Tamagnini Peter Hydrogenase Review 2007

REVIEW ARTICLE
Cyanobacterial hydrogenases: diversity, regulation and applications

Paula Tamagnini1,2, Elsa Leitao1, Paulo Oliveira3, Daniela Ferreira1,2, Filipe Pinto1, David James Harris4,5,
Thorsten Heidorn3 & Peter Lindblad3
1
IBMC 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; and
5
Departamento de Zoologia e Antropologia, Faculdade de Ciencias da Universidade do Porto, Porto, Portugal
Correspondence: Paula Tamagnini, IBMC Abstract

Instituto de Biologia Molecular e Celular, Rua
do Campo Alegre, 823. 4150-180 Porto,
Cyanobacteria may possess two distinct nickel-iron (NiFe)-hydrogenases: an
Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

Portugal. Tel.: 1351 2260 74900; fax: 1351 uptake enzyme found in N2-fixing strains, and a bidirectional one present in both
2260 99157; e-mail: pmtamagn@ibmc.up.pt 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
Received 5 January 2007; revised 12 July 2007; bidirectional enzyme (hoxEFUYH) probably plays a role in fermentation and/or
accepted 9 August 2007. acts as an electron valve during photosynthesis. hupSL constitute a transcriptional
First published online October 2007. unit, and are essentially transcribed under N2-fixing conditions. The bidirectional
hydrogenase consists of a hydrogenase and a diaphorase part, and the correspond-
DOI:10.1111/j.1574-6976.2007.00085.x
ing five hox genes are not always clustered or cotranscribed. The biosynthesis/
maturation of NiFe-hydrogenases is highly complex, requiring several core
Editor: Annick Wilmotte
proteins. In cyanobacteria, the genes that are thought to affect hydrogenases
Keywords
pleiotropically (hyp), as well as the genes presumably encoding the hydrogenase-
cyanobacteria; hydrogenase; hup ; hox ; hyp ; specific endopeptidases (hupW and hoxW) have been identified and characterized.
transcriptional regulator. 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.
Fossil traces of cyanobacteria are claimed to have been

Introduction found from around 3.5 billion years ago (Schopf, 2000), and
Cyanobacteria, one of the largest and most important ancestors of cyanobacteria most probably played a key role
groups of bacteria on Earth, are able to perform oxygenic in the formation of atmospheric oxygen, and are thought
photosynthesis using water as an electron donor and may be to have evolved into present-day chloroplasts of algae and
found in almost any ecological niche from fresh to salt water, green plants (Miyagishima, 2005; Mulkidjanian et al., 2006).
terrestrial and extreme environments (Whitton & Potts, Cyanobacteria display a relatively wide range of morpholo-
2000). The knowledge on such a diverse group of prokar- gical diversity, including unicellular, filamentous and colo-
yotic organisms has greatly increased since cyanobacterial nial forms. Some filamentous strains form differentiated
genomes became available. In 1996, the entire sequence of cells specialized in nitrogen fixation heterocysts, and
Synechocystis sp. PCC 6803 was published (Kaneko et al., spore-like resting cells akinetes. A number of nonhetero-
1996; Nakamura et al., 1998), and since then, many other cystous strains are also able to perform N2 fixation under
cyanobacterial genome projects have been completed and certain conditions. The fact that several cyanobacteria
released, including that of Nostoc punctiforme ATCC 29133/ are able to reduce nitrogen and carbon under aerobic
PCC 73102, one of the largest microbial genomes sequenced conditions may be responsible for their evolutionary and
so far (Meeks et al., 2001; Anderson et al., 2006). ecological success. In cyanobacteria, as in any diazotrophic

c 2007 Federation of European Microbiological Societies FEMS Microbiol Rev 31 (2007) 692720
Published by Blackwell Publishing Ltd. All rights reserved
Cyanobacterial hydrogenases 693
bacteria, the reduction of N2 to NH3 is accompanied by the of the HupL and the HupS proteins do not indicate any
formation of molecular hydrogen (Berman-Frank et al., transmembrane domains (Tamagnini et al., 2005), the
2003). The H2 produced by the nitrogenase is rapidly existence of a polypeptide that anchors the HupSL hetero-
consumed by an uptake hydrogenase, an enzyme that has dimer to the membrane seems likely. In fact, analysis of the
been found in almost all the N2-fixing cyanobacteria exam- available genomes revealed the presence of ORFs whose
ined so far, with one reported exception Synechococcus sp. products could potentially fulfill this anchoring role (Lind-
BG 043511 (Ludwig et al., 2006). Additionally, these strains berg, 2003). However, to date no definitive proof was
may contain a bidirectional hydrogenase, an enzyme that is obtained, and the existence of both a soluble and a mem-
generally present in the non nitrogen-fixing cyanobacteria brane-bound form of the enzyme cannot be excluded (see
(Tamagnini et al., 2002, 2005), but absent in Gloeobacter for e.g. Houchins & Burris, 1981b).
violaceus PCC 7421, a cyanobacterium that possesses a Immunolocalization studies, using antibodies produced
number of unique characteristics such as the absence of against hydrogenases from other bacteria, showed that the
thylakoids (Nakamura et al., 2003; Ludwig et al., 2006). The hydrogenase antigens are present in both the vegetative cells
distribution of genes related to hydrogenases among repre- and heterocysts of N. punctiforme, and several symbiotic
sentative cyanobacterial strains is displayed in Table 1. Both Nostoc strains (Lindblad & Sellstedt, 1990; Rai et al., 1992;
cyanobacterial hydrogenases are NiFe enzymes, which are Tamagnini et al., 1995). However, these studies do not

the most common hydrogenases found in bacteria and clarify whether the enzyme is in its active form in both
Archaea. The core enzyme consists of an ab heterodimer cell types. In Anabaena/Nostoc sp. PCC 7120, the uptake
with the large/a subunit hosting the bimetallic active site, hydrogenase activity was essentially associated with the
and the small/b-subunit containing the FeS clusters, which particulate fraction of the heterocysts (Houchins & Burris,
function as electron transfer domains between the electron 1981b); however, one must bear in mind that in this strain
acceptors/donors and the catalytic center of the enzyme the hupL gene undergoes a rearrangement, allowing its
(Fig. 1). In general, the NiFe hydrogenases are divided into expression in the heterocysts only, and that this process does
four groups, with the cyanobacterial uptake hydrogenases not occur in N. punctiforme (Oxelfelt et al., 1998). Moreover,
clustering together with the cytoplasmic H2 sensors of group the presence/levels of the cyanobacterial uptake hydrogenase
2, and the bidirectional enzymes belonging to group 3 are certainly dependent on the growth conditions. In
comprising the bidirectional heteromultimeric cytoplasmic heterocystous cyanobacteria grown in air and without
hydrogenases (for reviews on this subject, see Vignais et al., combined nitrogen, the uptake hydrogenase activity is
2001; Vignais & Colbeau, 2004). mainly confined to heterocysts, where it is protected from
In the present review, recent advances on cyanobacterial oxygen inactivation; however, the exact location of the
hydrogenases, have been summarized focusing on achieve- enzyme in cyanobacteria should be further investigated in
ments on the diversity and molecular regulation of both the both heterocystous and nonheterocystous strains.
uptake and the bidirectional enzyme. A strong correlation between the nitrogen-fixation pro-
Photobiological production of H2 by microorganisms is cess and the uptake hydrogenase activity has been demon-
of great public interest because it promises a renewable strated for cyanobacteria (Lambert & Smith, 1981;
energy carrier from natures most plentiful resources: solar Houchins, 1984; Wolk et al., 1994; Oxelfelt et al., 1995;
energy and water. Cyanobacteria and green algae are the Schutz et al., 2004), and this indicates that the main
only organisms known so far that are capable of both physiological function of the uptake hydrogenase is to
oxygenic photosynthesis and hydrogen production. In a reutilize and regain the H2/electrons produced by the H2
separate section, the possibilities and challenges in cyano- evolution through the nitrogenase. This recycling has been
bacterial-based hydrogen production are outlined. 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
Uptake hydrogenase
from nitrogenase, thereby protecting it from inactivation;
The cyanobacterial uptake hydrogenase, found exclusively in and (3) it supplies reducing equivalents (electrons) to
N2-fixing strains and encoded by the hup hydrogen uptake various cell functions (Bothe et al., 1977, 1991; Howarth &
genes, is at least a heterodimeric enzyme with a large Codd, 1985; Weisshaar & Boger, 1985; Smith, 1990).
subunit of about 60 kDa containing the active site (HupL)
and a small subunit of c. 35 kDa playing a role in electron
Physical organization of hup genes and the
transfer (HupS) (Fig. 1). Because the physiological and
corresponding proteins
biochemical data point to a membrane-bound enzyme
(Houchins & Burris, 1981b; Houchins, 1984; Lindblad & The physical arrangement of the structural genes encoding
Sellstedt, 1990; Rai et al., 1992), and the hydropathy profiles the uptake hydrogenase is very similar in all the
FEMS Microbiol Rev 31 (2007) 692720

c 2007 Federation of European Microbiological Societies
c

694
Table 1. Distribution of genes related to hydrogenases in representative cyanobacterial strains

Bidirectional Other GenBank
Bidirectional
Uptake hupL specific Uptake specific maturation accession
hydrogenase
hydrogenase recombinase endopeptidase endopeptidase genes number/
Organisms hoxFUYH hoxE hupSL XisC hoxW hupW hypFCDEAB References
Unicellular G. violaceus NC_005125
non-N2-fixing PCC 7421 Nakamura et al.

2007 Federation of European Microbiological Societies
(2003)
Synechocystis sp. 1 1 1 1 NC_000911
PCC 6803 Appel & Schulz Scattered Kaneko et al.
(1996) (1996)
Unicellular C. watsonii 1 1 1 NZ_ADV00000000
N2-fixing WH 8501 Scattered
Filamentous L. majuscula 1 ND 1 ND 1 1 Leitao et al. (2005,

nonheterocystousCCAP 1446/4 Operon 2006)
N2-fixing T. erythraeum 1 1 1 NC_008312
IMS 101
Filamentous A. variabilis 1 1 1 1 1 1 NC_007413

heterocystous ATCC 29413 Schmitz et al. Happe et al. (2000)
N2-fixing (1995)
Nostoc sp. 1 1 1 1 1 1 1 NC_003272
PCC 7120 Carrasco et al. (1995) Gubili & Borthakur Kaneko et al. (2001)
(1996, 1998)
N. punctiforme 1 1 1 NZ_AAAY00000000
PCC 73102 Oxelfelt et al. (1998) Operon
Hansel et al. (2001)
Rearragement occurring during the differentiation of a vegetative cell into a heterocyst.
ND, not determined.
P. Tamagnini et al.

Nitrogenase Bi-
Bi-directional hydrogenase
N2+ H+
NifK NifD H2 HoxH HoxE NADH

NifH HoxU

e NH
NifH NifD NifK 2H++ 2e HoxY HoxF NAD+

Dinitrogenase H2
reductase Dinitrogenase Hydrogenase Diaphorase
Hox(EFUYH)2
HupL
2H++ 2e
HupS

HupC
?
Uptake hydrogenase
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.
cyanobacteria studied so far: hupS and hupL are contiguous, Analysis of the predicted proteins encoded by the hupSL
with the gene encoding the smaller subunit located up- operon demonstrated that whereas HupS has the same
stream from the gene encoding the larger one (Carrasco number of amino acid residues in all the cyanobacteria
et al., 1995; Oxelfelt et al., 1998; Happe et al., 2000; Lindberg investigated [320 amino acids (aa)], HupL generally has
et al., 2000; Oliveira et al., 2004; Leitao et al., 2005) (Fig. 2). 531 aa with the exception of the filamentous nonheterocys-
Transcriptional start sites have been identified upstream of tous L. aestuarii CCY 9616 (six extra), L. majuscula (six extra),
the hupS start codon (Happe et al., 2000; Lindberg et al., and T. erythraeum (three extra). To date, the physiological
2000; Oliveira et al., 2004; Leitao et al., 2005), and a putative significance (if any) of these extra residues is still unknown.
transcriptional terminator, located immediately down- In the NiFe hydrogenases, the large subunit harbors the
stream of hupL, has been found in N. punctiforme (Lindberg active center that is deeply buried inside the protein, close to
et al., 2000). In agreement, reverse transcriptase (RT)-PCR the large interface between the two subunits, and the small
experiments, and the sizes of transcripts determined by subunit contains the FeS clusters that conduct electrons
Northern blot, indicate that hupSL constitute a transcrip- between the active center and the physiological electron
tional unit in Anabaena variabilis ATCC 29413, N. puncti- acceptor/donor (Vignais et al., 2001; Vignais & Colbeau,
forme and Lyngbya majuscula CCAP 1446/4 (Happe et al., 2004). In concordance, the cyanobacterial HupL sequences
2000; Lindberg et al., 2000; Leitao et al., 2005). In the contain the four conserved cysteine residues that are in-
unicellular Gloeothece sp. ATCC 27152 and in the filamen- volved in the coordination of the bimetallic NiFe center of
tous Trichodesmium erythraeum IMS 101 hupW the gene the active site, and HupS contains eight cysteine residues
encoding for the putative uptake hydrogenase-specific endo- clearly corresponding to those involved in the formation of
peptidase is the ORF located immediately downstream of the FeS clusters, and a ninth cysteine slightly shifted
hupL, and was shown to be cotranscribed with hupSL in compared with other bacteria (Tamagnini et al., 2002). In
Gloeothece sp. ATCC 27152 (Oliveira et al., 2004). In other addition, HupL contains the C-terminal region that is
strains, the position of hupW related to the hupSL varies presumably cleaved off, by a specific endopeptidase, as the
considerably, and in the strains examined they are tran- last step of the maturation of the large subunit. In contrast
scribed independently (Wunschiers et al., 2003) (Fig. 2). with other organisms, HupS lacks both the twin-arginine

c2007 Federation of European Microbiological Societies
696 P. Tamagnini et al.
(a)
Gloeothece sp. ATCC 27152 1000 bp
hup
S L W
Trichodesmium erythraeum IMS101

hyp hup
B A E D C F S L W
~ 589 kb
Tery0801 Tery0799 Tery0790

Tery0800

Lyngbya majuscula CCAP 1446/4
hyp hup
B A E D C F S L W
ORF1 ORF3 ORF4 ORF5 ORF6 ORF7 ORF9 ORF11

ORF2 ORF8 ORF10
Nostoc punctiforme ATCC 29133 / PCC 73102

hyp hup
BA E D C F S L W
NpR0363 NpR0367 NpR0370 NpF0371

NpR0364 NpF0372
NpR0365 NpF0373
NpR0366
Nostoc sp. PCC 7120

hup hyp hup
W BA E D C F S L
~ 880 kb
asr0697 alr0693 alr0692 asr0690 all0675

alr0691 asr0689
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).

