hydro-

genase, Fe is the first to be incorporated into the enzyme.

HypF and HypE are the proteins involved in the synthesis of

the CN, and maybe the CO, ligands of iron (Paschos et al.,

2001, 2002; Bock et al., 2006). HypF accepts carbamoyl

phosphate (CP) as a substrate, catalyzes a CP-dependent

hydrolysis of ATP into AMP and inorganic phosphate (PPi)

and forms an adenylated CP derivative. The carbamoyl

group of CP is transferred to the cysteine at the C-terminus

of HypE (Paschos et al., 2002; Reissmann et al., 2003). It was

demonstrated in vitro that the CN group from HypE-

thiocyanate can be transferred to the complex HypC plus

HypD (Blokesch et al., 2004a). Because the transfer of

the ligands to the iron requires the input of two electrons

(Blokesch & Bock, 2002), HypD is proposed to be the one

involved in this process, given that among all the maturation

proteins it is the only one with a redox-active cofactor

(Blokesch et al., 2004a; Roseboom et al., 2005). On the other

hand, HypC is a small chaperone-like protein that was

shown to form a complex with HypD (Blokesch & Bock,

2002) and to interact with the large subunit of the hydro-

genase (Magalon & Bock 2000a; Casalot & Rousset 2001).

Probably, the liganding of the iron takes place at the

HypC–HypD complex (Blokesch et al., 2002, 2004a), and

the interaction between HypC and the precursor of the large

subunit leads to the liberation of HypD (Blokesch et al.,

2004a; Blokesch & Bock, 2006). Subsequently, the liganded

Fe is transferred to the precursor of the hydrogenase large

subunit (Blokesch & Bock, 2006), and HypC remains

attached to the large subunit, maintaining it in an open

conformation, allowing the insertion of nickel. This step

requires the presence of HypA and HypB (Jacobi et al., 1992;

Olson et al., 2001). HypA is a zinc-containing protein that

binds nickel (Mehta et al., 2003; Blokesch et al., 2004b), and

HypB is a GTPase that probably plays a dual function: nickel

storage and nickel insertation (Maier et al., 1993, 1995). It is

thought that HypA functions as a nickel chaperone and that

HypB acts as a regulator, controlling the donation of the

metal to the apoprotein or the release of the nickel-free

chaperone (Blokesch et al., 2004b). After both metals have

been coordinated to the precursor of the large subunit, the

C–terminal extension is accessible and can be removed by

the specific endopeptidase. The cleavage can only occur after

HypC dissociation from the precursor of the large subunit

that already contains Ni and Fe(CO)(CN�)2 centers (Maga-

lon & Bock, 2000a, b), because the endopeptidase uses Ni

as a recognition motif. Following the cleavage of the

C-terminal tail from the large hydrogenase subunit, the

mature large subunit can be assembled, with the mature

small subunit forming the functional enzyme (Magalon &

Bock, 2000a). Maturation of the small subunit should occur

in parallel, and independently from the large subunit

maturation. The knowledge about this process is still scarce,

although recent studies highlighted at least four gene

products (encoded within the hup cluster, and downstream

of uptake hydrogenase structural genes) that are required for

the maturation of the small subunit of the NiFe

FEMS Microbiol Rev 31 (2007) 692–720c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

706 P. Tamagnini et al.

hydrogenases of Rhizobium leguminosarum bv. viciae (Man-

yani et al., 2005; Bock et al., 2006). In cyanobacteria, several

additional ORFs are commonly present near hyp or hup

genes (Leitao et al., 2006). The consistent location of these

ORFs might indicate that their proteins may have a role in

the uptake hydrogenase maturation process and/or its

regulation, notably regarding the small subunit.

hyp promoter regions and transcriptionalregulators

As mentioned above, the hyp genes can be found clustered or

scattered throughout the genome of cyanobacteria (Fig. 2).

Analysis of the hyp cluster promoter region of N. puncti-

forme revealed the presence of � 10 and � 35 elements, and

putative binding sites for NtcA (Hansel et al., 2001; Fig. 4).

Similarly, in the corresponding region of L. majuscula the

presence of a � 10 box, and two putative NtcA-binding sites

could be identified. In this organism, a clear � 35 box is not

present, but it should be taken into account that its sequence

is highly variable. Furthermore, a putative LexA-binding site

was also found in L. majuscula (Leitao et al., 2006; Fig. 4).

The transcriptional regulators NtcA and LexA were shown

to bind to the promoter regions of the hup and the hox

genes, suggesting their involvement in the regulation of the

uptake and bidirectional hydrogenase, respectively (see

above, Lindberg, 2003; Oliveira et al., 2004; Gutekunst

et al., 2005; Leitao et al., 2005; Oliveira & Lindblad, 2005).

