Cyanobacteria, one of the largest and most important groups of bacteria on Earth, are able to perform oxygenic photosynthesis using water as an electron donor and may be found in almost any ecological niche from fresh to salt water, terrestrial and extreme environments (Whitton & Potts, 2000). The knowledge on such a diverse group of prokaryotic organisms has greatly increased since cyanobacterial genomes became available. In 1996, the entire sequence of Synechocystis sp. PCC 6803 was published (Kaneko et al., 1996; Nakamura et al., 1998), and since then, many other cyanobacterial genome projects have been completed and released, including that of Nostoc punctiforme ATCC 29133/PCC 73102, one of the largest microbial genomes sequenced so far (Meeks et al., 2001; Anderson et al., 2006).
Fossil traces of cyanobacteria are claimed to have been found from around 3.5 billion years ago (Schopf, 2000), and ancestors of cyanobacteria most probably played a key role in the formation of atmospheric oxygen, and are thought to have evolved into present-day chloroplasts of algae and green plants (Miyagishima, 2005; Mulkidjanian et al., 2006). Cyanobacteria display a relatively wide range of morphological diversity, including unicellular, filamentous and colonial forms. Some filamentous strains form differentiated cells specialized in nitrogen fixation – heterocysts, and spore-like resting cells – akinetes. A number of nonheterocystous strains are also able to perform N2 fixation under certain conditions. The fact that several cyanobacteria are able to reduce nitrogen and carbon under aerobic conditions may be responsible for their evolutionary and ecological success. In cyanobacteria, as in any diazotrophic bacteria, the reduction of N2 to NH3 is accompanied by the formation of molecular hydrogen (Berman-Frank et al., 2003). The H2 produced by the nitrogenase is rapidly consumed by an uptake hydrogenase, an enzyme that has been found in almost all the N2-fixing cyanobacteria examined so far, with one reported exception –Synechococcus sp. BG 043511 (Ludwig et al., 2006). Additionally, these strains may contain a bidirectional hydrogenase, an enzyme that is generally present in the non nitrogen-fixing cyanobacteria (Tamagnini et al., 2002, 2005), but absent in Gloeobacter violaceus PCC 7421, a cyanobacterium that possesses a number of unique characteristics such as the absence of thylakoids (Nakamura et al., 2003; Ludwig et al., 2006). The distribution of genes related to hydrogenases among representative cyanobacterial strains is displayed in Table 1. Both cyanobacterial hydrogenases are NiFe enzymes, which are the most common hydrogenases found in bacteria and Archaea. The core enzyme consists of an αβ heterodimer with the large/α subunit hosting the bimetallic active site, and the small/β-subunit containing the FeS clusters, which function as electron transfer domains between the electron acceptors/donors and the catalytic center of the enzyme (Fig. 1). In general, the NiFe hydrogenases are divided into four groups, with the cyanobacterial uptake hydrogenases clustering together with the cytoplasmic H2 sensors of group 2, and the bidirectional enzymes belonging to group 3 comprising the bidirectional heteromultimeric cytoplasmic hydrogenases (for reviews on this subject, see Vignais et al., 2001; Vignais & Colbeau, 2004).
|Unicellular non-N2-fixing||G. violaceus PCC 7421||−||−||−||−||−||−||NC_005125 |
Nakamura et al. (2003)
|Synechocystis sp. PCC 6803||+|
Appel & Schulz (1996)
Kaneko et al. (1996)
|Unicellular N2-fixing||C. watsonii WH 8501||−||−||+||−||+||+|
|Filamentous nonheterocystous||L. majuscula CCAP 1446/4||+||ND||+||ND||+||+|
|Leitão et al. (2005, 2006)|
|N2-fixing||T. erythraeum IMS 101||−||−||+||−||+||+||NC_008312|
|Filamentous heterocystous N2-fixing||A. variabilis ATCC 29413||+|
Schmitz et al. (1995)
Happe et al. (2000)
|Nostoc sp. PCC 7120||+||+||+|
Carrasco et al. (1995)
Gubili & Borthakur (1996, 1998)
Kaneko et al. (2001)
|N. punctiforme PCC 73102||−||−||+|
Oxelfelt et al. (1998)
Hansel et al. (2001)
In the present review, recent advances on cyanobacterial hydrogenases, have been summarized focusing on achievements on the diversity and molecular regulation of both the uptake and the bidirectional enzyme.