(b)
1000 bp
Synechocystis sp. PCC 6803
hyp hox hyp
F A1 B1 C E F U Y H W E A2B2 D
~ 477 kb ~ 47 kb ~ 150 kb ~ 79 kb ~ 445 kb ~ 236 kb ~ 192 kb ~743 kb
sll1222 ssl2420
sll1225
Synechococcus elongatus PCC 7942

hox hox hyp hyp
F E U Y H W AB F C D E
~ 334 kb ~ 172 kb ~ 586 kb

Arthrospira platensis FACHB 341
hox
E F U Y H
unknown
Anabaena variabilis ATCC 29413

hyp hox
F C D E A B E F U Y H W
~ 52 kb
Ava4605 Ava4652 Ava4655 Ava4658 Ava4662 Ava4664

Ava4656 Ava4660 Ava4663
Nostoc sp. PCC 7120

hyp hox
F C D E A B E F U Y H W
~ 65 kb ~ 8.8 kb
asr0697 alr0750 alr0763 alr0765 all0767 all0769
asl0749 all0768
Fig. 2. Continued
signal peptide at the N-terminal, and the hydrophobic motif hydrogenases cluster them together with the soluble H2-
at the C-terminal proposed to be involved in translocation sensing enzymes (Vignais et al., 2001; Vignais & Colbeau,
and anchorage to the membrane, respectively. As mentioned 2004). However, the construction of hup mutants proved
previously, these general features of the cyanobacterial that the cyanobacterial uptake hydrogenase is indeed a

physiological functional enzyme rather than a regulatory bination in Nostoc sp. PCC 7120 (Carrasco et al., 2005). It
one (Happe et al., 2000; Lindberg et al., 2002; Lindblad et al., was also shown that the xisC-mutant forms heterocysts
2002; Masukawa et al., 2002). 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
hupL rearrangement in heterocystous strains H2 (Lindblad et al., 2002; Carrasco et al., 2005). Moreover,
Lindblad et al. (2002) showed that, in a competitive growth
Programmed DNA rearrangements have been described in
environment with increased light intensity, the wild-type
eukaryotes and prokaryotes but are relatively uncommon
strain has an advantage over the xisC-mutant, probably
events. In cyanobacteria, developmentally regulated DNA
because these specific conditions induced higher rates of
rearrangements have been reported to occur in heterocys-
H2 evolution that only the wild type has the capacity of
tous strains (for a review, see Golden, 1997). Generally, the
reutilizing through the oxyhydrogen reaction. These find-
ORF is interrupted in the vegetative cells by a 1060-kb
ings support the hypothesis that the uptake hydrogenase
DNA element, which is excised during the differentiation of
plays a role in minimizing the loss of energy caused by the
a photosynthetic vegetative cell into a N2-fixing heterocyst,
nitrogenase-dependent H2 formation.
restoring the structure of the gene/operon and allowing its
Despite the hupL element being absent from the two other
expression in heterocysts only.

heterocystous strains for which genome sequences are avail-
The rearrangement within hupL (large subunit of the
able, A. variabilis and N. punctiforme (see also Oxelfelt et al.,
uptake hydrogenase) was first described for Nostoc sp. PCC
1998; Happe et al., 2000), DNA hybridization studies
7120 (Carrasco et al., 1995). In the vegetative cells of this
showed that sequences similar to xisC were present in about
cyanobacterium, hupL is interrupted by a 9.5-kb element
half of the heterocystous strains tested (Tamagnini et al.,
that is excised late during the heterocyst differentiation
2000). These authors also showed that the presence of the
process by a site-specific recombination between the 16-bp
bidirectional hydrogenase is not ubiquitous among hetero-
direct repeats that flank the element (Fig. 3). The hupL
cystous cyanobacteria, although they could not establish a
element contains, in one of its borders, the gene that encodes
correlation between the presence/absence of the bidirec-
the recombinase necessary for the excision xisC (Carrasco
tional enzyme and hupL rearrangement.
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
hupSL intergenic region
unequivocally demonstrated that the inactivation of xisC
blocks the hupL rearrangement and that XisC alone is The regions between hupS and hupL in cyanobacteria are
sufficient to catalyze the hupL element site-specific recom- longer than in other microorganisms, differ considerably in
Vegetative cell Heterocyst
9.5 kb ?
9.5 kb element
containing xisC
hupS hupL
hupS 5
5 3hupL
Uptake hydrogenase
Uptake hydrogenase enzyme
enzyme
enzyme ?
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.

Table 2. Size and occurrence of repetitive sequences within the region between of hupS and hupL in cyanobacteria
Repetitive GenBank accession
Organism Size (bp) sequences 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.
size (ranging from 43 to 689 bp; see Table 2) and are not coupling between hupS and hupL by sequestering the ribo-
particularly conserved (except for Nostoc sp. PCC 7120 and some-binding site of hupL and thereby preventing the
A. variabilis). A prominent feature within the hupSL inter- initiation of translation of this gene (Lindberg et al., 2000).
genic region of heterocystous strains is the presence of Short However, although the sequestration of the hupL RBS may
Tandemly Repeated Repetive (STRR) sequences (with the be effective in N. punctiforme in which the hairpin folds the
exception of the relatively short 43-bp region of Nostoc sp. entire hupSL intergenic region (Lindberg et al., 2000), it
Mitsui 38901). STRR sequences have previously been shown does not occur in all hupSL intergenic hairpin structures
to be frequent in heterocyst-forming cyanobacteria and predicted. Only the construction of specific mutants will
relatively less frequent in unicellular strains (Asayama et al., help to clarify the function of these intergenic regions.
1996). Indeed, no STRR sequences could be discerned in the
hupSL intergenic region from nonheterocystous cyanobac-
teria. However, in the filamentous nonheterocystous L.
hup promoter regions and transcriptional
regulators
majuscula only about 10% of the intergenic region consists
of nonrepetitive nucleotides, with two distinct sets of Long As mentioned previously, in all cyanobacteria studied so far
Repeated Repetitive (LRR) sequences clearly identified (for the uptake hydrogenase structural genes are arranged in a
details see Leitao et al., 2005). Because the repetitive contiguous manner with the gene encoding the smaller
sequences within the hupSL intergenic region are highly subunit located upstream of the gene of the larger one. The
variable or even absent (Table 2), it is unlikely that these transcriptional start sites of the hup operons are localized
repeats play a direct role in the regulation of gene expres- 238, 59, 103 and 259 bp upstream from the hupS start codon
sion. However, in all strains, a putative stem-loop structure, for the unicellular Gloeothece sp. ATCC 27152, the filamen-
derived via 2D-computer modeling, might occur in the tous L. majuscula and the filamentous heterocystous A.
transcribed RNA (Lindberg et al., 2000; Tamagnini et al., variabilis and N. punctiforme, respectively (Happe et al.,
2002, 2005). The value of free energy (DG) was determined 2000; Lindberg et al., 2000; Oliveira et al., 2004; Leitao et al.,
for each secondary structure and it was negative in all cases 2005) (Fig. 4). The analysis of the regions upstream the
(ranging from 136.32 to 6.9 kcal mol1), meaning that transcriptional start point (tsp) revealed the presence
the formation of the hairpin is favored. It has been hypothe- of a 10 and a 35 box in both L. majuscula and
sized that the occurrence of the hairpin may increase the N. punctiforme, while in Gloeothece sp. ATCC 27152 and A.
stability of the transcript, and/or confer a translational variabilis only a 10 box could be clearly discerned. A putative

hox
hup
+1 +1
NtcA IHF LexA2 LexA1 LexA2 LexA1
259 bp hupS 168 bp hoxE
35 10 35 10
Nosctoc punctiforme PCC 73102 Synechocystis sp. PCC 6803
+1
hyp
Fnr
103 bp +1
hupS
10
NtcA2 NtcA1
Anabaena variabilis ATCC 29413 21 bp ORF
35 10
+1 Nostoc punctiforme PCC 73102

NtcA IHF

59 bp hupS +1
35 10
NtcA2 NtcA1
Lyngbya majuscula CCAP 1446/4 17 bp hypF
LexA 10
+1 Lyngbya majuscula CCAP 1446/4
NtcA
238 bp hupS
10
100 bp
Gloeothece sp. ATCC 27152
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).
binding site for NtcA (a protein that operates global nitro- 233.5 and 258.5, respectively, resembling class I CAP-
gen control in cyanobacteria) could be found in Gloeothece dependent promoters (Busby & Ebright, 1999; Herrero
sp. ATCC 27152, L. majuscula and N. punctiforme, although et al., 2001, 2004). These data indicate that the type of the
its relative position to the tsp varied depending on the NtcA-activated promoter (class I vs II) is not correlated to
strain. Moreover, in L. majuscula and N. punctiforme a the strategies used by heterocystous and nonheterocystous
possible binding site for the integration host factor (IHF) cyanobacteria to separate N2 fixation and photosynthesis. In
WATCAAN4TTR (Craig & Nash, 1984; Goodrich et al., the filamentous heterocystous A. variabilis, half of a se-
1990; Goodman et al., 1999) could be recognized in the quence motif identical to the consensus Fnr-binding se-
region between the NtcA motif and the tsp (Fig. 4). It has quence was identified 144-bp upstream of the tsp (Happe
been postulated that the possible binding of the IHF to the et al., 2000) (Fig. 4). Fnr is a regulator of a fumarate nitrate
promoter could bend the DNA (Friedman, 1988), and reductase, which has been found to be involved in the
consequently allow the contact of the NtcA with the RNA regulation of the hyp operon in Escherichia coli (Lutz et al.,
polymerase complex, activating the hupSL transcription. In 1991), and it is responsible for the induction of several
the unicellular Gloeothece sp. ATCC 27152, the potential operons in E. coli grown under anaerobic conditions (Spiro
NtcA-binding site is centered at 41.5 bp with respect to & Guest, 1990). In A. variabilis, although there is no
the tsp in place of the 35 box, like in the canonical NtcA- rearrangement of the hupL gene, hupSL are expressed in
activated promoters with the consensus sequence signature heterocysts only. These differentiated cells have very low
GTAN8TAC (Herrero et al., 2001), a structure similar to that intracellular O2 pressures which led Happe et al. (2000) to
of class II bacterial promoters activated by catabolite acti- suggest that the hupSL operon in A. variabilis could be
vator protein (CAP). In L. majuscula and N. punctiforme, the regulated in a manner similar to that of the anaerobically
NtcA-binding sites were found to be centered at positions induced operons in E. coli.

The possible interaction between NtcA and the hupSL(W) hydrogen uptake (dark) seems to be the most common
promoter regions in cyanobacteria was assessed by perform- strategy adopted by the later cyanobacteria (Bergman et al.,
ing band shift assays. These experiments indicate a specific 1997; Bohme, 1998; Berman-Frank et al., 2003). In fact, in
binding of NtcA to DNA sequences upstream of hupS in the the nonheterocystous Gloeothece sp. ATCC 27152 (unicellu-
three cyanobacterial strains tested (Gloeothece sp. ATCC lar) and L. majuscula (filamentous), grown under nitrogen-
27152, L. majuscula and N. punctiforme), suggesting, indeed, fixing conditions and 12 h light/12 h dark cycles, there is an
the involvement of NtcA in the transcription regulation of evident light/dark regulation with the highest levels of
the uptake hydrogenase gene cluster (Lindberg, 2003; hupSL(W) transcripts detected during the light phase or in
Oliveira et al., 2004; Leitao et al., 2005). The fact that the the transition between the light and dark phase, respectively
transcription of the uptake hydrogenase structural genes (Oliveira et al., 2004; Leitao et al., 2005). It has also been
is under the control of the transcriptional regulator that demonstrated that both organisms exhibit higher hydrogen-
operates global nitrogen control in cyanobacteria reinforces uptake activities during the dark period (in agreement with
the correlation observed between the activity of the the nitrogen fixation rates; see Reade et al., 1999; Lundgren
uptake hydrogenase and N2 fixation, already demonstrated et al., 2003). In L. majuscula, the increase of the HupL
in several filamentous heterocystous cyanobacteria (Hou- protein levels coincides with the increase of hydrogenase
chins, 1984; Wolk et al., 1994; Oxelfelt et al., 1995; Troshina uptake activity during the dark phase. In the beginning of

et al., 1996). 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
Transcription and expression patterns of
L. majuscula, a protein turnover occurs, with degradation
hup genes taking place during the light period and de novo synthesis
The first transcriptional data on cyanobacterial uptake taking place during the dark phase. The time difference
hydrogenases arose from RT-PCR experiments on Nostoc between the hupSL transcription and the hydrogen uptake
sp. PCC 7120, revealing that hupL is expressed only after a activity, both in Gloeothece sp. ATCC 27152 and L. majuscu-
photosynthetic vegetative cell differentiates into a N2-fixing la, might be due to the complexity of the maturation process
heterocyst (see above details about the DNA rearrangement of the uptake hydrogenase. Thus, it is possible that the
occurring within this strain, Carrasco et al., 1995, 2005). translation occurs as soon as the transcript is available, while
Subsequent studies with other filamentous heterocystous the enzyme becomes active only after the maturation
strains have shown that hupSL is a transcriptional unit process is completed. The temporal separation between
(Happe et al., 2000; Lindberg et al., 2000), present in cells the photosynthesis and nitrogen fixation/hydrogen uptake
grown under N2-fixing conditions (Axelsson et al., 1999; activity may also influence the time lag between transcrip-
Happe et al., 2000; Hansel et al., 2001). Non-N2-fixing tion and activity.
cultures of Nostoc muscorum, a strain without the hupL In the presence of combined nitrogen, hupSLW transcrip-
rearrangement, exhibit no in vivo H2-uptake activity (Ax- tion is totally repressed in Gloeothece sp. ATCC 27152, while
elsson et al., 1999). However, the transfer of N. muscorum in L. majuscula the levels of hupSL transcription and
cells from non-N2-fixing (ammonia) to N2-fixing condi- expression are significantly reduced but it is possible to
tions induced the appearance of a transcript (after c. 24 h), discern a pattern similar to the one observed in cells grown
and the relative amounts of transcript increased in parallel under N2-fixing conditions (Oliveira et al., 2004; Leitao
with the H2-uptake activity (Axelsson et al., 1999). A similar et al., 2005, Ferreira et al., 2007). The results obtained for
pattern of transcription was observed for A. variabilis L. majuscula under non-N2-fixing conditions could be ex-
and N. punctiforme, two other strains with noninterrupted plained by the mode of growth of this cyanobacterium, in
hupL genes (Happe et al., 2000; Hansel et al., 2001). These which the inner cells are probably not in the same conditions
authors demonstrated that hupSL transcripts were notably in terms of access to the combined nitrogen.
missing in A. variabilis and in N. punctiforme cells grown Besides the source of nitrogen, other factors were proven
with ammonia (and in A. variabilis cells grown with to influence the transcription/expression of the cyanobac-
nitrate), but were present in both organisms grown under terial uptake hydrogenases. Similar to any NiFe hydroge-
N2-fixing conditions. nase, the activity of the cyanobacterial uptake enzyme was
While the heterocyst provides a microaerobic environ- shown to be dependent on nickel availability, and the
ment protecting the oxygen-sensitive nitrogenases and up- addition of external nickel to the growth medium (up to a
take hydrogenases from the atmospheric and intracellulary certain concentration) increased the uptake hydrogenase
generated oxygen, the nonheterocystous cyanobacteria de- activity in several strains (Xiankong et al., 1984; Daday
veloped different approaches. The temporal separation et al., 1985; Kumar & Polasa, 1991; Oxelfelt et al., 1995;
between photosynthesis (light) and nitrogen-fixation/ Axelsson & Lindblad, 2002). Furthermore, the addition of

exogenous hydrogen was shown to induce hupSL transcrip- filamentous heterocystous strains (Nostoc spp. Tamagnini
tion and hydrogen uptake activity in N. muscorum and N. et al., 2000; Schutz et al., 2004). Furthermore, the increasing
punctiforme (Oxelfelt et al., 1995; Axelsson & Lindblad, number of cyanobacterial sequenced genomes is contribut-
2002), as well as hydrogen uptake activity in Nostoc sp. ing toward a better understanding of both the distribution
PCC 7120 (Houchins & Burris, 1981b). Both cyanobacterial and the diversity of this enzyme.
hydrogenases are affected by the oxygen partial pressure. The physiological function of the bidirectional hydroge-
Nostoc muscorum and N. punctiforme cultures transferred nase in cyanobacteria is not totally clear. It has been
from aerobic to anaerobic conditions showed an increase in suggested that the enzyme acts as an electron valve during
both the transcription of hupL and hydrogen uptake activity photosynthesis in Synechocystis sp. PCC 6803. This is based
(Axelsson & Lindblad, 2002). Similarly, the uptake hydro- on the fact that hoxH mutants are impaired in the oxida-
genase activity could be elicited by removing oxygen from tion of PSI, have higher fluorescence of PSII and have
the sparging gas of a culture of Nostoc sp. PCC 7120 different transcript levels of the photosynthetic genes psbA,
(Houchins & Burris, 1981b). The addition of organic carbon psaA and petB when compared with the wild type (Appel
to the culture medium can also influence the hydrogen et al., 2000). The enzyme has also been proposed to play a
uptake activity. Cells of N. punctiforme grown either photo- role in fermentation functioning as a mediator in the release
or chemoheterotrophically reach both higher nitrogenase of excess reducing power under anaerobic conditions (Stal &