The presence of putative binding sites for both transcrip-

tional factors NtcA and LexA within the hyp operon

promoter region, and preliminary results from electro-

phoretic mobility shift assays (Ferreira et al., 2007) suggest

the involvement of these proteins in the transcriptional

regulation of hyp genes in L. majuscula, a cyanobacterium

containing both hydrogenases. These data reinforce the

hypothesis that the Hyp proteins might be implicated in

the maturation/regulation of both hydrogenases, and raise

the hypothesis that the transcription of hyp genes in

cyanobacteria containing both hydrogenases could be under

the control of different transcriptional regulators, e.g. NtcA

and LexA.

Transcription and expression patterns of hypgenes

In the heterocystous N. punctiforme, the hup and hyp genes

are transcribed under N2-fixing but not under non-N2-

fixing growth conditions (Hansel et al., 2001). One should

bear in mind that N. punctiforme contains only one hydro-

genase (the uptake enzyme), and that in this organism both

the transcription of hupL and the H2 uptake activity are

repressed when combined nitrogen is present in the growth

medium (Oxelfelt et al., 1995; Hansel et al., 2001).

In the unicellular non-N2-fixing Synechocystis sp. PCC

6803, a cyanobacterium harboring only the bidirectional

hydrogenase, deletion and insertion mutants of hypA1, B1,

C, D, E and F showed no hydrogenase activity. Moreover, the

complementation of each of the above hyp- inactivated

genes restored the bidirectional hydrogenase activity to the

wild-type level in the respective mutants (Hoffmann et al.,

2006). In contrast, the deletion of the homologues hypA2

and hypB2 had no effect on the bidirectional hydrogenase

activity even though they are transcribed in the wild type,

demonstrating that the products of these genes are not

actively involved in the maturation process of the bidirec-

tional hydrogenase (Hoffmann et al., 2006).

Hydrogenase-specific endopeptidases geneshupW and hoxW, and corresponding proteins

The last step in the processing of the large subunit of NiFe-

hydrogenases is the cleavage of a C-terminal peptide, which,

most likely, allows a structural reorganization of the mole-

cule and the consequent assembly of the holoenzyme. After

both metals have been inserted into the apoprotein precur-

sor of the large subunit, the C-terminal extension is acces-

sible and can be removed by the specific endopeptidase

(Theodoratou et al., 2005; Bock et al., 2006). This process

triggers a conformational switch in which the free thiol of

the most C-terminally located cysteine residue closes the

bridge between the two metals resulting in the formation of

the complete heterobinuclear center (Maier & Bock, 1996;

Magalon & Bock, 2000a; Theodoratou et al., 2005; Bock

et al., 2006). The peptidase cleaves the hydrogenase large

subunit precursor after a histidine or an arginine residue at

the C-terminal consensus motif DPCxxCxx(H/R), liberating

a short polypeptide that varies considerably both in length

and sequence among different organisms (Wunschiers et al.,

2003). It has been postulated that the endopeptidase recog-

nizes its substrate, the nickel-containing hydrogenase pre-

cursor, at least in part via the metal that is coordinated by

three thiolates, and binds to the exposed C-terminal domain

(Theodoratou et al., 2000a, b, 2005 and Fig. 5). In addition,

the endopeptidase interacts with a structural domain to

which both the mature part of the large subunit and the C-

terminal extension contribute. Therefore, it is believed that

the recognition of the hydrogenase by the endopeptidase

does not depend on the cleavage site consensus sequence but

is mediated by the overall three-dimensional hydrogenase

and peptidase protein structures (Theodoratou et al.,

2000a, b). After the proteolytic cleavage, the mature large

hydrogenase subunit assembles with the small subunit and

eventually the enzyme becomes active.

In cyanobacteria hydrogenase large subunits, the C-

terminal consensus motif [DPCxxCxx(H/R)] was found in

all the deduced amino acid sequences; however, in the

FEMS Microbiol Rev 31 (2007) 692–720 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

707Cyanobacterial hydrogenases

uptake hydrogenase large subunits (HupL) the neutral pro-

line (P) at position 2 of the cutting site motif is exchanged

for an uncharged polar serine (S). The sequence of the

cutting site motif is totally conserved for each of the

cyanobacterial hydrogenases large subunits: HoxH (bidirec-

tional hydrogenase) – DPCLSCSTH; HupL (uptake hydro-

genase) – DSCLVCTVH (see Wunschiers et al., 2003 and

Fig. 5). The putative cleaved C-terminal polypeptide varies

in length (25–32 aa residues) and sequence (10–96% simi-

larity) for HoxH, while for HupL the polypeptide always has

the same length (16 aa residues) and is highly conserved for

all the deduced sequences [AHDAKTG(E/K)ELARFRT(A/

N/S)].