Photobiological production of H2 by microorganisms is of great public interest because it promises a renewable energy carrier from nature's most plentiful resources: solar energy and water. Cyanobacteria and green algae are the only organisms known so far that are capable of both oxygenic photosynthesis and hydrogen production. In a separate section, the possibilities and challenges in cyanobacterial-based hydrogen production are outlined.
The cyanobacterial uptake hydrogenase, found exclusively in N2-fixing strains and encoded by the hup– hydrogen uptake – genes, is at least a heterodimeric enzyme with a large subunit of about 60 kDa containing the active site (HupL) and a small subunit of c. 35 kDa playing a role in electron transfer (HupS) (Fig. 1). Because the physiological and biochemical data point to a membrane-bound enzyme (Houchins & Burris, 1981b; Houchins, 1984; Lindblad & Sellstedt, 1990; Rai et al., 1992), and the hydropathy profiles of the HupL and the HupS proteins do not indicate any transmembrane domains (Tamagnini et al., 2005), the existence of a polypeptide that anchors the HupSL heterodimer to the membrane seems likely. In fact, analysis of the available genomes revealed the presence of ORFs whose products could potentially fulfill this anchoring role (Lindberg, 2003). However, to date no definitive proof was obtained, and the existence of both a soluble and a membrane-bound form of the enzyme cannot be excluded (see for e.g. Houchins & Burris, 1981b).
Immunolocalization studies, using antibodies produced against hydrogenases from other bacteria, showed that the hydrogenase antigens are present in both the vegetative cells and heterocysts of N. punctiforme, and several symbiotic Nostoc strains (Lindblad & Sellstedt, 1990; Rai et al., 1992; Tamagnini et al., 1995). However, these studies do not clarify whether the enzyme is in its active form in both cell types. In Anabaena/Nostoc sp. PCC 7120, the uptake hydrogenase activity was essentially associated with the particulate fraction of the heterocysts (Houchins & Burris, 1981b); however, one must bear in mind that in this strain the hupL gene undergoes a rearrangement, allowing its expression in the heterocysts only, and that this process does not occur in N. punctiforme (Oxelfelt et al., 1998). Moreover, the presence/levels of the cyanobacterial uptake hydrogenase are certainly dependent on the growth conditions. In heterocystous cyanobacteria grown in air and without combined nitrogen, the uptake hydrogenase activity is mainly confined to heterocysts, where it is protected from oxygen inactivation; however, the exact location of the enzyme in cyanobacteria should be further investigated in both heterocystous and nonheterocystous strains.
A strong correlation between the nitrogen-fixation process and the uptake hydrogenase activity has been demonstrated for cyanobacteria (Lambert & Smith, 1981; Houchins, 1984; Wolk et al., 1994; Oxelfelt et al., 1995; Schütz et al., 2004), and this indicates that the main physiological function of the uptake hydrogenase is to reutilize and regain the H2/electrons produced by the H2 evolution through the nitrogenase. This recycling has been suggested to have at least three beneficial functions to the organism: (1) it provides ATP via the oxyhydrogen reaction, minimizing the loss of energy; (2) it removes the oxygen from nitrogenase, thereby protecting it from inactivation; and (3) it supplies reducing equivalents (electrons) to various cell functions (Bothe et al., 1977, 1991; Howarth & Codd, 1985; Weisshaar & Böger, 1985; Smith, 1990).
Physical organization of hup genes and the corresponding proteins
The physical arrangement of the structural genes encoding the uptake hydrogenase is very similar in all the cyanobacteria studied so far: hupS and hupL are contiguous, with the gene encoding the smaller subunit located upstream from the gene encoding the larger one (Carrasco et al., 1995; Oxelfelt et al., 1998; Happe et al., 2000; Lindberg et al., 2000; Oliveira et al., 2004; Leitão et al., 2005) (Fig. 2). Transcriptional start sites have been identified upstream of the hupS start codon (Happe et al., 2000; Lindberg et al., 2000; Oliveira et al., 2004; Leitão et al., 2005), and a putative transcriptional terminator, located immediately downstream of hupL, has been found in N. punctiforme (Lindberg et al., 2000). In agreement, reverse transcriptase (RT)-PCR experiments, and the sizes of transcripts determined by Northern blot, indicate that hupSL constitute a transcriptional unit in Anabaena variabilis ATCC 29413, N. punctiforme and Lyngbya majuscula CCAP 1446/4 (Happe et al., 2000; Lindberg et al., 2000; Leitão et al., 2005). In the unicellular Gloeothece sp. ATCC 27152 and in the filamentous Trichodesmium erythraeum IMS 101 hupW– the gene encoding for the putative uptake hydrogenase-specific endopeptidase – is the ORF located immediately downstream of hupL, and was shown to be cotranscribed with hupSL in Gloeothece sp. ATCC 27152 (Oliveira et al., 2004). In other strains, the position of hupW related to the hupSL varies considerably, and in the strains examined they are transcribed independently (Wünschiers et al., 2003) (Fig. 2).