and hydrogen uptake activities than photoautotrophically Moezelaar, 1997; Troshina et al., 2002). Furthermore, it has
grown cells (Oxelfelt et al., 1995). However, the effect of been suggested previously that the bidirectional hydroge-
carbon substrates on the cyanobacterial uptake hydrogenase nase could be part of the respiratory complex I (Appel &
activity is difficult to assess, and apparently contradictory Schulz, 1996; Schmitz & Bothe, 1996), because only 11
results are reported in the literature (Houchins, 1984; subunits out of 14 conserved subunits of the prokaryotic
Kumar et al., 1986; Chen et al., 1989; Margheri et al., 1991). 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
Bidirectional hydrogenase respiratory complex I (Schmitz et al., 1995). However, the
The soluble or loosely membrane associated cyanobacterial bidirectional hydrogenase has been demonstrated to be
bidirectional hydrogenase might be present in both N2- and absent from several cyanobacterial strains (Tamagnini et al.,
non-N2-fixing strains (Tamagnini et al., 2000, 2002). Initi- 1997, 2000; Schutz et al., 2004; Ludwig et al., 2006). More-
ally, the bidirectional hydrogenase was thought to be com- over, N. punctiforme, a strain naturally lacking the bidirec-
posed of four subunits (encoded by the hox hydrogen tional hydrogenase (Tamagnini et al., 1997), has rates of
oxidation genes), in which HoxFU constitute the diaphor- respiration comparable to cyanobacteria containing the
ase part, and HoxYH constitute the hydrogenase part bidirectional hydrogenase (Boison et al., 1999). In addition,
(Schmitz et al., 1995; Appel & Schulz, 1996; Boison et al., mutants of hoxU in Synechococcus sp. PCC 6301 (former
1996, 1998; Sheremetieva et al., 2002). However, because Anacystis nidulans) (Boison et al., 1998) and hoxEF in
HoxE was shown to copurify with the active bidirectional Synechocystis sp. PCC 6803 (Howitt & Vermaas, 1999)
enzyme, the cyanobacterial bidirectional hydrogenase is showed nonimpaired respiratory O2 uptake while being
considered to be a heteropentameric enzyme encoded by affected in H2 evolution. Furthermore, inactivation of hoxH
hoxEFUYH, HoxE belonging to the diaphorase part in Synechocystis sp. PCC 6803 and Nostoc sp. PCC 7120
(Schmitz et al., 2002). Bidirectional hydrogenases with more resulted only in a small decrease in the growth rate com-
than four subunits have also been identified in other pared with the respective wild types (Appel et al.,
bacteria, such as the photosynthetic purple sulfur bacteria 2000; Masukawa et al., 2002). Taking into account all the
Thiocapsa roseopersicina and Allochromatium vinosum which data, it seems that in general the bidirectional hydrogenase
contain heteropentameric cyanobacterial-type bidirectional does not play an essential role for cell survival in the strains
hydrogenases (Rakhely et al., 2004; Long et al., 2007), and where it is present.
Ralstonia eutropha, which possess two HoxI subunits besides Attempting to shed some light on the physiological
HoxFUYH (Burgdorf et al., 2005). In recent years, the function of the bidirectional hydrogenase, Cournac et al.
number of reports showing the presence of a functional (2004) demonstrated that the bidirectional hydrogenase in
and active bidirectional hydrogenase in cyanobacteria has Synechocystis sp. PCC 6803 is insensitive to light, reversibly
increased significantly, ranging from unicellular strains inactivated by O2 and can be quickly reactivated by NADH
(Gloeocapsa alpicola CALU 743 Sheremetieva et al., or NADPH. This work also reported H2 evolution by cells
2002; Troshina et al., 2002) to filamentous nonheterocystous incubated anaerobically in the dark, after an adaptation
(L. majuscula Schutz et al., 2004; Leitao et al., 2005; period. This dark H2 evolution was enhanced by exogen-
Arthrospira and Spirulina spp. Zhang et al., 2005a, b), and ously added glucose and resulted from the oxidation of

NAD(P)H produced by fermentation reactions. Upon illu- although interspersed with different ORFs at diverse posi-
mination, a short (o 30 s) burst of H2 output was observed, tions. In other cases, the hox genes are found in two different
followed by rapid H2 uptake, and a concomitant decrease in clusters separated by several kilobase (c. 333 and 8.8 kb in
CO2 concentration in the cyanobacterial cell suspension, Synechococcus sp. PCC 6301 and Nostoc sp. PCC 7120,
which were both linked to photosynthetic electron transport respectively). Despite this fact, the similarities at the de-
in the thylakoids (Cournac et al., 2004). Moreover, in this duced amino acid level of their homologous hydrogenase
experimental setup, in anoxia (or microaerobiosis) and in proteins range between 55% and 81%.
the presence of H2, H2 uptake was of the same magnitude as The bidirectional hydrogenase has been purified from
photosynthetic activity and could therefore contribute sig- several cyanobacterial strains: A. cylindrica (Hallenbeck &
nificantly to CO2 fixation. Therefore, although the bidirec- Benemann, 1978), Spirulina maxima (Llama et al., 1979),
tional hydrogenase in Synechocystis sp. PCC 6803 is Microcystis aeruginosa (Asada et al., 1987), Synechococcus sp.
constitutively expressed in the presence of O2 (Appel et al., PCC 6301 (Schmitz et al., 1995, 2002) and Synechocystis sp.
2000), it probably plays a role mainly under anaerobic or PCC 6803 (Schmitz et al., 2002), but the data collected by
microaerobic conditions, and at the onset of light before the Schmitz et al. (2002) finally helped to clarify the picture of
enzyme is inactivated by photosynthetic O2. In the ndhB the subunit composition and molecular mass of the cyano-
mutant M55, which is defective in the type I NADPH- bacterial bidirectional hydrogenase. Thus, it is widely ac-

dehydrogenase complex (NDH-1) and produces only low cepted that the bidirectional hydrogenase is composed of
amounts of O2 in the light, H2 uptake was negligible during five subunits, HoxE, HoxF, HoxU, HoxY and HoxH, with
dark-to-light transitions, allowing several minutes of con- apparent molecular weights of c. 20, 61, 28, 24 and 49 kDa,
tinuous H2 production. It was further shown that two respectively. The molecular weight of the native protein
pathways of electron supply for H2 production operate in (375 kDa) indicates a dimeric assembly of the enzyme
M55, namely photolysis of water at the level of photosystem complex, Hox(EFUYH)2 (Schmitz et al., 2002).
II and carbohydrate-mediated reduction of the plastoqui- Similar to the uptake hydrogenase, the large subunit of the
none pool. When comparing the features of the Synechocys- hydrogenase dimer (HoxH) harbors the active metal center
tis sp. PCC 6803 hydrogenase with those of the homologous containing nickel and iron. The two metal atoms are held in
NAD1-dependent hydrogenase of R. eutropha, despite se- close proximity by two disulfide bridges provided by two
quence homologies between the two enzymes, their char- cysteine residues of the protein. The iron has two cyanide ions
acteristics are not identical, which might indicate that this and one carbon monoxide as ligands, whereas the nickel ion
enzyme might have slightly different functions in different is coordinated by two additional cysteines (Volbeda et al.,
organisms (Cournac et al., 2004). 1995). The small subunit of the hydrogenase dimer (HoxY),
If the function of the bidirectional hydrogenase is still open and the different components of the diaphorase part of the
to debate, its subcellular localization is not less controversial. bidirectional hydrogenase (HoxF and HoxU) also contain
The bidirectional hydrogenase can be found in both the several conserved cysteine residues putatively involved in the
heterocysts and the vegetative cells (Hallenbeck & Benemann, coordination of FeS clusters (Schmitz et al., 2002; for a review,
1978; Houchins & Burris, 1981a), and in Nostoc sp. PCC 7120 see Tamagnini et al., 2002). In addition, in the middle region
appears in the soluble fraction after cell disruption, and of HoxF, typical glycine-rich binding sites for NAD1
consequently has been considered to be a soluble enzyme (GxGxxGxxxG) and flavin mononucleotide (GxGxxxxGx10
(Houchins & Burris, 1981b). Nevertheless, investigations in GxxG) can be identified (Schmitz et al., 1995). HoxE may be
other cyanobacteria suggest a weak association of the bidirec- involved as a bridging subunit in membrane attachment.
tional hydrogenase with cell membranes: in A. variabilis and Moreover, a functional role in electron transport directed to
Synechocystis sp. PCC 6803, an association with the thylakoid membrane components, as demonstrated experimentally for
membrane was proposed (Serebriakova et al., 1994; Appel the Hox-hydrogenase of Thiocapsa roseopersicina (Rakhely
et al., 2000), while in Synechococcus sp. PCC 6301 immuno- et al., 2004), could be considered because sequence motifs for
logical data implied an association with the cytoplasmic binding of an additional FeS cluster are present in this gene
membrane (Kentemich et al., 1989, 1991). (Schmitz et al., 2002).
Physical organization of hox genes and the hox promoter regions and transcriptional
corresponding proteins regulators
In cyanobacteria, the structural genes encoding the bidirec- The information about the transcription and regulation of
tional hydrogenase are organized in a dissimilar way (see the hox genes is limited in cyanobacteria, but the under-
Fig. 2). In some strains (e.g. Synechocystis sp. PCC 6803 and standing of these mechanisms is now emerging. Recent
A. variabilis), the hox genes are localized in one cluster, studies showed that the hox genes in Synechocystis

sp. PCC 6803 are transcribed as a single operon (Gutekunst signal transduction pathways directly or indirectly involved
et al., 2005; Oliveira & Lindblad, 2005; Antal et al., 2006) in the regulation of LexA, and consequently its downstream
with the transcription start point located 168-bp upstream targets, definitely require further investigation.
of the hoxE start codon (Gutekunst et al., 2005; Oliveira &
Lindblad, 2005).
Transcription and expression patterns of hox
Up to now, only one regulator LexA has been proven
genes
to bind and regulate the transcription of the hox genes in
cyanobacteria. Two independent studies (Gutekunst et al., The number of studies focusing on the transcription and
2005; Oliveira & Lindblad, 2005) demonstrated an interac- regulation of the hox genes in cyanobacteria is scarce.
tion between LexA and the promoter region of the bidirec- Nevertheless, transcripts of the bidirectional hydrogenase
tional hydrogenase in Synechocystis sp. PCC 6803. However, have been shown to be present in NH1 4 -grown filaments,
two distinct regions were analyzed and both were demon- and in both vegetative cells and heterocysts under nitrogen-
strated to be targets for this interaction. Oliveira & Lindblad fixing conditions in A. variabilis (Boison et al., 2000). In
(2005) showed that LexA binds to a region located between addition, hoxFUYH were shown to be transcribed as a single
the nucleotides 198 and 338 bp, respective to transla- unit together with other two ORFs with unknown function.
tional start point, while Gutekunst et al. (2005) found that However, it should be kept in mind that these experiments

LexA interacts further upstream on the hox promoter, at the were performed using RT-PCR and do not exclude addi-
positions 592 to 690 bp, in relation to the hoxE ATG tional promoters within the operon (Boison et al., 2000). On
codon (see Fig. 4). Furthermore, a LexA-depleted mutant the other hand, in the unicellular Synechococcus sp. PCC
showed a reduced hydrogenase activity compared with the 6301 and Synechococcus sp. PCC 7942 the hox genes are
wild-type, suggesting that LexA works as a transcription located apart and give rise to two different transcripts
activator of the hox genes in Synechocystis sp. PCC 6803 (Boison et al., 2000; Schmitz et al., 2001). While hoxEF are
(Gutekunst et al., 2005). Synechocystis sp. PCC 6803 LexA cotranscribed in both strains, the second transcript is
has been detected in different proteomic studies (Wang constituted by hoxUYH together with hoxW, hypA and hypB
et al., 2000; Gan et al., 2005; Srivastava et al., 2005; Fulda in Synechococcus sp. PCC 6301 (Boison et al., 2000), and by
et al., 2006; Kurian et al., 2006; Slabas et al., 2006), and its hoxUYHW only in Synechococcus sp. PCC 7942 (Schmitz
transcript has also been identified in microarray experi- et al., 2001). For the last strain, using real-time PCR and
ments (Hihara et al., 2001; Kamei et al., 2001; Li et al., 2004; reporter gene constructs, it was suggested that a second
Singh et al., 2004; Tu et al., 2004; Shapiguzov et al., 2005). promoter might be present between hoxH and hoxW
Interestingly, in some proteomic studies, LexA has been (Schmitz et al., 2001). Furthermore, it was demonstrated
identified in association with thylakoid membrane fractions that the hox genes have a circadian clock expression
(Wang et al., 2000; Srivastava et al., 2005), which represents (Schmitz et al., 2001), a fact that has also been demonstrated
an unexpected location for a transcription regulator. for hoxE in Synechocystis sp. PCC 6803 (Kucho et al., 2005).
Based on the observations that the bidirectional hydro- Very few studies focusing on the regulation of hox genes
genase activity is directly affected by the redox status of the transcription have been performed in cyanobacteria. Analy-
cell, either in photosynthesis or in fermentation, and that sis of the transcription of hoxY and hoxH in G. alpicola,
the regulation of the hox gene expression can be operated by under combined nitrogen-limiting growth conditions, de-
LexA, hypothesis was recently put forward on the direct monstrated an increase in the enzyme activity, but no
involvement of the transcription regulator LexA as a med- regulation at the transcript level (Sheremetieva et al., 2002).
iator of the redox-responsive regulation of the hox gene In contrast, Northern blot analyses of the hox genes expres-
expression in Synechocystis sp. PCC 6803 (Antal et al., 2006). sion in Synechocystis sp. PCC 6803 under combined nitro-
Interestingly, the expression of the cyanobacterial DEAD- gen-limiting growth conditions demonstrated an increase in
box RNA helicase, crhR, which is regulated in response to transcription (Antal et al., 2006), followed by an increase in
conditions that elicit reduction of the photosynthetic elec- enzyme activity (T.K. Antal, P. Oliveira & P. Lindblad,
tron transport chain, was recently shown as being directly unpublished data). A similar increase in the hox genes
controlled by LexA in Synechocystis sp. PCC 6803 (Patter- transcription has also been observed with microarray in
son-Fortin et al., 2006). Transcript analysis indicated that Synechocystis sp. PCC 6803 cells undergoing nitrogen starva-
lexA and crhR are divergently expressed, with the respective tion for 4 h (Osanai et al., 2006 supplementary material).
transcripts accumulating differently under conditions, Furthermore, a transfer to a low level of oxygen in
which, respectively, oxidize and reduce the electron trans- A. variabilis induced both the enzyme activity as well as the
port chain, suggesting that LexA works as a repressor of the relative amount of hoxH (Sheremetieva et al., 2002). It has
crhR transcription (Patterson-Fortin et al., 2006). Although long been demonstrated that microaerobic/anaerobic con-
these results are in agreement with the initial hypothesis, the ditions influence hox transcription and bidirectional

hydrogenase activity in heterocystous cyanobacteria (Hou- via hydrogenase, when grown under sulfur starvation con-
chins & Burris, 1981a; Houchins, 1984; Serebryakova et al., ditions.
1994; Schmitz & Bothe, 1996; Axelsson & Lindblad, 2002; Although the understanding of the regulation and the
Sheremetieva et al., 2002). The bidirectional hydrogenase in physiological role of the bidirectional hydrogenase is becom-
Nostoc sp. PCC 7120 is active in both vegetative cells and in ing clearer, intriguing recent results on the hydrogenase
heterocysts in aerobically grown filaments, with heterocysts activity from two substrains of Synechocystis sp. PCC 6803
having several fold more activity than vegetative cells. When have shown that they do not have comparable values
the filaments were transferred to anaerobic conditions, the (Gutekunst et al., 2006). The authors suggested that these
activity of the bidirectional hydrogenase increased by about phenotypic differences in the hydrogenase activity might be
two orders of magnitude with approximately the same due to divergences in their metabolism. In fact, maintenance
activity levels in both types of cells (Houchins & Burris, of these strains in culture collections, or under various
1981a). Similar results have been observed in A. variabilis laboratory conditions, may have led to spontaneous muta-
(Serebryakova et al., 1994). In contrast to the filamentous tions and unintended selective pressures, resulting in the
cyanobacteria, the activity of the bidirectional hydrogenase observable variations in each subculture (Ikeuchi & Tabata,
in the unicellular G. alpicola is not directly dependent on 2001). Therefore, special care must be taken when interpret-
oxygen (Troshina et al., 2002). Higher activity is observed ing results coming from different laboratories and different