In cyanobacteria, the genes encoding for the putative

hydrogenase-specific C-terminal endopeptidases were iden-

tified and named hupW and hoxW for the gene encoding the

enzyme processing the uptake and the bidirectional hydro-

genase, respectively (Kaneko et al., 1995, 2001; Boison et al.,

2000; Schmitz et al., 2001; Wunschiers et al., 2003; Oliveira

et al., 2004; Leitao et al., 2005).

The position of hupW and hoxW in the cyanobacterial

chromosome is rather variable; however, in several cases

hupW is in the vicinity and in the same direction of hupSL

(uptake hydrogenase structural genes). In the nonheterocys-

tous Gloeothece sp. ATCC 27152 and T. erythraeum, hupW is

even the ORF located immediately downstream of hupL, and

was shown to be cotranscribed with hupSL in Gloeothece

sp. ATCC 27152 (Oliveira et al., 2004). In contrast, in the

heterocystous strains A. variabilis, Nostoc sp. PCC 7120 and

N. punctiforme, hupW is not part of any known hydrogenase

cluster (Fig. 2), and it was shown to be transcribed under

N2- and non-N2-fixing conditions in the last two strains

(Wunschiers et al., 2003). These authors postulated that the

transcription of hupW under conditions in which the

transcripts of the uptake hydrogenase structural genes could

not be detected (presence of ammonia) could imply that

hupW is constitutively expressed. Taking into account all the

available data, it is not yet possible to establish whether the

expression of hupW is or is not constitutive or whether this

depends on the strain/existence of cell differentiation.

Similar to what happens for hupW, analysis of the

available cyanobacterial genomic sequences revealed that

the position and orientation of hoxW in the chromosome is

also variable but, in most of the cases, hoxW is downstream

of hoxH, one of the bidirectional hydrogenase structural

genes (Fig. 2). RT-PCR experiments indicate that in the

unicellular non-N2-fixing Synechococcus sp. PCC 6301,

hoxW is part of a polycistronic message containing hoxUYH-

WhypAB (Boison et al., 2000), while in Synechococcus sp.

PCC 7942 it was demonstrated that although hoxW consti-

tute a unit together with hoxUYH, it is mainly expressed by

its own promoter (Schmitz et al., 2001). In the heterocystous

Nostoc sp. PCC 7120, similar to hupW, hoxW is transcribed

under both N2- and non-N2-fixing conditions (Wunschiers

et al., 2003). Although some data indicate that endo-

peptidases transcripts are present when the corresponding

hydrogenase large subunit transcript is absent, and it has

been proposed that their expression is independently

regulated from the expression of both the hydrogenase

structural and the other accessory genes in cyanobacteria

(Wunschiers et al., 2003), it is premature to make any

general conclusion.

To date, two different hydrogenase specific-endopepti-

dases have been purified and studied, namely HycI and

HybD from E. coli (Rossmann et al., 1995; Fritsche et al.,

1999). Both are monomeric proteins of a molecular mass of

Endo-peptidase

C - S

DPCxx

Cxx

(H/R

)

S

HoxH DPCLSCSTH 25-32 a.a.

HupL D

Endo-

CO CN CN

C - S

DPCxx

Cxx

(H/R

)

COOH

Large Subunit

C

CO CN CN

C S - C

S - C

SmallSubunit

Large Subunit

S

DSCLVCTVH 16 a.a.

- S

- S

Fig. 5. Schematic representation of the putative final step of the maturation process of the Ni–Fe hydrogenases large subunit: cleavage of a small

peptide by a specific endopeptidase, followed by a conformational change that encloses the bimetallic center. This structural reorganization of the large

subunit will allow the consequent assembly of the holoenzyme. In the large subunits of cyanobacterial hydrogenases – HoxH (bidirectional hydrogenase)

and HupL (uptake hydrogenase) – the C-terminal consensus motif DPCxxCxx(H/R) was found in all the deduced sequences, but in HupL the proline is

exchanged by a serine (see box). The putative cleaved polypeptide varies in length and sequence for HoxH, while for HupL is always has the same length

and is highly conserved.

FEMS Microbiol Rev 31 (2007) 692–720c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

708 P. Tamagnini et al.

c. 17 kDa, and they are devoid of metal or other cofactors.

Alignments of the amino acid sequences showed that

hydrogenase- specific C-terminal endopeptidases share low

sequence similarity, with only a few positions fully con-

served (Theodoratou et al., 2005). As a general feature, they

have three highly conserved amino acid residues (Glu, Asp

and His) that, most likely, have the function of interacting

with the nickel in the hydrogenase large subunit precursor

(Theodoratou et al., 2000a). The alignment of the putative

cyanobacterial endopeptidases with the corresponding pro-

teins from E. coli clearly shows that although the amino acid

sequence identity is low, they are indeed structurally related

(67–77% structural identity) (Wunschiers et al., 2003).