under nitrogen starvation and low light, and it was sug- cyanobacterial strains, even from the same strain, but
gested that the bidirectional hydrogenase could act as an cultured in different laboratories.
alternative electron donor to PSI after inactivation of PSII
due to nitrogen starvation. Under dark anoxic conditions,
Maturation of cyanobacterial
the unicellular cyanobacterium G. alpicola produces H2
catalyzed by the bidirectional hydrogenase (Troshina et al.,
hydrogenases
2002). In addition, the unicellular strain Chroococcidiopsis The biosynthesis/maturation of NiFe-hydrogenases is a
thermalis CALU 758 contains a bidirectional hydrogenase highly complex process requiring at least seven core proteins
with some catalytic properties more related to an uptake for the incorporation of the metal ions and CO and CN
hydrogenase, i.e. not inducible under anaerobic conditions ligands in to the active center, the orientation of the FeS
or under nitrate-starving conditions (Serebryakova et al., clusters within the small subunit and the cleavage of the C-
2000). terminus as the final step in the maturation of the large
Because the bidirectional hydrogenase in cyanobacteria is subunit (for a recent review on this subject, see Bock et al.,
a metal-dependent enzyme, containing nickel and iron in 2006, and also Casalot & Rousset, 2001; Blokesch et al., 2002;
its active center and FeS clusters involved in electron Mulrooney & Hausinger, 2003; Kuchar & Hausinger 2004;
transfer, the availability of these elements in the growing Vignais & Colbeau, 2004; Theodoratou et al., 2005). The
medium has been a subject of research. Axelsson & Lindblad genes encoding the proteins involved in the maturation of
(2002) showed that in the heterocystous N. muscorum hydrogenases were firstly characterized for E. coli, and while
CCAP 1453/12, the addition of external nickel to the most of the Hyp proteins affect hydrogenases pleiotropically,
growing medium increased the mRNA abundance of hoxH the large subunit of each hydrogenase is proteolytically
(monitored by RT-PCR). Making use of reporter gene processed by a specific endopeptidase (Lutz et al., 1991;
constructs, Gutekunst et al. (2006) were able to show that Jacobi et al., 1992; Menon et al., 1994; Rossmann et al., 1995;
the transcription of the bidirectional hydrogenase genes in Theodoratou et al., 2005; Bock et al., 2006). Homologues of
Synechocystis sp. PCC 6803 increased with lower concentra- the hyp genes are present in all organisms capable of forming
tions of iron, the signal being 10 times higher in cells grown NiFe hydrogenases. Although little is known about the
with 0.22 mM iron compared with nonstarved cells. In the biosynthesis/maturation of the cyanobacterial hydrogenases,
same work, measurements of the hydrogenase activity several genes presumably involved in this process have been
revealed a reduction of the enzyme activity alongside the identified clustered or scattered throughout the genomes of
decrease in the iron concentration. The increase in tran- several cyanobacterial strains (Boison et al., 1996; Gubili &
scription of the hox genes, when the cells undergo iron Borthakur, 1996, 1998; Kaneko et al., 1996; Sakamoto et al.,
starvation, might be a feedback mechanism to compensate 1998; Hansel et al., 2001; Tamagnini et al., 2002; Wunschiers
for the lack of functionally active enzyme (Gutekunst et al., et al., 2003; Hoffmann et al., 2006; Leitao et al., 2006). The
2006). The availability of sulfur in the growth medium has presence of a single copy of most of the hyp genes (hypFC-
also been shown to influence the bidirectional hydrogenase DEAB) in the genome of cyanobacteria, regardless of
activity in Synechocystis sp. PCC 6803 and G. alpicola (Antal possessing only the uptake hydrogenase (e.g. N. puncti-
& Lindblad, 2005). Both strains showed an enhanced (more forme), the bidirectional hydrogenase (e.g. Synechocystis sp.
than fourfold) H2 production capacity during fermentation PCC 6803) or both enzymes (e.g. Nostoc sp. PCC 7120)

suggests that they might be responsible for the maturation 2001, 2002; Bock et al., 2006). HypF accepts carbamoyl
of both hydrogenases. In contrast, the genes encoding for the phosphate (CP) as a substrate, catalyzes a CP-dependent
putative hydrogenase C-terminal endopeptidases hupW hydrolysis of ATP into AMP and inorganic phosphate (PPi)
and hoxW were identified and seem to be specific for the and forms an adenylated CP derivative. The carbamoyl
cyanobacterial uptake and the bidirectional hydrogenase, group of CP is transferred to the cysteine at the C-terminus
respectively, resembling the situation in other organisms of HypE (Paschos et al., 2002; Reissmann et al., 2003). It was
(Wunschiers et al., 2003; Oliveira et al., 2004; Leitao et al., demonstrated in vitro that the CN group from HypE-
2006). 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
Physical organization of hyp genes and the
(Blokesch & Bock, 2002), HypD is proposed to be the one
corresponding proteins
involved in this process, given that among all the maturation
The hyp genes in cyanobacteria are frequently clustered and proteins it is the only one with a redox-active cofactor
in the vicinity of the structural genes of one of the hydro- (Blokesch et al., 2004a; Roseboom et al., 2005). On the other
genases (Fig. 2), with a well-known exception the uni- hand, HypC is a small chaperone-like protein that was
cellular non-N2-fixing Synechocystis sp. PCC 6803 in which shown to form a complex with HypD (Blokesch & Bock,

the hypABCDEF genes are scattered throughout the genome. 2002) and to interact with the large subunit of the hydro-
Still, in this organism the homologs hypA2 and hypB2 are genase (Magalon & Bock 2000a; Casalot & Rousset 2001).
clustered (Kaneko et al., 1996), but these two do not seem to Probably, the liganding of the iron takes place at the
play a key role in the maturation of the bidirectional HypCHypD complex (Blokesch et al., 2002, 2004a), and
hydrogenase (Hoffmann et al., 2006). In three Synechococ- the interaction between HypC and the precursor of the large
cus, closely related strains (Synechococcus elongatus PCC subunit leads to the liberation of HypD (Blokesch et al.,
6301, Synechococcus elongatus PCC 7942 and Synechococcus 2004a; Blokesch & Bock, 2006). Subsequently, the liganded
sp. PCC 7002) hypABFC are together and downstream of Fe is transferred to the precursor of the hydrogenase large
hox genes, while hypD and hypE are apart in the two first subunit (Blokesch & Bock, 2006), and HypC remains
organisms (Boison et al., 1996). In the heterocystous strains, attached to the large subunit, maintaining it in an open
N. punctiforme, Nostoc sp. PCC 7120 and A. variabilis and in conformation, allowing the insertion of nickel. This step
the N2-fixing but nonheterocystous L. majuscula, the hyp requires the presence of HypA and HypB (Jacobi et al., 1992;
genes are located in a cluster with all genes orientd in the Olson et al., 2001). HypA is a zinc-containing protein that
same direction, and relatively close to the uptake hydro- binds nickel (Mehta et al., 2003; Blokesch et al., 2004b), and
genase structural genes, although in the opposite direction HypB is a GTPase that probably plays a dual function: nickel
(Gubili & Borthakur, 1998; Hansel et al., 2001; Leitao et al., storage and nickel insertation (Maier et al., 1993, 1995). It is
2006). However, this organization does not constitute a thought that HypA functions as a nickel chaperone and that
pattern for N2-fixing strains, because it contrasts with the HypB acts as a regulator, controlling the donation of the
organization observed for other nonheterocystous strains, metal to the apoprotein or the release of the nickel-free
such as the filamentous T. erythraeum, in which hyp genes chaperone (Blokesch et al., 2004b). After both metals have
are located much further upstream of hupSL (ca. 589 kb), been coordinated to the precursor of the large subunit, the
and the unicellular Crocosphaera watsonii WH 8501, in Cterminal extension is accessible and can be removed by
which the genes are scattered over the genome resembling the specific endopeptidase. The cleavage can only occur after
the non-N2-fixing Synechocystis sp. PCC 6803. When the HypC dissociation from the precursor of the large subunit
genes are grouped, the order varies in non-N2-fixing com- that already contains Ni and Fe(CO)(CN)2 centers (Maga-
pared with N2-fixing strains being hypABFC and hypFC- lon & Bock, 2000a, b), because the endopeptidase uses Ni
DEAB, respectively. In the former case, ORFs interspersed as a recognition motif. Following the cleavage of the
with the hyp genes can be found in several organisms. C-terminal tail from the large hydrogenase subunit, the
The putative cyanobacterial Hyp proteins possess con- mature large subunit can be assembled, with the mature
served motifs and may fulfill functions similar to the small subunit forming the functional enzyme (Magalon &
corresponding proteins in other organisms (Tamagnini Bock, 2000a). Maturation of the small subunit should occur
et al., 2002; Vignais & Colbeau, 2004; Hoffmann et al., in parallel, and independently from the large subunit
2006; Leitao et al., 2006). It is believed that from the two maturation. The knowledge about this process is still scarce,
metal ions present in the active center of a NiFe hydro- although recent studies highlighted at least four gene
genase, Fe is the first to be incorporated into the enzyme. products (encoded within the hup cluster, and downstream
HypF and HypE are the proteins involved in the synthesis of of uptake hydrogenase structural genes) that are required for
the CN, and maybe the CO, ligands of iron (Paschos et al., the maturation of the small subunit of the NiFe

hydrogenases of Rhizobium leguminosarum bv. viciae (Man- In the unicellular non-N2-fixing Synechocystis sp. PCC
yani et al., 2005; Bock et al., 2006). In cyanobacteria, several 6803, a cyanobacterium harboring only the bidirectional
additional ORFs are commonly present near hyp or hup hydrogenase, deletion and insertion mutants of hypA1, B1,
genes (Leitao et al., 2006). The consistent location of these C, D, E and F showed no hydrogenase activity. Moreover, the
ORFs might indicate that their proteins may have a role in complementation of each of the above hyp- inactivated
the uptake hydrogenase maturation process and/or its genes restored the bidirectional hydrogenase activity to the
regulation, notably regarding the small subunit. 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
hyp promoter regions and transcriptional activity even though they are transcribed in the wild type,
regulators
demonstrating that the products of these genes are not
As mentioned above, the hyp genes can be found clustered or actively involved in the maturation process of the bidirec-
scattered throughout the genome of cyanobacteria (Fig. 2). tional hydrogenase (Hoffmann et al., 2006).
Analysis of the hyp cluster promoter region of N. puncti-
forme revealed the presence of 10 and 35 elements, and
Hydrogenase-specific endopeptidases genes
putative binding sites for NtcA (Hansel et al., 2001; Fig. 4).

Similarly, in the corresponding region of L. majuscula the
hupW and hoxW , and corresponding proteins
presence of a 10 box, and two putative NtcA-binding sites The last step in the processing of the large subunit of NiFe-
could be identified. In this organism, a clear 35 box is not hydrogenases is the cleavage of a C-terminal peptide, which,
present, but it should be taken into account that its sequence most likely, allows a structural reorganization of the mole-
is highly variable. Furthermore, a putative LexA-binding site cule and the consequent assembly of the holoenzyme. After
was also found in L. majuscula (Leitao et al., 2006; Fig. 4). both metals have been inserted into the apoprotein precur-
The transcriptional regulators NtcA and LexA were shown sor of the large subunit, the C-terminal extension is acces-
to bind to the promoter regions of the hup and the hox sible and can be removed by the specific endopeptidase
genes, suggesting their involvement in the regulation of the (Theodoratou et al., 2005; Bock et al., 2006). This process
uptake and bidirectional hydrogenase, respectively (see triggers a conformational switch in which the free thiol of
above, Lindberg, 2003; Oliveira et al., 2004; Gutekunst the most C-terminally located cysteine residue closes the
et al., 2005; Leitao et al., 2005; Oliveira & Lindblad, 2005). bridge between the two metals resulting in the formation of
The presence of putative binding sites for both transcrip- the complete heterobinuclear center (Maier & Bock, 1996;
tional factors NtcA and LexA within the hyp operon Magalon & Bock, 2000a; Theodoratou et al., 2005; Bock
promoter region, and preliminary results from electro- et al., 2006). The peptidase cleaves the hydrogenase large
phoretic mobility shift assays (Ferreira et al., 2007) suggest subunit precursor after a histidine or an arginine residue at
the involvement of these proteins in the transcriptional the C-terminal consensus motif DPCxxCxx(H/R), liberating
regulation of hyp genes in L. majuscula, a cyanobacterium a short polypeptide that varies considerably both in length
containing both hydrogenases. These data reinforce the and sequence among different organisms (Wunschiers et al.,
hypothesis that the Hyp proteins might be implicated in 2003). It has been postulated that the endopeptidase recog-
the maturation/regulation of both hydrogenases, and raise nizes its substrate, the nickel-containing hydrogenase pre-
the hypothesis that the transcription of hyp genes in cursor, at least in part via the metal that is coordinated by
cyanobacteria containing both hydrogenases could be under three thiolates, and binds to the exposed C-terminal domain
the control of different transcriptional regulators, e.g. NtcA (Theodoratou et al., 2000a, b, 2005 and Fig. 5). In addition,
and LexA. 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
Transcription and expression patterns of hyp
the recognition of the hydrogenase by the endopeptidase
genes
does not depend on the cleavage site consensus sequence but
In the heterocystous N. punctiforme, the hup and hyp genes is mediated by the overall three-dimensional hydrogenase
are transcribed under N2-fixing but not under non-N2- and peptidase protein structures (Theodoratou et al.,
fixing growth conditions (Hansel et al., 2001). One should 2000a, b). After the proteolytic cleavage, the mature large
bear in mind that N. punctiforme contains only one hydro- hydrogenase subunit assembles with the small subunit and
genase (the uptake enzyme), and that in this organism both eventually the enzyme becomes active.
the transcription of hupL and the H2 uptake activity are In cyanobacteria hydrogenase large subunits, the C-
repressed when combined nitrogen is present in the growth terminal consensus motif [DPCxxCxx(H/R)] was found in
medium (Oxelfelt et al., 1995; Hansel et al., 2001). all the deduced amino acid sequences; however, in the

CO CN CN Small
Subunit
CO CN CN
C -S
Endo-
COOH C -S S-C
peptidase
)
/R
(H
xx
C -S S C
xx
PC C -S S-C
D
HoxH DPCLSCSTH 25-32 a.a.
Large Subunit Large Subunit
HupL DSCLVCTVH
D 16 a.a.
Fig. 5. Schematic representation of the putative final step of the maturation process of the NiFe 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.
uptake hydrogenase large subunits (HupL) the neutral pro- transcription of hupW under conditions in which the
line (P) at position 2 of the cutting site motif is exchanged transcripts of the uptake hydrogenase structural genes could
for an uncharged polar serine (S). The sequence of the not be detected (presence of ammonia) could imply that
cutting site motif is totally conserved for each of the hupW is constitutively expressed. Taking into account all the
cyanobacterial hydrogenases large subunits: HoxH (bidirec- available data, it is not yet possible to establish whether the
tional hydrogenase) DPCLSCSTH; HupL (uptake hydro- expression of hupW is or is not constitutive or whether this
genase) DSCLVCTVH (see Wunschiers et al., 2003 and depends on the strain/existence of cell differentiation.
Fig. 5). The putative cleaved C-terminal polypeptide varies Similar to what happens for hupW, analysis of the
in length (2532 aa residues) and sequence (1096% simi- available cyanobacterial genomic sequences revealed that
larity) for HoxH, while for HupL the polypeptide always has the position and orientation of hoxW in the chromosome is
the same length (16 aa residues) and is highly conserved for also variable but, in most of the cases, hoxW is downstream
all the deduced sequences [AHDAKTG(E/K)ELARFRT(A/ of hoxH, one of the bidirectional hydrogenase structural
N/S)]. genes (Fig. 2). RT-PCR experiments indicate that in the
In cyanobacteria, the genes encoding for the putative unicellular non-N2-fixing Synechococcus sp. PCC 6301,
hydrogenase-specific C-terminal endopeptidases were iden- hoxW is part of a polycistronic message containing hoxUYH-
tified and named hupW and hoxW for the gene encoding the WhypAB (Boison et al., 2000), while in Synechococcus sp.
enzyme processing the uptake and the bidirectional hydro- PCC 7942 it was demonstrated that although hoxW consti-
genase, respectively (Kaneko et al., 1995, 2001; Boison et al., tute a unit together with hoxUYH, it is mainly expressed by
2000; Schmitz et al., 2001; Wunschiers et al., 2003; Oliveira its own promoter (Schmitz et al., 2001). In the heterocystous
et al., 2004; Leitao et al., 2005). Nostoc sp. PCC 7120, similar to hupW, hoxW is transcribed
The position of hupW and hoxW in the cyanobacterial under both N2- and non-N2-fixing conditions (Wunschiers
chromosome is rather variable; however, in several cases et al., 2003). Although some data indicate that endo-
hupW is in the vicinity and in the same direction of hupSL peptidases transcripts are present when the corresponding
(uptake hydrogenase structural genes). In the nonheterocys- hydrogenase large subunit transcript is absent, and it has
tous Gloeothece sp. ATCC 27152 and T. erythraeum, hupW is been proposed that their expression is independently
even the ORF located immediately downstream of hupL, and regulated from the expression of both the hydrogenase
was shown to be cotranscribed with hupSL in Gloeothece structural and the other accessory genes in cyanobacteria
sp. ATCC 27152 (Oliveira et al., 2004). In contrast, in the (Wunschiers et al., 2003), it is premature to make any
heterocystous strains A. variabilis, Nostoc sp. PCC 7120 and general conclusion.
N. punctiforme, hupW is not part of any known hydrogenase To date, two different hydrogenase specific-endopepti-
cluster (Fig. 2), and it was shown to be transcribed under dases have been purified and studied, namely HycI and
N2- and non-N2-fixing conditions in the last two strains HybD from E. coli (Rossmann et al., 1995; Fritsche et al.,
(Wunschiers et al., 2003). These authors postulated that the 1999). Both are monomeric proteins of a molecular mass of

c. 17 kDa, and they are devoid of metal or other cofactors. either missing data or as a fifth state. Support for nodes was
Alignments of the amino acid sequences showed that estimated by bootstrapping with 10 000 replicates.
hydrogenase- specific C-terminal endopeptidases share low Both analyses gave widely congruent estimates of phylo-
sequence similarity, with only a few positions fully con- geny (Fig. 6). The three heterocystous strains form a clade
served (Theodoratou et al., 2005). As a general feature, they with 100% support, separated from the nonheterocystous
have three highly conserved amino acid residues (Glu, Asp strains by between 16% and 21% divergence. Within the
and His) that, most likely, have the function of interacting nonheterocystous strains, two pairs of taxa Cyanothece
with the nickel in the hydrogenase large subunit precursor with Crocosphaera and the two Lyngbya species are well
(Theodoratou et al., 2000a). The alignment of the putative supported. Other relationships are poorly supported.
cyanobacterial endopeptidases with the corresponding pro- Although the analysis with gaps treated as missing data
teins from E. coli clearly shows that although the amino acid suggests that the filamentous taxa are not a clade, analysis
sequence identity is low, they are indeed structurally related with gaps treated as a fifth character supported a relation-
(6777% structural identity) (Wunschiers et al., 2003). 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
Phylogenetic analysis sister-taxa relationship between T. erythraeum and L. ma-

Recently, the phylogenetic origin of cyanobacterial and algal juscula is strongly supported through analysis of the Hyp
hydrogenases was analyzed (Ludwig et al., 2006), leading to sequences. These results are not in conflict with those
the conclusion that Chloroflexus is probably the closest suggested by Ludwig et al. (2006), in which the only well-
ancestor of cyanobacteria. In all cyanobacterial genomes supported node within cyanobacteria is that of the three
sequenced to date, and in the genome of Chloroflexus, the heterocystous strains. Evidence for the position of the
two hydrogenase operons hup and hox are widely ancestral root within the cyanobacteria is weak, although T.
separated on the chromosome, rendering simultaneous gene erythraeum may be sister taxa to the remaining sampled
transfer unlikely. The authors claim that the current dis- cyanobacteria (Ludwig et al., 2006).
tribution of the hydrogenases in cyanobacterial strains Phylogenetic analysis of cyanobacterial hydrogenases ac-
probably reflects a differential loss of the genes from their cessory proteins (Hyp A,B,C,D,E and F) and the bidirec-
last common ancestor, and that the two sets of genes, tional hydrogenases structural proteins (Hox) is
encoding the uptake and bidirectional, were either kept in complicated by the higher level of variation between species,
the genome or lost differentially in the different strains and in particular greater length variation that leads to
according to their ecological needs or constraints. Although uncertain alignment for many positions. Further, not all of
the phylogenetic analysis of Ludwig et al. (2006) clearly the amino acid sequences of hydrogenase accessory proteins
demonstrated the monophyly of cyanobacteria, and their are available for all the species analyzed for the uptake
relationship with other photosynthetic bacteria, relation- hydrogenase structural genes. However, unweighted parsi-
ships within the cyanobacteria were poorly resolved using mony analyses indicate that supported estimates of relation-
HupL sequences. This is probably related to the difficulties ships recovered for these proteins do not conflict with the
of aligning the cyanobacteria with the other highly divergent estimate of phylogeny shown in Fig. 6 (analyses not shown).
lineages, and acerbated by the low number of sequences
available and long branches leading to terminal nodes. The
Genetic engineering/cyanobacterial H2
high variability of the sequences also means that more
distant bacterial outgroups cannot be unambiguously
production
aligned. However, analysis solely within the cyanobacteria Cyanobacteria can be used for the production of molecular
for both HupS and HupL is less complex, because analysis of hydrogen (H2), a possible future energy carrier, which has
the predicted proteins demonstrates that in HupS the been the subject of several recent reviews (Levin et al., 2004;
number of residues in all known cyanobacteria is constant Dutta et al., 2005; Kruse et al., 2005; Prince & Kheshgi, 2005;
(320 aa), while HupL generally has 531 aa, with the excep- Sakurai & Masukawa, 2007). As the main advantages,
tion of the filamentous nonheterocystous strains L. majus- cyanobacteria can use sunlight as an energy source, water
cula and L. aestuarii with six extra (one insertion of 5 aa and as an electron source and air as a carbon (CO2) and a
another of one), and T. erythraeum with three extra, coin- nitrogen (N2) source. Therefore, no complicated or expen-
ciding with the position of the five inserts in Lyngbya spp. sive media are needed for the cultivation of cyanobacteria,
Owing to this relatively conserved identity, alignment of the and the overall theoretical energy conversion efficiency
amino acids for phylogenetic analysis was facile. (from solar energy sun to H2) may be the highest possible.
Amino acid sequences were analyzed under the criterion In cyanobacteria, two natural pathways for H2 produc-
of maximum parsimony, with gaps treated separately as tion can be used.


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.
H2 production as a by-product during nitrogen vanadium is also limited, some N2-fixing microorganisms
fixation by nitrogenases are able to synthesize an alternative iron-nitrogenase.
Depending on the type of nitrogenase (molybdenum,
In N2-fixing strains, H2 is produced as a by-product by the
vanadium or iron), different amounts of electrons are
nitrogenase enzymatic complex. As this reaction needs the
allocated for N2 fixation or H2 production. The general
input of ATP (at least two ATP per electron), the overall
equation for the nitrogenase-catalyzed reaction is as follows
energy efficiency for hydrogen production is rather low. The
(Rees et al., 2005):
turnover of the nitrogenase enzyme is not very high
(o 10 s1), and the H2 produced is efficiently taken up N2 2n 6e 2n 6H p2n 6ATP
by an uptake hydrogenase. The overall oxygen sensitive ! 2NH3 nH2 p2n 6ADP p2n 6Pi
N2-fixation process is occurring in an anaerobic environ-
ment achieved using a number of different strategies includ- It has been reported that n is 1 for the molybdenum-
ing spatial or/and temporal separation of N2 fixation and containing enzymes, 3 for the vanadium-nitrogenases and
7.5 for the iron-only nitrogenases, respectively. As a conse-
oxygenic photosynthesis and increased respiration.
Cyanobacterial nitrogenases contain molybdenum (Mo), quence, the alternative nitrogenases, although still very little
vanadium (V) or iron (Fe) in the active site, with different is known in cyanobacteria, may be better H2 producers
genes and gene products making up the different nitro- compared with the more conventional molybdenum-nitro-
genases (Eady, 1996; Zhao et al., 2006). With sufficient genases.
amounts of molybdenum available, the active site harbors
molybdenum and iron. Under molybdenum-deprived con- H2 production by the bidirectional hydrogenase
ditions, the conventional molybdenum-nitrogenase is re- The cyanobacterial bidirectional hydrogenase may, under
placed by an alternative vanadium-nitrogenase, and if anaerobic conditions, produce and evolve significant

amounts of H2. Because this reaction is not dependent on 2004), Nostoc sp. PCC 7120 (Lindblad et al., 2002; Masuka-
ATP, it is energetically more efficient and favorable for H2 wa et al., 2002; Carrasco et al., 2005) and Nostoc sp. PCC
production, with a much higher turnover (1 million turn- 7422 (Yoshino et al., 2007) have been shown to be signifi-
overs per second) compared with the nitrogenase-based H2 cantly better H2 producers compared with the respective wild
production. At the same time, the enzyme is not specifically types. In general, the H2 produced by a nitrogenase in the
located in an oxygen-protected environment, and the reac- wild type will be quickly reoxidized by the uptake hydro-
tion turns into the opposite direction (H2 uptake) above a genase, whereas in an uptake hydrogenase-deficient mutant
certain H2 partial pressure. Therefore, a continuous and very the H2 produced will leave the cells. One should bear in mind
effective removal of both O2 and H2 from the cells and the that all these strains, with the exception of N. punctiforme,
culture is necessary to lower the overall energy conversion also possess a bidirectional hydrogenase. However, only for
efficiency significantly. Furthermore, an accumulation of Nostoc sp. PCC 7120 (Masukawa et al., 2002) the effect of a
ATP could inhibit the electron flow, because it is produced hox-defective mutant (DhoxH) has been investigated. A
during the linear or cyclic electron flow around PSI, but is Nostoc sp. PCC 7120 mutant deficient in both hydrogenases
not used by the electron acceptor hydrogenase. (DhupL/DhoxH) showed the same increase in H2 evolution as
Besides the specific challenges for H2 production con- the uptake hydrogenase-deficient mutant (DhupL), whereas
nected to the H2-evolving enzymes, there are additional the bidirectional hydrogenase-deficient mutant (DhoxH) pro-

unsolved issues for photoautotrophical H2 production in duced less H2 compared with the wild type.
general. These are related to the low quantum efficiency, due In gas exchange experiments with an uptake hydrogenase-
to the naturally large antenna systems of the photosystems, deficient mutant of Nostoc punctiforme (Lindberg et al.,
and to electron consuming pathways directly competing 2004), the amount of H2 produced per molecule of N2 fixed
with e.g. nitrogenases and hydrogenases. varied with the light conditions. The ratio of H2 produced/
In summary, to achieve a sustainable, renewable cyano- N2 fixed under low light and high light was 1.4 and 6.1,
bacterial-based H2 production, the following challenges respectively. This showed that, under the specific conditions,
have to be addressed: the energy flow through the nitrogenase may be directed
(1) efficient H2 uptake by the cells, towards the H2 production rather than the N2 fixation.
(2) low energy efficiency and turnover of the nitrogenase (2) Low energy efficiency and turnover of the nitrogenase
and/or the hydrogenase, and/or the hydrogenase: H2-evolving enzymes with the high-
(3) limiting amounts of active H2-evolving enzymes, est reported turnover are the Fe-hydrogenases (Houchins,
(4) high oxygen sensitivity of the nitrogenase and/or the 1984; Adams, 1990). These enzymes are irreversibly inacti-
hydrogenase, vated by oxygen, and are present in e.g. fermentative bacteria
(5) electron flow inhibition by accumulation of ATP in a (e.g. Clostridium) and green algae (e.g. Chlamydomonas) but
hydrogenase-driven system, not in cyanobacteria. An elegant strategy for the creation of
(6) low quantum efficiency due to too large antennas in an efficient H2 producer, which will not be inhibited by the
both Photosystem II (PSII) and PSI and surrounding oxygen, would be the expression of a highly
(7) electron-consuming pathways competing with an effi- active Fe-hydrogenase in the heterocysts of filamentous
cient electron transfer to the H2 enzymes. cyanobacteria unable to reoxidase any H2 (i.e. an uptake
In recent years, there have been attempts to overcome hydrogenase-deficient strain). The heterologous expression
these barriers and problems, mainly by targeted genetic of different iron-hydrogenases in various organisms such as
engineering of cyanobacterial strains: Synechococcus (Asada et al., 2000), E. coli (Posewitz et al.,
(1) Efficient H2 uptake by the cells: Cyanobacteria have 2004; King et al., 2006) and Clostridium (Girbal et al., 2005)
evolved an effective mechanism to recycle the H2 evolved has already been achieved. Recently, the accessory genes
during nitrogen fixation: an uptake hydrogenase that oxi- necessary for the maturation of iron-hydrogenases into
dizes the H2 evolved, and transfers electrons to e.g. the active enzymes were identified (Posewitz et al., 2004; Bock
respiratory-chain. As this reaction significantly lowers the et al., 2006; King et al., 2006). Therefore, the heterologous
H2 production efficiency of a nitrogenase-based system, expression of an active iron-hydrogenase in a cyanobacterial
targeted mutants with reduced or deficient uptake hydro- host, e.g. in the heterocyst of a strain for which the genome
genase activity have been produced. This was first achieved has been sequenced, is an interesting and realistic project.
by chemical mutagenesis (Kumar & Kumar, 1991; Mikheeva Moreover, because the iron-hydrogenases are able to use a
et al., 1995), and later, since the molecular biology tools for wide variety of primary electron donors (Vignais et al.,
genetic engineering were established, by targeted knock-out 2001), including ferredoxin, which is the electron donor of
of structural or accessory genes of the uptake hydrogenase. the cyanobacterial nitrogenase, it may be possible to link the
Uptake hydrogenase-deficient mutants of A. variabilis introduced iron-hydrogenase to an existing electron transfer
(Happe et al., 2000), N. punctiforme (Lindberg et al., 2002, pathway within the cyanobacterial cell.

(3) Limiting amounts of active H2-evolving enzymes: should be directed to the H2-evolving enzyme (nitrogenase
Because the H2-evolving enzymes (nitrogenase(s) and/or or hydrogenase) to reach maximal energy conversion effi-
bidirectional hydrogenases) are strictly regulated on several ciency. In addition to the electron flow in the photosynthetic
different levels (transcription, translation and maturation), electron transport chain, a transmembrane potential is built
one possible way to enhance the production of H2 may be up that is used for generating ATP through an ATP synthase.
the overexpression of these enzymes. For this purpose, the In a nitrogenase-based system, the ATP is clearly needed for
genes encoding the selected enzyme/protein to be over- N2 fixation. However, in the hydrogenase-catalyzed reaction
expressed are placed under the control of an artificial no ATP is consumed and the electron flow could, in a
promoter and ribosomal-binding site (RBS) combination photosynthetic microorganism, be inhibited by the accu-
on an expression vector or placed directly in the genome. mulated transmembrane potential across the thylakoid
The choice of a constitutive or an inducible promoter, membrane (Lee & Greenbaum, 2003). It has been observed
together with a strong RBS, takes the enzyme biosynthesis in the green algae Chlamydomonas reinhardtii that oxygen
out of the control of the organisms natural regulation serves as an electron sink, competing for electrons with the
system, allowing a significant increase in the amount of H2-producing pathway, resulting in a new oxygen sensitiv-
enzyme produced. ity (Lee & Greenbaum, 2003). A possible solution may be
In heterocystous cyanobacteria grown under N2-fixing the introduction of a synthetic, polypeptide based on the

conditions, c. 510% of the vegetative cells differentiate into proton channel into the thylakoid membranes, with its
heterocysts. These specialized cells are compartments with transcription controlled by a hydrogenase promoter (Lee &
reduced O2 pressure, and thus suitable for H2 production, Greenbaum, 2003). The proton channel may be expressed
either via nitrogenase or via an introduced hydrogenase. under H2-producing (anaerobic) conditions, to dissipate the
Therefore, the increase of the heterocyst frequency should proton gradient across the thylakoids membrane. A similar
result in a higher overall H2 production capacity by the strategy may work for cyanobacteria.
organism. Interestingly, it has been shown that the hetero- (6) Low quantum efficiency due to too large antennas in
cyst frequency can be increased, e.g. by overexpression of both PSII and PSI: In nature, phototrophic organisms have
hetR or by inactivation of patS, or hetN (Buikema & developed to handle and compete during very low light
Haselkorn, 2001; Golden & Yoon, 2003; Borthakur et al., intensities, and, therefore, large antenna systems have been
2005). However, this has not been coupled to H2 production evolved. However, for biotechnological applications in
or increased H2 production. photobioreactors, where an optimal supply with light can
(4) High oxygen sensitivity of the nitrogenase and/or the be engineered, only a small part of the light will be used by
hydrogenase: One main obstacle in H2 production using the microorganisms. In addition, self-shading may become
photosynthetic microorganisms is the high sensitivity of the a severe limitation causing reduced efficiency in utilizing
H2-evolving enzymes, and some attempts have been made to incoming solar energy. To overcome this problem, a reduc-
introduce less oxygen sensitive hydrogenases into cyanobac- tion of the antenna size has been proposed (Melis et al.,
teria. At the Craig Venter Institute (US), work is being 1998; Nakajima & Ueda, 1999; Lee et al., 2002). The
carried out aiming at transferring the O2-tolerant NiFe- phycocyanin-deficient mutant PD1 of Synechocystis PCC
hydrogenase of the purple-sulfur photosynthetic bacterium 6714, generated by chemical mutagenesis, showed up to
T. roseopersicina (Kovacs et al., 2005) into a Synechococcus 50% higher maximal photosynthesis activity under high
strain. In addition, several other putative O2-tolerant NiFe- light conditions compared with the wild type (Nakajima &
hydrogenases have been identified from the marine environ- Ueda, 1997, 1999). Antenna-deficient green algal mutants,
ment that could be alternative candidates to be introduced created by chemical mutagenesis (Lee et al., 2002) or genetic
into a cyanobacterial background (Xu et al., 2005). Also in engineering (Polle et al., 2003; Tetali et al., 2007), also
the US, the genes encoding the more O2-tolerant NiFe showed remarkably greater solar conversion efficiencies and
hydrogenase of the purple nonsulfur photosynthetic bacter- a higher photosynthetic productivity than the respective
ium Rubrivivax gelatinosus CBS (Ghirardi et al., 2005), and wild type under mass culture conditions.
its accessory proteins, are being introduced into Synechocys- (7) Electron-consuming pathways competing with an effi-
tis. The in vivo half-life of this hydrogenase is 21 h in air and cient electron transfer to the H2 enzymes: For the production
6 h in air when the protein is partially purified (Ghirardi of H2 through the action of an active nitrogenase or
et al., 2005). However, to the authors knowledge, the hydrogenase electrons are required to combine with protons
heterologous expression of a more oxygen-tolerant hydro- to form H2. Because protons are abundant within the cell,
genase in any cyanobacterium remains to be shown. the main limitation is the available number of electrons. The
(5) Electron flow inhibition by accumulation of ATP in a primary electron donors for the H2-producing enzymes
hydrogenase-driven system: In an optimal H2 production are ferredoxin (nitrogenase, Fe-hydrogenases) and NADH/
system, all electrons derived from water splitting in PSII NADPH (NiFe-hydrogenases). However, the electrons are