Phylogenetic analysis

Recently, the phylogenetic origin of cyanobacterial and algal

hydrogenases was analyzed (Ludwig et al., 2006), leading to

the conclusion that Chloroflexus is probably the closest

ancestor of cyanobacteria. In all cyanobacterial genomes

sequenced to date, and in the genome of Chloroflexus, the

two hydrogenase operons – hup and hox – are widely

separated on the chromosome, rendering simultaneous gene

transfer unlikely. The authors claim that the current dis-

tribution of the hydrogenases in cyanobacterial strains

probably reflects a differential loss of the genes from their

last common ancestor, and that the two sets of genes,

encoding the uptake and bidirectional, were either kept in

the genome or lost differentially in the different strains

according to their ecological needs or constraints. Although

the phylogenetic analysis of Ludwig et al. (2006) clearly

demonstrated the monophyly of cyanobacteria, and their

relationship with other photosynthetic bacteria, relation-

ships within the cyanobacteria were poorly resolved using

HupL sequences. This is probably related to the difficulties

of aligning the cyanobacteria with the other highly divergent

lineages, and acerbated by the low number of sequences

available and long branches leading to terminal nodes. The

high variability of the sequences also means that more

distant bacterial outgroups cannot be unambiguously

aligned. However, analysis solely within the cyanobacteria

for both HupS and HupL is less complex, because analysis of

the predicted proteins demonstrates that in HupS the

number of residues in all known cyanobacteria is constant

(320 aa), while HupL generally has 531 aa, with the excep-

tion of the filamentous nonheterocystous strains L. majus-

cula and L. aestuarii with six extra (one insertion of 5 aa and

another of one), and T. erythraeum with three extra, coin-

ciding with the position of the five inserts in Lyngbya spp.

Owing to this relatively conserved identity, alignment of the

amino acids for phylogenetic analysis was facile.

Amino acid sequences were analyzed under the criterion

of maximum parsimony, with gaps treated separately as

either missing data or as a fifth state. Support for nodes was

estimated by bootstrapping with 10 000 replicates.

Both analyses gave widely congruent estimates of phylo-

geny (Fig. 6). The three heterocystous strains form a clade

with 100% support, separated from the nonheterocystous

strains by between 16% and 21% divergence. Within the

nonheterocystous strains, two pairs of taxa – Cyanothece

with Crocosphaera and the two Lyngbya species are well

supported. Other relationships are poorly supported.

Although the analysis with gaps treated as missing data

suggests that the filamentous taxa are not a clade, analysis

with gaps treated as a fifth character supported a relation-

ship between T. erythraeum and Lyngbya spp., although with

weak support (51%). Thus, exact relationships within this

group cannot be ascertained by these sequences, although a

sister-taxa relationship between T. erythraeum and L. ma-

juscula is strongly supported through analysis of the Hyp

sequences. These results are not in conflict with those

suggested by Ludwig et al. (2006), in which the only well-

supported node within cyanobacteria is that of the three

heterocystous strains. Evidence for the position of the

ancestral root within the cyanobacteria is weak, although T.

erythraeum may be sister taxa to the remaining sampled

cyanobacteria (Ludwig et al., 2006).

Phylogenetic analysis of cyanobacterial hydrogenases ac-

cessory proteins (Hyp A,B,C,D,E and F) and the bidirec-

tional hydrogenases structural proteins (Hox) is

complicated by the higher level of variation between species,

and in particular greater length variation that leads to

uncertain alignment for many positions. Further, not all of

the amino acid sequences of hydrogenase accessory proteins

are available for all the species analyzed for the uptake

hydrogenase structural genes. However, unweighted parsi-

mony analyses indicate that supported estimates of relation-

ships recovered for these proteins do not conflict with the

estimate of phylogeny shown in Fig. 6 (analyses not shown).

Genetic engineering/cyanobacterial H2

production

Cyanobacteria can be used for the production of molecular

hydrogen (H2), a possible future energy carrier, which has

been the subject of several recent reviews (Levin et al., 2004;

Dutta et al., 2005; Kruse et al., 2005; Prince & Kheshgi, 2005;

Sakurai & Masukawa, 2007). As the main advantages,

cyanobacteria can use sunlight as an energy source, water

as an electron source and air as a carbon (CO2) and a

nitrogen (N2) source. Therefore, no complicated or expen-

sive media are needed for the cultivation of cyanobacteria,

and the overall theoretical energy conversion efficiency

(from solar energy sun to H2) may be the highest possible.

In cyanobacteria, two natural pathways for H2 produc-

tion can be used.

FEMS Microbiol Rev 31 (2007) 692–720 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

709Cyanobacterial hydrogenases

H2 production as a by-product during nitrogenfixation by nitrogenases

In N2-fixing strains, H2 is produced as a by-product by the

nitrogenase enzymatic complex. As this reaction needs the

input of ATP (at least two ATP per electron), the overall

energy efficiency for hydrogen production is rather low. The

turnover of the nitrogenase enzyme is not very high

(o 10 s�1), and the H2 produced is efficiently taken up

by an uptake hydrogenase. The overall oxygen sensitive

N2-fixation process is occurring in an anaerobic environ-

ment achieved using a number of different strategies includ-

ing spatial or/and temporal separation of N2 fixation and

oxygenic photosynthesis and increased respiration.