mainly used by other pathways, the so-called competing As a future perspective, the development of synthetic
pathways, e.g. respiration and the Calvin cycle. Therefore, biology reveals new possibilities for the direct construction
one strategy for an enhanced H2 production is to direct the of efficient H2-evolving cyanobacterial strains. Both in the
electron flow towards the H2-producing enzymes and away US (e.g. the Craig Venter Institute) and in Europe (e.g. the
from any other competing pathway. EU/NEST project BioModularH2), the first attempts have
Experiments with the ndhB mutant M55 of Synechocystis been initiated to use this new concept aiming at designing
PCC 6803, which is defective in the type I NADPH- reusable, standardized molecular building blocks that will
dehydrogenase complex (NDH-1) (Cournac et al., 2004), produce a photosynthetic bacterium containing engineered
showed that this mutant produces only low amounts of O2 chemical pathways for competitive, clean and sustainable H2
in the light, has a poor capacity to fix CO2 and evolves H2 production.
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. Concluding remarks
In another study, the level of reduced NADP shifted from The fundamental aspects of cyanobacterial hydrogenases,
50% in the wild type to 100% in the NDH-1 mutant (Cooley and their more applied potential use as future producers of

& Vermaas, 2001). As the cyanobacterial bidirectional hy- renewable H2 from sun and water, are receiving increased
drogenase evolves H2 at a relatively high level of reduced international attention. At the same time, significant progress
NAD(P), the construction of mutants with blocked electron is being made in the understanding of the molecular regula-
transfer in selected key pathways, increasing the relative level tion of the genes encoding both the enzymes as well as the
of reduced NAD(P), may be a promising strategy to increase accessory proteins needed for the correct assembly of an
the H2 production capacity. active hydrogenase. In the last few years, the transcription
Another strategy for directing electrons toward the hy- factors directly involved in the regulation of cyanobacterial
drogenase is to directly link the hydrogenase to PSI. hydrogenases have been identified. Moreover, the first steps to
Ihara et al. (2006b) fused the membrane-bound NiFe use isolated components from cyanobacteria and other
hydrogenase from Ralstonia eutropha H16 to the peripheral microorganisms in order to create a functional H2-producing
PSI subunit PsaE of the cyanobacterium Thermosynechococ- unit are being taken. With the increasing scientific commu-
cus elongatus (Hyd-PsaE), and used a PsaE-free PSI nity and public interest in clean and renewable energy
(PSI) extract from a PsaE-deficient mutant of Synechocystis sources, and consequent funding opportunities, rapid pro-
sp. PCC 6803. The resulting hydrogenase/PSI complex gress will be made in the fundamental understanding of the
showed light-driven hydrogen production in vitro, which regulation and maturation of cyanobacterial hydrogenases at
was five times higher compared with a control without a both genetic and protein levels. Unique and unexpected
direct coupling of the hydrogenase to PSI. However, as the results in the transcriptional regulation of cyanobacterial
activity of the hydrogenase-PsaE fusion protein was only hydrogenases will emerge during the coming years. Moreover,
16% of that of the wild-type hydrogenase protein and was the more applied aspects will be highlighted with progress in
totally suppressed by adding ferredoxin (Fd) and ferredox- generating genetically modified strains with an increased
in-NADP1-reductase (FNR), the authors concluded that the capacity for renewable H2 from sun and water. The possibi-
linker between the hydrogenase and PsaE has to be opti- lities and challenges within synthetic biology, including the
mized. In another work by the same group (Ihara et al., use of isolated proteins and parts, will be explored, aiming at
2006a), PsaE from Synechocystis sp. PCC 6803 was creating both cyanobacteria with a high potential for H2
chemically cross-linked with cytochrome c3 (cytc3) from production as well as functional in vitro systems.
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-
Acknowledgements
genase from Desulfovibrio vulgaris was enhanced sevenfold. This work was financially supported by FCT (POCTI/BIO/
This clearly demonstrated that it is possible, in vitro, to 44592/2002; SFRH/BD/4912/2001, SFRH/BD/16954/2004),
direct the electron-flow toward the hydrogenase by changing ESF (III Quadro Comunitario de Apoio), the Swedish
the environment of the electron-donating PSI. The Research Council, the Swedish Energy Agency, the Nordic
next challenge will be to develop an in vivo, or even Energy Research Program (project BioHydrogen), the EU/
in situ, functional system based on the assembly of different NEST Projects SOLAR-H (contract # 516510) and BioMo-
enzymes and proteins. dularH2 (contract # 043340).

References Blokesch M, Albracht SP, Matzanke BF, Drapal NM, Jacobi A &
Bock A (2004a) The complex between hydrogenase-
Adams MWW (1990) The structure and mechanism of iron-
maturation proteins HypC and HypD is an intermediate in the
hydrogenases. Biochim Biophys Acta 1020: 115145.
supply of cyanide to the active site iron of [NiFe]-
Anderson DC, Campbell EL & Meeks JC (2006) A soluble 3D LC/
hydrogenases. J Mol Biol 344: 155167.
MS/MS proteome of the filamentous cyanobacterium Nostoc
Blokesch M, Rohrmoser M, Rode S & Bock A (2004b) HybF, a
punctiforme. J Prot Res 5: 30963104.
zinc-containing protein involved in NiFe hydrogenase
Antal TK & Lindblad P (2005) Production of H2 by sulphur-
deprived cells of the unicellular cyanobacteria Gloeocapsa maturation. J Bacteriol 186: 26032611.
Bock A, King PW, Blokesch M & Posewitz MC (2006) Maturation
alpicola and Synechocystis sp. PCC 6803 during dark
incubation with methane or at various extracellular pH. J Appl of hydrogenases. Adv Microb Physiol 51: 171.
Bohme H (1998) Regulation of nitrogen fixation in heterocyst-
Microbiol 98: 114120.
Antal TK, Oliveira P & Lindblad P (2006) The bidirectional forming cyanobacteria. Trends Plant Sci 3: 346351.
hydrogenase in the cyanobacterium Synechocystis sp. strain Boison G, Schmitz O, Mikheeva L, Shestakov S & Bothe H (1996)
PCC 6803. Int J Hydrogen Energy 31: 14391444. Cloning, molecular analysis and insertional mutagenesis of the
Appel J & Schulz R (1996) Sequence analysis of an operon of bidirectional hydrogenase genes from the cyanobacterium
NAD(P)-reducing nickel hydrogenase from the Anacystis nidulans. FEBS Lett 394: 153158.
Boison G, Schmitz O, Schmitz B & Bothe H (1998) Unusual gene

cyanobacterium Synechocystis sp. PCC 6803 gives additional
evidence for direct coupling of the enzyme to NADP(H)- arrangement of the bidirectional hydrogenase and functional
dehydrogenase (complex I). Biochim Biophys Acta 1298: analysis of its diaphorase subunit HoxU in respiration of the
141147. unicellular cyanobacterium Anacystis nidulans. Curr Microbiol
Appel J, Phunpruch S, Steinmuller K & Schulz R (2000) The 36: 253258.
bidirectional hydrogenase of Synechocystis sp. PCC 6803 works Boison G, Bothe H, Hansel A & Lindblad P (1999) Evidence
as an electron valve during photosynthesis. Arch Microbiol 173: against a common use of the diaphorase subunits by the
333338. bidirectional hydrogenase and by the respiratory complex I in
Asada Y, Kawamura S & Ho K-K (1987) Hydrogenase from the cyanobacteria. FEMS Microbiol Lett 174: 159165.
unicellular cyanobacterium, Microcystis aeruginosa. Boison G, Bothe H & Schmitz O (2000) Transcriptional analysis
Phytochemistry 26: 637640. of hydrogenase genes in the cyanobacteria Anacystis nidulans
Asada Y, Koike Y, Schnackenberg J, Miyake M, Uemura I & and Anabaena variabilis monitored by RT-PCR. Curr Microbiol
Miyake J (2000) Heterologous expression of clostridial 40: 315321.
hydrogenase in the cyanobacterium Synechococcus PCC7942. Borthakur PB, Orozco CC, Young-Robbins SS, Haselkorn R &
Biochim Biophys Acta 1490: 269278. Callahan SM (2005) Inactivation of patS and hetN causes
Asayama M, Kabasawa M, Takahashi I, Aida T & Shirai M (1996) lethal levels of heterocyst differentiation in the filamentous
Highly repetitive sequences and characteristics of genomic cyanobacterium Anabaena sp. PCC 7120. Mol Microbiol 57:
DNA in unicellular cyanobacterial strains. FEMS Microbiol Lett 111123.
137: 175181. Bothe H, Tennigkeit J & Eisbrenner G (1977) The utilization of
Axelsson R & Lindblad P (2002) Transcriptional regulation of molecular hydrogen by the blue-green alga Anabaena
Nostoc hydrogenases: effects of oxygen, hydrogen, and nickel. cylindrica. Arch Microbiol 114: 4349.
Appl Environ Microbiol 68: 444447. Bothe H, Kentemich T & Dai H (1991) Recent aspects on the
Axelsson R, Oxelfelt F & Lindblad P (1999) Transcriptional hydrogenasenitrogenase relationship in cyanobacteria. Dev
regulation of Nostoc uptake hydrogenase. FEMS Microbiol Lett Plant Soil Sci 48: 367375.
170: 7781. Buikema WJ & Haselkorn R (2001) Expression of the Anabaena
Bergman B, Gallon JR, Rai AN & Stal LJ (1997) N2 fixation by hetR gene from a copper-regulated promoter leads to
non-heterocystous cyanobacteria. FEMS Microbiol Rev 19: heterocyst differentiation under repressing conditions. PNAS
139185. 98: 27292734.
Berman-Frank I, Lundgren P & Falkowski P (2003) Nitrogen Burgdorf T, van der Linden E, Bernhard M, Yin QY, Back JW,
fixation and photosynthetic oxygen evolution in Hartog AF, Muijsers AO, de Koster CG, Albracht SP &
cyanobacteria. Res Microbiol 154: 15764. Friedrich B (2005) The soluble NAD1-reducing [NiFe]-
Blokesch M & Bock A (2002) Maturation of [NiFe]-hydrogenases hydrogenase from Ralstonia eutropha H16 consists of six
in Escherichia coli: the HypC cycle. J Mol Biol 324: 28796. subunits and can be specifically activated by NADPH. J
Blokesch M & Bock A (2006) Properties of the [NiFe]- Bacteriol 187: 31223132.
hydrogenase maturation protein HypD. FEBS Lett 580: Busby S & Ebright RH (1999) Transcription activation by
40654068. catabolite activator protein (CAP). J Mol Biol 293: 199213.
Blokesch M, Paschos A, Theodoratou E, Bauer A, Hube M, Huth Carrasco CD, Buettner JA & Golden JW (1995) Programed DNA
S & Bock A (2002) Metal insertion into NiFe-hydrogenases. rearrangment of a cyanobacterial hupL gene in heterocysts.
Biochem Soc Trans 30: 674680. Proc Natl Acad Sci USA 92: 791795.

Carrasco CD, Garcia JS & Golden JW (1998) Programmed DNA Girbal L, von Abendroth G, Winkler M, Benton PM, Meynial-
rearrangement of a hydrogenase gene during Anabaena Salles I, Croux C, Peters JW, Happe T & Soucaille P (2005)
heterocyst development. BioHydrogen (Zaborsky OR, Homologous and heterologous overexpression in Clostridium
Benemann JR, Matsunaga T, Miyake J & San Pietro A, eds), pp. acetobutylicum and characterization of purified clostridial and
203207. Plenum Press, New York. algal Fe-only hydrogenases with high specific activities. Appl
Carrasco CD, Holliday SD, Hansel A, Lindblad P & Golden JW Environ Microb 71: 27772781.
(2005) Heterocyst-specific excision of the Anabaena sp. strain Golden JW (1997) Programmed DNA Rearrangements in
PCC 7120 hupL element requires xisC. J Bacteriol 187: Cyanobacteria. Bacterial Genomes: Physical Structure
60316038. and Analysis (de Bruijn FR, Lupski JR & Weinstok GM),
Casalot L & Rousset M (2001) Maturation of the [NiFe] pp. 162163. Chapman & Hall, New York.
hydrogenases. Trends Microbiol 9: 228237. Golden JW & Yoon H-S (2003) Heterocyst development in
Chen PC, Almon H & Boger P (1989) Physiological factors Anabaena. Curr Opin Microbiol 6: 557563.
determining hydrogenase activity in nitrogen-fixing Goodman SD, Velten NG, Gao Q, Robinson S & Segall AM (1999)
heterocystous cyanobacteria. Plant Physiol 89: 10351038. In vitro selection of integration host factor binding sites. J
Cooley JW & Vermaas WFJ (2001) Succinate dehydrogenase and Bacteriol 181: 32463255.
other respiratory pathways in thylakoid membranes of Goodrich JA, Schwartz ML & McClure WR (1990) Searching for
Synechocystis sp. strain PCC 6803: capacity comparisons and and predicting the activity of sites for DNA binding proteins:

physiological function. J Bacteriol 183: 42514258. compilation and analysis of the binding sites for Escherichia
Cournac L, Guedeney G, Peltier G & Vignais PM (2004) coli integration host factor (IHF). Nucleic Acids Res 18:
Sustained photoevolution of molecular hydrogen in a mutant 49935000.
of Synechocystis sp. strain PCC 6803 deficient in the type I Gubili J & Borthakur D (1996) The use of a PCR cloning and
NADPH-dehydrogenase complex. J Bacteriol 186: 17371746. screening strategy to identify lambda clones containing the
Craig NL & Nash AH (1984) E. coli integration host factor binds hupB gene of Anabaena sp. strain PCC 7120. J Microbiol
to specific sites in DNA. Cell 39: 707716. Methods 27: 175182.
Daday A, Mackerras AH & Smith GD (1985) The effect of nickel Gubili J & Borthakur D (1998) Organization of the hupDEAB
on hydrogen metabolism and nitrogen fixation in the genes within the hydrogenase gene cluster of Anabaena sp.
cyanobacterium Anabaena cylindrica. J Gen Microbiol 131: strain PCC 7120. J Appl Phycol 10: 163167.
231238. Gutekunst K, Phunpruch S, Schwarz C, Schuchardt S, Schulz-
Dutta D, De D, Chaudhuri S & Bhattacharya SK (2005) Hydrogen Friedrich R & Appel J (2005) LexA regulates the bidirectional
production by cyanobacteria. Microb Cell Fact 4: 36. hydrogenase in the cyanobacterium Synechocystis sp. PCC
Eady RR (1996) Structurefunction relationships of alternative 6803 as a transcription activator. Mol Microbiol 58: 810823.
nitrogenases. Chem Rev 96: 30133030. Gutekunst K, Hoffmann D, Lommer M, Egert M, Suzuki I,
Ferreira D, Leitao E, Sjoholm J, Oliveira P, Lindblad P, Moradas- Schulz-Friedrich R & Appel J (2006) Metal dependence and
Ferreira P & Tamagnini P (2007) Transcription and regulation intracellular regulation of the bidirectional NiFe-hydrogenase
of the hydrogenase(s) accessory genes, hypFCDEAB, in the in Synechocystis sp. PCC 6803. Int J Hydrogen Energ 31:
cyanobacterium Lyngbya majuscula CCAP 1446/4. Arch 14521459.
Microbiol (Epub ahead of print, PMID: 17639348). Hallenbeck PC & Benemann JR (1978) Characterization and
Friedman DI (1988) Integration host factor: a protein for all partial purification of the reversible hydrogenase of Anabaena
reasons. Cell 55: 545554. cylindrica. FEBS Lett 94: 261264.
Fritsche E, Paschos A, Beisel HG, Bock A & Huber R (1999) Hansel A, Axelsson R, Lindberg P, Troshina OY, Wunschiers R &
Crystal structure of the hydrogenase maturating Lindblad P (2001) Cloning and characterisation of a hyp gene
endopeptidase HYBD from Escherichia coli. J Mol Biol 288: cluster in the filamentous cyanobacterium Nostoc sp. strain
989998. PCC 73102. FEMS Microbiol Let 201: 5964.
Fulda S, Mikkat S, Huang F, Huckauf J, Marin K, Norling B & Happe T, Schutz K & Bohme H (2000) Transcriptional and
Hagemann M (2006) Proteome analysis of salt stress response mutational analysis of the uptake hydrogenase of the
in the cyanobacterium Synechocystis sp. strain PCC 6803. filamentous cyanobacterium Anabaena variabilis ATCC 29413.
Proteomics 6: 27332745. J Bacteriol 182: 16241631.
Gan CS, Reardon KF & Wright PC (2005) Comparison of protein Herrero A, Muro-Pastor AM & Flores E (2001) Nitrogen control
and peptide prefractionation methods for the shotgun in cyanobacteria. J Bacteriol 183: 411425.
proteomic analysis of Synechocystis sp. PCC 6803. Proteomics 5: Herrero A, Muro-Pastor AM, Valladares A & Flores E (2004)
24682478. Cellular differentiation and the NtcA transcription factor in
Ghirardi ML, King PW, Posewitz MC, Maness PC, Fedorov A, filamentous cyanobacteria. FEMS Microbiol Rev 28: 469487.
Kim K, Cohen J, Schulten K & Seibert M (2005) Approaches to Hihara Y, Kamei A, Kanehisa M, Kaplan A & Ikeuchi M (2001)
developing biological H2-photoproducing organisms and DNA microarray analysis of cyanobacterial gene expression
processes. Biochem Soc Trans 33: 7072. during acclimation to high light. Plant Cell 13: 793806.

Hoffmann D, Gutekunst K, Klissenbauer M, Schluz-Friedrich R Kaneko T, Nakamura Y, Wolk CP et al. (2001) Complete genomic
& Appel J (2006) Mutagenesis of hydrogenase accessory genes sequence of the filamentous nitrogen-fixing cyanobacterium
of Synechocystis sp. PCC 6803. Additional homologues of hypA Anabaena sp. strain PCC 7120. DNA Res 8: 205213.
and hypB are not active in hydrogenase maturation. FEBS J Kentemich T, Bahnweg M, Mayer F & Bothe H (1989)
273: 45164527. Localization of the reversible hydrogenase in cyanobacteria. Z
Houchins JP (1984) The physiology and biochemistry of Naturforsch 44c: 384391.
hydrogen metabolism in cyanobacteria. Biochim Biophys Acta Kentemich T, Casper M & Bothe H (1991) The reversible
768: 227255. hydrogenase in Anacystis nidulans is a component of the
Houchins JP & Burris RH (1981a) Comparative characterization cytoplasmic membrane. Naturwissenschaften 78: 559560.
of two distinct hydrogenases from Anabaena sp. strain 7120. King PW, Posewitz MC, Ghirardi ML & Seibert M (2006)
J Bacteriol 146: 215221. Functional studies of [FeFe] hydrogenase maturation in an
Houchins JP & Burris RH (1981b) Occurrence and localization of Escherichia coli biosynthetic system. J Bacteriol 188:
two distinct hydrogenases in the heterocystous 21632172.
cyanobacterium Anabaena sp. strain 7120. J Bacteriol 146: Kovacs KL, Kovacs AT, Maroti G, Meszaros LS, Balogh J,
209214. Latinovics D, Fulop A, David R, Doroghazi E & Rakhely G
Howarth DC & Codd GA (1985) The uptake and production of (2005) The hydrogenases of Thiocapsa roseopersicina. Biochem
Soc Trans 33: 6163.

molecular hydrogen by unicellular cyanobacteria. J Gen
Microbiol 131: 15611569. Kruse O, Rupprecht J, Mussgnug JH, Dismukes GC & Hankamer
Howitt CA & Vermaas W (1999) Subunits of the NAD(P)- B (2005) Photosynthesis: a blueprint for solar energy capture
reducing nickel-containing hydrogenase do not act as part of and biohydrogen production technologies. Photochem
the type-1 NAD(P)H-dehydrogenase in the cyanobacterium Photobiol Sci 4: 957970.
Kuchar J & Hausinger RP (2004) Biosynthesis of metal sites.
Synechocystis sp. PCC 6803. The Phototrophic Prokaryotes
Chem Rev 104: 509525.
(Peschek GA, Loffelhardt W & Schmetterer G, eds), pp.
Kucho K-i, Okamoto K, Tsuchiya Y, Nomura S, Nango M,
595601. Kluwer Academic/Plenum Publishers, New York.
Kanehisa M & Ishiura M (2005) Global analysis of circadian
Ihara M, Nakamoto H, Kamachi T, Okura I & Maeda M (2006a)
expression in the cyanobacterium Synechocystis sp. strain PCC
Photoinduced hydrogen production by direct electron transfer
6803. J Bacteriol 187: 21902199.
from photosystem I cross-linked with cytochrome c3 to
Kumar D & Kumar HD (1991) Effect of monochromatic lights on
[NiFe]-hydrogenase. Photochem Photobiol 82: 16771685.
nitrogen-fixation and hydrogen evolution in the isolated
Ihara M, Nishihara H, Yoon K-S, Lenz O, Friedrich B, Nakamoto
heterocysts of Anabaena sp. strain CA. Int J Hydrogen Energy
H, Kojima K, Honma D, Kamachi T & Okura I (2006b)
16: 397401.
Light-driven hydrogen production by a hybrid complex of a
Kumar S & Polasa H (1991) Influence of nickel and copper on
[NiFe]-hydrogenase and the cyanobacterial photosystem I.
photobiological hydrogen production and uptake in
Photochem Photobiol 82: 676682.
Oscillatoria subbrevis strain 111. Proc Indian Nat Sci Acad B57:
Ikeuchi M & Tabata S (2001) Synechocystis sp. PCC 6803 a
281285.
useful tool in the study of the genetics of cyanobacteria. Kumar AP, Perraju BTVV & Singh HN (1986) Carbon nutrition
Photosynth Res 70: 7383. and the regulation of uptake hydrogenase activity in free-living
Jacobi A, Rossmann R & Bock A (1992) The hyp operon gene and symbiotic Anabaena cycadeae. New Phytol 104: 115120.
products are required for the maturation of catalytically active Kurian D, Jansen T & Maenpaa P (2006) Proteomic analysis of
hydrogenase isoenzymes in Escherichia coli. Arch Microbiol heterotrophy in Synechocystis sp. PCC 6803. Proteomics 6:
158: 444451. 14831494.
Kamei A, Hihara Y, Yoshihara S, Geng X, Kanehisa M & Ikeuchi Lambert GR & Smith GD (1981) The hydrogen metabolism of
M (2001) Functional Analysis of lexA-like gene, sll1626 in cyanobacteria (blue-green algae). Biol Rev 56: 589660.
Synechocystis sp. PCC 6803 using DNA microarray. Plant Cell Lee JW & Greenbaum E (2003) A new oxygen sensitivity and its
Physiol 32suppl: S95. potential application in photosynthetic H2 production. Appl
Kaneko T, Tanaka A, Sato S, Kotani H, Sazuka T, Miyajima N, Biochem Biotechnol 105: 303313.
Sugiura M & Tabata S (1995) Sequence analysis of the genome Lee JW, Mets L & Greenbaum E (2002) Improvement of
of the unicellular cyanobacterium Synechocystis sp. strain PCC photosynthetic CO2 fixation at high light intensity through
6803. I. Sequence features in the 1 Mb region from map reduction of chlorophyll antenna size. Appl Biochem Biotechnol
positions 64% to 92% of the genome. DNA Res 2: 153166. 98: 3748.
Kaneko T, Sato S, Kotani H et al. (1996) Sequence analysis of the Leitao E, Oxelfelt F, Oliveira P, Moradas-Ferreira P & Tamagnini
genome of the unicellular cyanobacterium Synechocystis sp. P (2005) Analysis of the hupSL operon of the
strain PCC6803. II. Sequence determination of the entire nonheterocystous cyanobacterium Lyngbya majuscula CCAP
genome and assignment of potential protein-coding regions. 1446/4: regulation of transcription and expression under a
DNA Res 3: 185209. light-dark regime. Appl Environ Microbiol 71: 45674576.

Leitao E, Pereira S, Bondoso J, Ferreira D, Pinto F, Moradas- the activity of the three hydrogenase isoenzymes in Escherichia
Ferreira P & Tamagnini P (2006) Genes involved in the coli. Mol Microbiol 5: 123135.
maturation of hydrogenase(s) in the nonheterocystous Magalon A & Bock A (2000a) Analysis of the HypCHycE
cyanobacterium Lyngbya majuscula CCAP 1446/4. Int J complex, a key intermediate in the assembly of the metal
Hydrogen Energy 31: 14691477. center of the Escherichia coli hydrogenase 3. J Biol Chem 275:
Levin DB, Lawrence P & Murry L (2004) Biohydrogen 2111421120.
production: prospects and limitations to practical application. Magalon A & Bock A (2000b) Dissection of the maturation
Int J Hydrogen Energy 29: 173185. reactions of the [NiFe] hydrogenase 3 from Escherichia coli
Li H, Singh AK, McIntyre LM & Sherman LA (2004) Differential taking place after nickel incorporation. FEBS Lett 473:
gene expression in response to hydrogen peroxide and the 254258.
putative PerR regulon of Synechocystis sp. strain PCC 6803. Maier T & Bock A (1996) Generation of active [NiFe]
J Bacteriol 186: 33313345. hydrogenase in vitro from a nickel-free precursor form.
Lindberg P (2003) Cyanobacterial hydrogen metabolism uptake
Biochemistry 35: 1008910093.
hydrogenase and hydrogen production by nitrogenase in
Maier T, Jacobi A, Sauter M & Bock A (1993) The product of the
filamentous cyanobacteria. PhD Thesis, Faculty of Science
hypB gene, which is required for nickel incorporation into
and Technology, Uppsala University, Sweden (ISBN
hydrogenases, is a novel guanine nucleotide-binding protein. J
91-554-5708-8).

Bacteriol 175: 630635.
Lindberg P, Hansel A & Lindblad P (2000) hupS and hupL
Maier T, Lottspeich F & Bock A (1995) GTP hydrolysis by HypB is
constitute a transcription unit in the cyanobacterium Nostoc
essential for nickel insertion into hydrogenases of Escherichia
sp. PCC 73102. Arch Microbiol 174: 129133.
Lindberg P, Schutz K, Happe T & Lindblad P (2002) A hydrogen- coli. Eur J Biochem 230: 133138.
producing, hydrogenase-free mutant strain of Nostoc Manyani H, Rey L, Palacios JM, Imperial J & Ruiz-Argueso T
punctiforme ATCC 29133. Int J Hydrogen Energy 27: (2005) Gene products of the hupGHIJ operon are involved in
12911296. maturation of the ironsulfur subunit of the [NiFe]
Lindberg P, Lindblad F & Cournac L (2004) Gas exchange in the hydrogenase from Rhizobium leguminosarum bv. viciae. J
filamentous cyanobacterium Nostoc punctiforme strain ATCC Bacteriol 187: 70187026.
29133 and its hydrogenase-deficient mutant strain NHM5. Margheri MC, Tredici MR, Allotta G & Vagnoli L (1991)
Appl Environ Microbiol 70: 21372145. Heterotrophic metabolism and regulation of uptake
Lindblad P & Sellstedt A (1990) Occurrence and localization of an hydrogenase activity in symbiotic cyanobacteria. Plant Soil
uptake hydrogenase in the filamentous heterocystous 137: 139144.
cyanobacterium Nostoc PCC 73102. Protoplasma 159: 915. Masukawa H, Mochimaru M & Sakurai H (2002) Disruption of
Lindblad P, Christensson K, Lindberg P, Fedorov A, Pinto F & the uptake hydrogenase gene, but not of the bi-directional
Tsygankov A (2002) Photoproduction of H2 by wildtype hydrogenase gene, leads to enhanced photobiological
Anabaena PCC 7120 and a hydrogen uptake deficient mutant: hydrogen production by the nitrogen-fixing cyanobacterium
from laboratory experiments to outdoor culture. Int J Anabaena sp. PCC 7120. Appl Microbiol Biotechnol 58:
Hydrogen Energy 27: 12711281. 618624.
Llama MJ, Serra JL, Rao KK & Hall DO (1979) Isolation and Meeks JC, Elhai J, Thiel T, Potts M, Larimer F, Lamerdin J, Predki
characterization of the hydrogenase activity from the non- P & Atlas R (2001) An overview of the genome of Nostoc
heterocystous cyanobacterium Spirulina maxima. FEBS Lett
punctiforme, a multicellular, symbiotic cyanobacterium.
98: 342346.
Photosynth Res 70: 85106.
Long M, Liu J, Chen Z, Bleijlevens B, Roseboom W & Albracht SP
Mehta N, Olson JW & Maier RJ (2003) Characterization of
(2007) Characterization of a HoxEFUYH type of [NiFe]
Helicobacter pylori nickel metabolism accessory proteins
hydrogenase from Allochromatium vinosum and some EPR
needed for maturation of both urease and hydrogenase. J
and IR properties of the hydrogenase module. J Biol Inorg
Bacteriol 185: 726734.
Chem 12: 6278.
Melis A, Neidhardt J & Benemann JR (1998) Dunaliella salina
Ludwig M, Schulz-Friedrich R & Appel J (2006) Occurrence of
hydrogenases in cyanobacteria and anoxygenic photosynthetic (Chlorophyta) with small chlorophyll antenna sizes exhibit
bacteria: implications for the phylogenetic origin of higher photosynthetic productivities and photon use
cyanobacterial and algal hydrogenases. J Mol Evol 63: 758768. efficiencies than normally pigmented cells. J Appl Phycol 10:
Lundgren P, Bauer K, Lugomela C, Soderback E & Bergman B 515525.
(2003) Reevaluation of the nitrogen fixation behaviour in the Menon NK, Chatelus CY, Dervartanian M, Wendt JC,
marine non-heterocystous cyanobacterium Lyngbya Shanmugam KT, Peck HD Jr & Przybyla AE (1994) Cloning,
majuscula. J Phycol 39: 310314. sequencing, and mutational analysis of the hyp operon
Lutz S, Jacobi A, Schlensog V, Bohm R, Sawers G & Bock A (1991) encoding Escherichia coli hydrogenase 2. J Bacteriol 176:
Molecular characterization of an operon (hyp) necessary for 44164423.