Cyanobacterial nitrogenases contain molybdenum (Mo),

vanadium (V) or iron (Fe) in the active site, with different

genes and gene products making up the different nitro-

genases (Eady, 1996; Zhao et al., 2006). With sufficient

amounts of molybdenum available, the active site harbors

molybdenum and iron. Under molybdenum-deprived con-

ditions, the conventional molybdenum-nitrogenase is re-

placed by an alternative vanadium-nitrogenase, and if

vanadium is also limited, some N2-fixing microorganisms

are able to synthesize an alternative iron-nitrogenase.

Depending on the type of nitrogenase (molybdenum,

vanadium or iron), different amounts of electrons are

allocated for N2 fixation or H2 production. The general

equation for the nitrogenase-catalyzed reaction is as follows

(Rees et al., 2005):

N2 þ ð2nþ 6Þe� þ ð2nþ 6ÞHþ þ pð2nþ 6ÞATP! 2NH3 þ nH2 þ pð2nþ 6ÞADPþ pð2nþ 6ÞPi

It has been reported that n is 1 for the molybdenum-

containing enzymes, 3 for the vanadium-nitrogenases and

7.5 for the iron-only nitrogenases, respectively. As a conse-

quence, the alternative nitrogenases, although still very little

is known in cyanobacteria, may be better H2 producers

compared with the more conventional molybdenum-nitro-

genases.

H2 production by the bidirectional hydrogenase

The cyanobacterial bidirectional hydrogenase may, under

anaerobic conditions, produce and evolve significant

Fig. 6. Unrooted single most parsimonious tree recovered from an MP analysis with gaps treated as missing data of combined small (HupS) and large

(HupL) subunit amino acid sequences of cyanobacterial uptake hydrogenases. 210 characters were parsimony informative, and a single tree of 552

steps was recovered (CI = 0.83, RI = 0.76). NJ recovered an identical topology. Treating gaps as a fifth state altered the topology as indicated in the text.

Values beside nodes indicate bootstrap support for MP/NJ. 100� indicates 100% support in both analyses.

FEMS Microbiol Rev 31 (2007) 692–720c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

710 P. Tamagnini et al.

amounts of H2. Because this reaction is not dependent on

ATP, it is energetically more efficient and favorable for H2

production, with a much higher turnover (1 million turn-

overs per second) compared with the nitrogenase-based H2

production. At the same time, the enzyme is not specifically

located in an oxygen-protected environment, and the reac-

tion turns into the opposite direction (H2 uptake) above a

certain H2 partial pressure. Therefore, a continuous and very

effective removal of both O2 and H2 from the cells and the

culture is necessary to lower the overall energy conversion

efficiency significantly. Furthermore, an accumulation of

ATP could inhibit the electron flow, because it is produced

during the linear or cyclic electron flow around PSI, but is

not used by the electron acceptor hydrogenase.

Besides the specific challenges for H2 production con-

nected to the H2-evolving enzymes, there are additional

unsolved issues for photoautotrophical H2 production in

general. These are related to the low quantum efficiency, due

to the naturally large antenna systems of the photosystems,

and to electron consuming pathways directly competing

with e.g. nitrogenases and hydrogenases.

In summary, to achieve a sustainable, renewable cyano-

bacterial-based H2 production, the following challenges

have to be addressed:

(1) efficient H2 uptake by the cells,

(2) low energy efficiency and turnover of the nitrogenase

and/or the hydrogenase,

(3) limiting amounts of active H2-evolving enzymes,

(4) high oxygen sensitivity of the nitrogenase and/or the

hydrogenase,

(5) electron flow inhibition by accumulation of ATP in a

hydrogenase-driven system,

(6) low quantum efficiency due to too large antennas in

both Photosystem II (PSII) and PSI and

(7) electron-consuming pathways competing with an effi-

cient electron transfer to the H2 enzymes.

In recent years, there have been attempts to overcome

these barriers and problems, mainly by targeted genetic

engineering of cyanobacterial strains:

(1) Efficient H2 uptake by the cells: Cyanobacteria have

evolved an effective mechanism to recycle the H2 evolved

during nitrogen fixation: an uptake hydrogenase that oxi-

dizes the H2 evolved, and transfers electrons to e.g. the

respiratory-chain. As this reaction significantly lowers the

H2 production efficiency of a nitrogenase-based system,

targeted mutants with reduced or deficient uptake hydro-

genase activity have been produced. This was first achieved

by chemical mutagenesis (Kumar & Kumar, 1991; Mikheeva

et al., 1995), and later, since the molecular biology tools for

genetic engineering were established, by targeted knock-out

of structural or accessory genes of the uptake hydrogenase.