Mikheeva LE, Schmitz O, Shestakov SV & Bothe H (1995) Paschos A, Bauer A, Zimmermann A, Zehelein E & Bock A (2002)
Mutants of the cyanobacterium Anabaena variabilis altered in HypF, a carbamoyl phosphate-converting enzyme involved in
hydrogenase activities. Z Naturforsch 50c: 505510. [NiFe] hydrogenase maturation. J Biol Chem 277:
Miyagishima SY (2005) Origin and evolution of the chloroplast 4994549951.
division machinery. J Plant Res 118: 295306. Patterson-Fortin LM, Colvin KR & Owttrim GW (2006) A LexA-
Mulkidjanian AY, Koonin EV, Makarova KS et al. (2006) The related protein regulates redox-sensitive expression of the
cyanobacterial genome core and the origin of photosynthesis. cyanobacterial RNA helicase, crhR. Nucl Acids Res 34:
Proc Natl Acad Sci USA 103: 1312613131. 34463454.
Mulrooney SB & Hausinger RP (2003) Nickel uptake and Polle JEW, Kanakagiri SD & Melis A (2003) tla1, a DNA
utilization by microorganisms. FEMS Microbiol Rev 27: insertional transformant of the green alga Chlamydomonas
239261. reinhardtii with a truncated light-harvesting chlorophyll
Nakajima Y & Ueda R (1997) Improvement of photosynthesis in antenna size. Planta 217: 4959.
dense microalgal suspension by reduction of light harvesting Posewitz MC, King PW, Smolinski SL, Zhang LP, Seibert M &
pigments. J Appl Phycol 9: 503510. Ghirardi ML (2004) Discovery of two novel radical S-
Nakajima Y & Ueda R (1999) Improvement of microalgal adenosylmethionine proteins required for the assembly of an
photosynthetic productivity by reducing the content of light active [Fe] hydrogenase. J Biol Chem 279: 2571125720.

harvesting pigment. J Appl Phycol 11: 195201. Prince RC & Kheshgi HS (2005) The photobiological production
Nakamura Y, Kaneko T, Hirosawa M, Miyajima N & Tabata S of hydrogen: potential efficiency and effectiveness as a
(1998) CyanoBase, a www database containing the complete renewable fuel. Crit Rev Microbiol 31: 1931.
nucleotide sequence of the genome of Synechocystis sp. strain Rai AN, Borthakur M, Soderback E & Bergman B (1992)
PCC6803. Nucleic Acids Res 26: 6367. Immunogold localization of hydrogenase in the
Nakamura Y, Kaneko T, Sato S et al. (2003) Complete genome cyanobacterialplant symbioses Peltigera canina, Anthoceros
structure of Gloeobacter violaceus PCC 7421, a cyanobacterium punctatus and Gunnera magellanica. Symbiosis 12: 131144.
that lacks thylakoids. DNA Res 10: 137145. Rakhely G, Kovacs A, Maroti G, Fodor BD, Csanadi G, Latinovics
Oliveira P & Lindblad P (2005) LexA, a transcription regulator D & Kovacs KL (2004) Cyanobacterial-type,
binding in the promoter region of the bidirectional heteropentameric, NAD1-reducing NiFe hydrogenase in the
hydrogenase in the cyanobacterium Synechocystis sp. PCC purple sulfur photosynthetic bacterium Thiocapsa
6803. FEMS Microbiol Lett 251: 5966. roseopersicina. Appl Environ Microbiol 70: 722728.
Oliveira P, Leitao E, Tamagnini P, Moradas-Ferreira P & Oxelfelt F Reade JPH, Dougherty LJ, Rogers LJ & Gallon JR (1999) Synthesis
(2004) Characterization and transcriptional analysis of and proteolytic degradation of nitrogenase in cultures of the
hupSLW in Gloeothece sp. ATCC 27152: an uptake hydrogenase unicellular cyanobacterium Gloeothece strain ATCC 27152.
from a unicellular cyanobacterium. Microbiology 150: Microbiology 145: 17491758.
36473655. Rees DC, Akif Tezcan F, Haynes CA, Walton MY, Andrade S,
Olson JW, Mehta NS & Maier RJ (2001) Requirement of nickel Einsle O & Howard JB (2005) Structural basis of biological
metabolism proteins HypA and HypB for full activity of both nitrogen fixation. Philos Trans A Math Phys Eng Sci 363:
hydrogenase and urease in Helicobacter pylori. Mol Microbiol 971984.
39: 176182. Reissmann S, Hochleitner E, Wang H, Paschos A, Lottspeich F,
Osanai T, Imamura S, Asayama M, Shirai M, Suzuki I, Murata N Glass RS & Bock A (2003) Taming of a poison: biosynthesis of
& Tanaka K (2006) Nitrogen induction of sugar catabolic gene the NiFe-hydrogenase cyanide ligands. Science 299:
expression in Synechocystis sp. PCC 6803. DNA Res 13: 10671070.
185195. Roseboom W, Blokesch M, Bock A & Albracht SP (2005) The
Oxelfelt F (1998) Hydrogenases in the cyanobacterium Nostoc sp. biosynthetic routes for carbon monoxide and cyanide in the
strain PCC 73102. PhD Thesis, Faculty of Science and NiFe active site of hydrogenases are different. FEBS Lett 579:
Technology, Uppsala University, Sweden (ISBN 91-554-4290- 469472.
0). Rossmann R, Maier T, Lottspeich F & Bock A (1995)
Oxelfelt F, Tamagnini P, Salema R & Lindblad P (1995) Hydrogen Characterisation of a protease from Escherichia coli involved in
uptake in Nostoc strain PCC 73102: effects of nickel, hydrogen, hydrogenase maturation. Eur J Biochem 227: 545550.
carbon and nitrogen. Plant Physiol Biochem 33: 617623. Sakamoto T, Delgaizo VB & Bryant DA (1998) Growth on urea
Oxelfelt F, Tamagnini P & Lindblad P (1998) Hydrogen uptake in can trigger death and peroxidation of the cyanobacterium
Nostoc sp. strain PCC 73102. Cloning and characterization of a Synechococcus sp. strain PCC 7002. Appl Environ Microbiol 64:
hupSL homologue. Arch Microbiol 169: 267274. 23612366.
Paschos A, Glass RS & Bock A (2001) Carbamoylphosphate Sakurai H & Masukawa H (2007) Promoting R&D in
requirement for synthesis of the active center of [NiFe]- Photobiological hydrogen production utilizing mariculture-
hydrogenases. FEBS Lett 488: 912. raised cyanobacteria. Mar Biotechnol 9: 128145.

Schmitz O & Bothe H (1996) The diaphorase subunit HoxU of Srivastava R, Pisareva T & Norling B (2005) Proteomic studies of
the bidirectional hydrogenase as electron transferring protein the thylakoid membrane of Synechocystis sp. PCC 6803.
in cyanobacterial respiration? Naturwissenschaften 83: Proteomics 5: 49054916.
525527. Stal LJ & Moezelaar R (1997) Fermentation in cyanobacteria.
Schmitz O, Boison G, Hilscher R, Hundeshagen B, Zimmer W, FEMS Microbiol Rev 21: 179211.
Lottspeich F & Bothe H (1995) Molecular biological analysis of Tamagnini P, Oxelfelt F, Salema R & Lindblad P (1995)
a bidirectional hydrogenase from cyanobacteria. Eur J Biochem Immunological characterization of hydrogenases in the
233: 266276. nitrogen-fixing cyanobacterium Nostoc sp. strain PCC 73102.
Schmitz O, Boison G & Bothe H (2001) Quantitative analysis of Curr Microbiol 31: 102107.
expression of two circadian clock-controlled gene clusters Tamagnini P, Troshina O, Oxelfelt F, Salema R & Lindblad P
coding for the bidirectional hydrogenase in the (1997) Hydrogenases in Nostoc sp. strain PCC 73102, a strain
cyanobacterium Synechococcus sp. PCC7942. Mol Microbiol 41: lacking a bidirectional enzyme. Appl Environ Microbiol 63:
14091417. 18011807.
Schmitz O, Boison G, Salzmann H, Bothe H, Schutz K, Wang S & Tamagnini P, Costa J-L, Almeida L, Oliveira M-J, Salema R &
Happe T (2002) HoxE a subunit specific for the pentameric Lindblad P (2000) Diversity of cyanobacterial hydrogenases, a
bidirectional hydrogenase complex (HoxEFUYH) of molecular approach. Curr Microbiol 40: 356361.
cyanobacteria. Biochim Biophys Acta 1554: 6674. Tamagnini P, Axelsson R, Lindberg P, Oxelfelt F, Wunschiers R &

Schopf JW (2000) The fossil record: tracing the roots of the Lindblad P (2002) Hydrogenases and hydrogen metabolism of
cyanobacterial lineage. The Ecology of Cyanobacteria (Whitton cyanobacteria. Microbiol Mol Biol Rev 66: 120.
BA & Potts M, eds), pp. 1335. Kluwer Academic Publishers, Tamagnini P, Leitao E & Oxelfelt F (2005) Uptake hydrogenase in
Dordrecht, the Netherlands. cyanobacteria: novel input from non-heterocystous strains.
Schutz K, Happe T, Troshina O, Lindblad P, Leitao E, Oliveira P & Biochem Soc Trans 33: 6769.
Tamagnini P (2004) Cyanobacterial H2 production a Tetali SD, Mitra M & Melis A (2007) Development of the light-
comparative analysis. Planta 218: 350359. harvesting chlorophyll antenna in the green alga
Serebriakova LT, Zorin NA & Lindblad P (1994) Reversible Chlamydomonas reinhardtii is regulated by the novel Tla1
hydrogenase in Anabaena variabilis ATCC 29413: presence and gene. Planta 225: 813829.
localization in non-N2-fixing cells. Arch Microbiol 161: Theodoratou E, Paschos A, Magalon A, Fritsche E, Huber R &
140144. Bock A (2000a) Nickel serves as a substrate recognition motif
Serebryakova LT, Sheremetieva ME & Lindblad P (2000) H2- for the endopeptidase involved in hydrogenase maturation.
uptake and evolution in the unicellular cyanobacterium Eur J Biochem 267: 19951999.
Chroococcidiopsis thermalis CALU 758. Plant Physiol Biochem Theodoratou E, Paschos A, Mintz-Weber S & Bock A (2000b)
38: 525530. Analysis of the cleavage site specificity of the endopeptidase
Shapiguzov A, Lyukevich AA, Allakhverdiev SI, Sergeyenko TV, involved in the maturation of the large subunit of hydrogenase
Suzuki I, Murata N & Los DA (2005) Osmotic shrinkage of 3 from Escherichia coli. Arch Microbiol 173: 110116.
cells of Synechocystis sp. PCC 6803 by water efflux via Theodoratou E, Huber R & Bock A (2005) [NiFe]-hydrogenase
aquaporins regulates osmostress-inducible gene expression. maturation endopeptidase: structure and function. Biochem
Microbiology 151: 447455. Soc Trans 33: 108111.
Sheremetieva ME, Troshina OY, Serebryakova LT & Lindblad P Troshina OY, Serebryakova LT & Lindblad P (1996) Induction of
(2002) Identification of hox genes and analysis of their H2-uptake and nitrogenase activities in the cyanobacterium
transcription in the unicellular cyanobacterium Gloeocapsa Anabaena variabilis ATCC 29413: effects of hydrogen and
alpicola CALU 743 growing under nitrate-limiting conditions. organic substrate. Curr Microbiol 33: 1115.
FEMS Microbiol Lett 214: 229233. Troshina O, Serebryakova LT, Sheremetieva ME & Lindblad P
Singh AK, Li H & Sherman LA (2004) Microarray analysis and (2002) Production of H2 by the unicellular cyanobacterium
redox control of gene expression in the cyanobacterium Gloeocapsa alpicola CALU 743 during fermentation. Int J
Synechocystis sp. PCC 6803. Physiol Plantarum 120: 2735. Hydrogen Energy 27: 12831289.
Slabas AR, Suzuki I, Murata N, Simon WJ & Hall JJ (2006) Tu C-J, Shrager J, Burnap RL, Postier BL & Grossman AR (2004)
Proteomic analysis of the heat shock response in Synechocystis Consequences of a deletion in dspA on transcript
PCC6803 and a thermally tolerant knockout strain lacking the accumulation in Synechocystis sp. strain PCC 6803. J Bacteriol
histidine kinase 34 gene. Proteomics 6: 845864. 186: 38893902.
Smith GD (1990) Hydrogen metabolism in cyanobacteria. Vignais PM & Colbeau A (2004) Molecular biology of microbial
Phycotalk (Kumar EHD, ed), pp. 131143. Rastogi & Co, hydrogenases. Curr Issues Mol Biol 6: 159188.
Meerut, India. Vignais PM, Billoud B & Meyer J (2001) Classification and
Spiro S & Guest JR (1990) FNR and its role in oxygen-regulated phylogeny of hydrogenases. FEMS Microbiol Rev 25: 455501.
gene expression in Escherichia coli. FEMS Microbiol Rev 6: Volbeda A, Charon M-H, Piras C, Hatchikian EC, Frey M &
399428. Fontecilla-Camps JC (1995) Crystal structure of the

nickeliron hydrogenase from Desulfovibrio gigas. Nature 373: Xiankong Z, Tabita FR & Van Baalen C (1984) Nickel control of
580587. hydrogen production and uptake in Anabaena spp. strains CA
Wang Y, Sun J & Chitnis PR (2000) Proteomic study of the and 1F. J Gen Microbiol 130: 18151818.
peripheral proteins from thylakoid membranes of the Xu Q, Yooseph S, Smith HO & Venter JC (2005) Development of
a novel recombinant cyanobacterial system for hydrogen
cyanobacterium Synechocystis sp. PCC 6803. Electrophoresis 21:
production from water. DOE Genomics: GTL Contractor-
17461754.
Grantee Workshop III 63.
Weisshaar H & Boger P (1985) Pathways of hydrogen uptake in
Yoshino F, Ikeda H, Masukawa H & Sakurai H (2007) High
the cyanobacterium Nostoc muscorum. Arch Microbiol 142: photobiological hydrogen production activity of a Nostoc sp.
349353. PCC 7422 uptake hydrogenase-deficient mutant with high
Whitton BA & Potts M (2000) Introduction to the cyanobacteria. nitrogenase activity. Mar Biotechnol 9: 101112.
The Ecology of Cyanobacteria (Whitton BA & Potts M, eds), pp. Zhang X-H, Shiraiwa Y, Sui Z-H & Zhang X-C (2005a) Cloning
111. Kluwer Academic Publishers, Dordrecht, the and characterization of hoxY gene from Arthrospira and
Netherlands. Spirulina and its application in phylogenetic studies. Periodical
Wolk CP, Ernest A & Elhai J (1994) Heterocyst metabolism and Ocean Univ China 35: 10211025.
Zhang X, Zhang X, Shiraiwa Y, Mao Y, Sui Z & Liu J (2005b)
development. The Molecular Biology of Cyanobacteria (Bryant
Cloning and characterization of hoxH genes from Arthrospira

DA, ed), pp. 769823. Kluwer Academic Publishers,
and Spirulina and application in phylogenetic study. Mar
Dordrecht, the Netherlands. Biotechnol 7: 287296.
Wunschiers R, Batur M & Lindblad P (2003) Presence and Zhao Y, Bian S-M, Zhou H-N & Huang J-F (2006) Diversity of
expression of hydrogenase specific C-terminal endopeptidases nitrogenase systems in diazotrophs. J Int Plant Biol 48:
in cyanobacteria. BMC Microbiol 3: 8. 745755.


Tamagnini Peter Hydrogenase Review 2007

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Tamagnini Peter Hydrogenase Review 2007

Uploaded by

Copyright:

Available Formats

REVIEW ARTICLE

Cyanobacterial hydrogenases: diversity, regulation and applications

Correspondence: Paula Tamagnini, IBMC Abstract

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

Fossil traces of cyanobacteria are claimed to have been

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

FEMS Microbiol Rev 31 (2007) 692720

Table 1. Distribution of genes related to hydrogenases in representative cyanobacterial strains

Published by Blackwell Publishing Ltd. All rights reserved

Filamentous L. majuscula 1 ND 1 ND 1 1 Leitao et al. (2005,

Filamentous A. variabilis 1 1 1 1 1 1 NC_007413

FEMS Microbiol Rev 31 (2007) 692720

NifK NifD H2 HoxH HoxE NADH

NifH NifD NifK 2H++ 2e HoxY HoxF NAD+

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

FEMS Microbiol Rev 31 (2007) 692720

Gloeothece sp. ATCC 27152 1000 bp

Trichodesmium erythraeum IMS101

Tery0801 Tery0799 Tery0790

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

ORF1 ORF3 ORF4 ORF5 ORF6 ORF7 ORF9 ORF11

Nostoc punctiforme ATCC 29133 / PCC 73102

NpR0363 NpR0367 NpR0370 NpF0371

Nostoc sp. PCC 7120

asr0697 alr0693 alr0692 asr0690 all0675

Synechococcus elongatus PCC 7942

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

Anabaena variabilis ATCC 29413

Ava4605 Ava4652 Ava4655 Ava4658 Ava4662 Ava4664

Nostoc sp. PCC 7120

asr0697 alr0750 alr0763 alr0765 all0767 all0769

FEMS Microbiol Rev 31 (2007) 692720

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

Vegetative cell Heterocyst

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

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

FEMS Microbiol Rev 31 (2007) 692720

Nosctoc punctiforme PCC 73102 Synechocystis sp. PCC 6803

+1 Nostoc punctiforme PCC 73102

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

FEMS Microbiol Rev 31 (2007) 692720

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

FEMS Microbiol Rev 31 (2007) 692720

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

FEMS Microbiol Rev 31 (2007) 692720

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

FEMS Microbiol Rev 31 (2007) 692720

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

FEMS Microbiol Rev 31 (2007) 692720

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

FEMS Microbiol Rev 31 (2007) 692720

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

FEMS Microbiol Rev 31 (2007) 692720

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015

FEMS Microbiol Rev 31 (2007) 692720

Downloaded from http://femsre.oxfordjournals.org/ by guest on December 30, 2015