Uptake hydrogenase-deficient mutants of A. variabilis

(Happe et al., 2000), N. punctiforme (Lindberg et al., 2002,

2004), Nostoc sp. PCC 7120 (Lindblad et al., 2002; Masuka-

wa et al., 2002; Carrasco et al., 2005) and Nostoc sp. PCC

7422 (Yoshino et al., 2007) have been shown to be signifi-

cantly better H2 producers compared with the respective wild

types. In general, the H2 produced by a nitrogenase in the

wild type will be quickly reoxidized by the uptake hydro-

genase, whereas in an uptake hydrogenase-deficient mutant

the H2 produced will leave the cells. One should bear in mind

that all these strains, with the exception of N. punctiforme,

also possess a bidirectional hydrogenase. However, only for

Nostoc sp. PCC 7120 (Masukawa et al., 2002) the effect of a

hox-defective mutant (DhoxH) has been investigated. A

Nostoc sp. PCC 7120 mutant deficient in both hydrogenases

(DhupL/DhoxH) showed the same increase in H2 evolution as

the uptake hydrogenase-deficient mutant (DhupL), whereas

the bidirectional hydrogenase-deficient mutant (DhoxH) pro-

duced less H2 compared with the wild type.

In gas exchange experiments with an uptake hydrogenase-

deficient mutant of Nostoc punctiforme (Lindberg et al.,

2004), the amount of H2 produced per molecule of N2 fixed

varied with the light conditions. The ratio of H2 produced/

N2 fixed under low light and high light was 1.4 and 6.1,

respectively. This showed that, under the specific conditions,

the energy flow through the nitrogenase may be directed

towards the H2 production rather than the N2 fixation.

(2) Low energy efficiency and turnover of the nitrogenase

and/or the hydrogenase: H2-evolving enzymes with the high-

est reported turnover are the Fe-hydrogenases (Houchins,

1984; Adams, 1990). These enzymes are irreversibly inacti-

vated by oxygen, and are present in e.g. fermentative bacteria

(e.g. Clostridium) and green algae (e.g. Chlamydomonas) but

not in cyanobacteria. An elegant strategy for the creation of

an efficient H2 producer, which will not be inhibited by the

surrounding oxygen, would be the expression of a highly

active Fe-hydrogenase in the heterocysts of filamentous

cyanobacteria unable to reoxidase any H2 (i.e. an uptake

hydrogenase-deficient strain). The heterologous expression

of different iron-hydrogenases in various organisms such as

Synechococcus (Asada et al., 2000), E. coli (Posewitz et al.,

2004; King et al., 2006) and Clostridium (Girbal et al., 2005)

has already been achieved. Recently, the accessory genes

necessary for the maturation of iron-hydrogenases into

active enzymes were identified (Posewitz et al., 2004; Bock

et al., 2006; King et al., 2006). Therefore, the heterologous

expression of an active iron-hydrogenase in a cyanobacterial

host, e.g. in the heterocyst of a strain for which the genome

has been sequenced, is an interesting and realistic project.

Moreover, because the iron-hydrogenases are able to use a

wide variety of primary electron donors (Vignais et al.,

2001), including ferredoxin, which is the electron donor of

the cyanobacterial nitrogenase, it may be possible to link the

introduced iron-hydrogenase to an existing electron transfer

pathway within the cyanobacterial cell.

FEMS Microbiol Rev 31 (2007) 692–720 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

711Cyanobacterial hydrogenases

(3) Limiting amounts of active H2-evolving enzymes:

Because the H2-evolving enzymes (nitrogenase(s) and/or

bidirectional hydrogenases) are strictly regulated on several

different levels (transcription, translation and maturation),

one possible way to enhance the production of H2 may be

the overexpression of these enzymes. For this purpose, the

genes encoding the selected enzyme/protein to be over-

expressed are placed under the control of an artificial

promoter and ribosomal-binding site (RBS) combination

on an expression vector or placed directly in the genome.

The choice of a constitutive or an inducible promoter,

together with a strong RBS, takes the enzyme biosynthesis

out of the control of the organism’s natural regulation

system, allowing a significant increase in the amount of

enzyme produced.

In heterocystous cyanobacteria grown under N2-fixing

conditions, c. 5–10% of the vegetative cells differentiate into

heterocysts. These specialized cells are compartments with

reduced O2 pressure, and thus suitable for H2 production,

either via nitrogenase or via an introduced hydrogenase.

Therefore, the increase of the heterocyst frequency should

result in a higher overall H2 production capacity by the

organism. Interestingly, it has been shown that the hetero-

cyst frequency can be increased, e.g. by overexpression of

hetR or by inactivation of patS, or hetN (Buikema &

Haselkorn, 2001; Golden & Yoon, 2003; Borthakur et al.,

2005). However, this has not been coupled to H2 production

or increased H2 production.

(4) High oxygen sensitivity of the nitrogenase and/or the

hydrogenase: One main obstacle in H2 production using

photosynthetic microorganisms is the high sensitivity of the

H2-evolving enzymes, and some attempts have been made to

introduce less oxygen sensitive hydrogenases into cyanobac-

teria. At the Craig Venter Institute (US), work is being

carried out aiming at transferring the O2-tolerant NiFe-

hydrogenase of the purple-sulfur photosynthetic bacterium

T. roseopersicina (Kovacs et al., 2005) into a Synechococcus

strain. In addition, several other putative O2-tolerant NiFe-

hydrogenases have been identified from the marine environ-

ment that could be alternative candidates to be introduced

into a cyanobacterial background (Xu et al., 2005). Also in

the US, the genes encoding the more O2-tolerant NiFe

hydrogenase of the purple nonsulfur photosynthetic bacter-

ium Rubrivivax gelatinosus CBS (Ghirardi et al., 2005), and

its accessory proteins, are being introduced into Synechocys-

tis. The in vivo half-life of this hydrogenase is 21 h in air and

6 h in air when the protein is partially purified (Ghirardi

et al., 2005). However, to the authors’ knowledge, the

heterologous expression of a more oxygen-tolerant hydro-

genase in any cyanobacterium remains to be shown.

(5) Electron flow inhibition by accumulation of ATP in a

hydrogenase-driven system: In an optimal H2 production

system, all electrons derived from water splitting in PSII

should be directed to the H2-evolving enzyme (nitrogenase

or hydrogenase) to reach maximal energy conversion effi-

ciency. In addition to the electron flow in the photosynthetic

electron transport chain, a transmembrane potential is built

up that is used for generating ATP through an ATP synthase.

In a nitrogenase-based system, the ATP is clearly needed for

N2 fixation. However, in the hydrogenase-catalyzed reaction

no ATP is consumed and the electron flow could, in a

photosynthetic microorganism, be inhibited by the accu-

mulated transmembrane potential across the thylakoid

membrane (Lee & Greenbaum, 2003). It has been observed

in the green algae Chlamydomonas reinhardtii that oxygen

serves as an electron sink, competing for electrons with the

H2-producing pathway, resulting in a ‘new oxygen sensitiv-

ity’ (Lee & Greenbaum, 2003). A possible solution may be

the introduction of a synthetic, polypeptide based on the

proton channel into the thylakoid membranes, with its

transcription controlled by a hydrogenase promoter (Lee &

Greenbaum, 2003). The proton channel may be expressed

under H2-producing (anaerobic) conditions, to dissipate the

proton gradient across the thylakoids membrane. A similar

strategy may work for cyanobacteria.

(6) Low quantum efficiency due to too large antennas in

both PSII and PSI: In nature, phototrophic organisms have

developed to handle and compete during very low light

intensities, and, therefore, large antenna systems have been

evolved. However, for biotechnological applications in

photobioreactors, where an optimal supply with light can

be engineered, only a small part of the light will be used by

the microorganisms. In addition, self-shading may become

a severe limitation causing reduced efficiency in utilizing

incoming solar energy. To overcome this problem, a reduc-

tion of the antenna size has been proposed (Melis et al.,

1998; Nakajima & Ueda, 1999; Lee et al., 2002). The

phycocyanin-deficient mutant PD1 of Synechocystis PCC

6714, generated by chemical mutagenesis, showed up to

50% higher maximal photosynthesis activity under high

light conditions compared with the wild type (Nakajima &

Ueda, 1997, 1999). Antenna-deficient green algal mutants,

created by chemical mutagenesis (Lee et al., 2002) or genetic

engineering (Polle et al., 2003; Tetali et al., 2007), also

showed remarkably greater solar conversion efficiencies and

a higher photosynthetic productivity than the respective

wild type under mass culture conditions.

(7) Electron-consuming pathways competing with an effi-

cient electron transfer to the H2 enzymes: For the production

of H2 through the action of an active nitrogenase or

hydrogenase electrons are required to combine with protons

to form H2. Because protons are abundant within the cell,

the main limitation is the available number of electrons. The

primary electron donors for the H2-producing enzymes

are ferredoxin (nitrogenase, Fe-hydrogenases) and NADH/

NADPH (NiFe-hydrogenases). However, the electrons are

FEMS Microbiol Rev 31 (2007) 692–720c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

712 P. Tamagnini et al.

mainly used by other pathways, the so-called ‘competing

pathways’, e.g. respiration and the Calvin cycle. Therefore,

one strategy for an enhanced H2 production is to direct the

electron flow towards the H2-producing enzymes and away

from any other competing pathway.

Experiments with the ndhB mutant M55 of Synechocystis

PCC 6803, which is defective in the type I NADPH-

dehydrogenase complex (NDH-1) (Cournac et al., 2004),

showed that this mutant produces only low amounts of O2

in the light, has a poor capacity to fix CO2 and evolves H2

for several minutes during dark-to-light transitions, while

the H2 uptake was negligible. The electrons used to produce

H2 were mainly coming from water splitting in PSII and

from the carbohydrate-mediated reduction of the PQ pool.

In another study, the level of reduced NADP shifted from

50% in the wild type to 100% in the NDH-1 mutant (Cooley

& Vermaas, 2001). As the cyanobacterial bidirectional hy-

drogenase evolves H2 at a relatively high level of reduced

NAD(P), the construction of mutants with blocked electron

transfer in selected key pathways, increasing the relative level

of reduced NAD(P), may be a promising strategy to increase

the H2 production capacity.

Another strategy for directing electrons toward the hy-

drogenase is to directly link the hydrogenase to PSI.

Ihara et al. (2006b) fused the membrane-bound NiFe

hydrogenase from Ralstonia eutropha H16 to the peripheral

PSI subunit PsaE of the cyanobacterium Thermosynechococ-

cus elongatus (Hyd-PsaE), and used a PsaE-free PSI

(PSI�) extract from a PsaE-deficient mutant of Synechocystis

sp. PCC 6803. The resulting hydrogenase/PSI complex

showed light-driven hydrogen production in vitro, which

was five times higher compared with a control without a

direct coupling of the hydrogenase to PSI. However, as the

activity of the hydrogenase-PsaE fusion protein was only

16% of that of the wild-type hydrogenase protein and was

totally suppressed by adding ferredoxin (Fd) and ferredox-

in-NADP1-reductase (FNR), the authors concluded that the

linker between the hydrogenase and PsaE has to be opti-

mized. In another work by the same group (Ihara et al.,

2006a), PsaE from Synechocystis sp. PCC 6803 was

chemically cross-linked with cytochrome c3 (cytc3) from

Desulfovibrio vulgaris and stoichiometrically assembled with

PsaE-free PSI to form a cytc3/PSI complex. The NADPH

production by this complex coupled with Fd and FNR

decreased to c. 10% of the original activity, whereas the H2

production by the cytc3/PSI complex coupled with hydro-

genase from Desulfovibrio vulgaris was enhanced sevenfold.

This clearly demonstrated that it is possible, in vitro, to

direct the electron-flow toward the hydrogenase by changing

the environment of the electron-donating PSI. The

next challenge will be to develop an in vivo, or even

in situ, functional system based on the assembly of different

enzymes and proteins.

As a future perspective, the development of synthetic

biology reveals new possibilities for the direct construction

of efficient H2-evolving cyanobacterial strains. Both in the

US (e.g. the Craig Venter Institute) and in Europe (e.g. the

EU/NEST project ‘BioModularH2’), the first attempts have

been initiated to use this new concept aiming at designing

reusable, standardized molecular building blocks that will

produce a photosynthetic bacterium containing engineered

chemical pathways for competitive, clean and sustainable H2

production.

Concluding remarks

The fundamental aspects of cyanobacterial hydrogenases,

and their more applied potential use as future producers of

renewable H2 from sun and water, are receiving increased

international attention. At the same time, significant progress

is being made in the understanding of the molecular regula-

tion of the genes encoding both the enzymes as well as the

accessory proteins needed for the correct assembly of an

active hydrogenase. In the last few years, the transcription

factors directly involved in the regulation of cyanobacterial

hydrogenases have been identified. Moreover, the first steps to

use isolated components from cyanobacteria and other

microorganisms in order to create a functional H2-producing

unit are being taken. With the increasing scientific commu-

nity and public interest in clean and renewable energy

sources, and consequent funding opportunities, rapid pro-

gress will be made in the fundamental understanding of the

regulation and maturation of cyanobacterial hydrogenases at

both genetic and protein levels. Unique and unexpected

results in the transcriptional regulation of cyanobacterial

hydrogenases will emerge during the coming years. Moreover,

the more applied aspects will be highlighted with progress in

generating genetically modified strains with an increased

capacity for renewable H2 from sun and water. The possibi-

lities and challenges within synthetic biology, including the

use of isolated proteins and parts, will be explored, aiming at

creating both cyanobacteria with a high potential for H2

production as well as functional in vitro systems.

Acknowledgements

This work was financially supported by FCT (POCTI/BIO/

44592/2002; SFRH/BD/4912/2001, SFRH/BD/16954/2004),

ESF (III Quadro Comunitario de Apoio), the Swedish

Research Council, the Swedish Energy Agency, the Nordic

Energy Research Program (project BioHydrogen), the EU/

NEST Projects SOLAR-H (contract # 516510) and BioMo-

dularH2 (contract # 043340).

FEMS Microbiol Rev 31 (2007) 692–720 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

713Cyanobacterial hydrogenases

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