Analysis of the predicted proteins encoded by the hupSL operon demonstrated that whereas HupS has the same number of amino acid residues in all the cyanobacteria investigated [320 amino acids (aa)], HupL generally has 531 aa with the exception of the filamentous nonheterocystous L. aestuarii CCY 9616 (six extra), L. majuscula (six extra), and T. erythraeum (three extra). To date, the physiological significance (if any) of these extra residues is still unknown.
In the NiFe hydrogenases, the large subunit harbors the active center that is deeply buried inside the protein, close to the large interface between the two subunits, and the small subunit contains the FeS clusters that conduct electrons between the active center and the physiological electron acceptor/donor (Vignais et al., 2001; Vignais & Colbeau, 2004). In concordance, the cyanobacterial HupL sequences contain the four conserved cysteine residues that are involved in the coordination of the bimetallic NiFe center of the active site, and HupS contains eight cysteine residues clearly corresponding to those involved in the formation of the FeS clusters, and a ninth cysteine slightly shifted compared with other bacteria (Tamagnini et al., 2002). In addition, HupL contains the C-terminal region that is presumably cleaved off, by a specific endopeptidase, as the last step of the maturation of the large subunit. In contrast with other organisms, HupS lacks both the twin-arginine signal peptide at the N-terminal, and the hydrophobic motif at the C-terminal proposed to be involved in translocation and anchorage to the membrane, respectively. As mentioned previously, these general features of the cyanobacterial hydrogenases cluster them together with the soluble H2-sensing enzymes (Vignais et al., 2001; Vignais & Colbeau, 2004). However, the construction of hup− mutants proved that the cyanobacterial uptake hydrogenase is indeed a physiological functional enzyme rather than a regulatory one (Happe et al., 2000; Lindberg et al., 2002; Lindblad et al., 2002; Masukawa et al., 2002).
hupL rearrangement in heterocystous strains
Programmed DNA rearrangements have been described in eukaryotes and prokaryotes but are relatively uncommon events. In cyanobacteria, developmentally regulated DNA rearrangements have been reported to occur in heterocystous strains (for a review, see Golden, 1997). Generally, the ORF is interrupted in the vegetative cells by a 10–60-kb DNA element, which is excised during the differentiation of a photosynthetic vegetative cell into a N2-fixing heterocyst, restoring the structure of the gene/operon and allowing its expression in heterocysts only.
The rearrangement within hupL (large subunit of the uptake hydrogenase) was first described for Nostoc sp. PCC 7120 (Carrasco et al., 1995). In the vegetative cells of this cyanobacterium, hupL is interrupted by a 9.5-kb element that is excised late during the heterocyst differentiation process by a site-specific recombination between the 16-bp direct repeats that flank the element (Fig. 3). The hupL element contains, in one of its borders, the gene that encodes the recombinase necessary for the excision –xisC (Carrasco et al., 1995, 1998, 2005). Site-directed mutagenesis revealed that the XisC protein has a functional similarity to the phage integrase family of recombinases. Recently, it has been unequivocally demonstrated that the inactivation of xisC blocks the hupL rearrangement and that XisC alone is sufficient to catalyze the hupL element site-specific recombination in Nostoc sp. PCC 7120 (Carrasco et al., 2005). It was also shown that the xisC-mutant forms heterocysts without any obvious developmental defects and that the mutant grown under N2-fixing conditions (BG110) was not only defective for hydrogen uptake activity but evolves H2 (Lindblad et al., 2002; Carrasco et al., 2005). Moreover, Lindblad et al. (2002) showed that, in a competitive growth environment with increased light intensity, the wild-type strain has an advantage over the xisC-mutant, probably because these specific conditions induced higher rates of H2 evolution that only the wild type has the capacity of reutilizing through the oxyhydrogen reaction. These findings support the hypothesis that the uptake hydrogenase plays a role in minimizing the loss of energy caused by the nitrogenase-dependent H2 formation.
Despite the hupL element being absent from the two other heterocystous strains for which genome sequences are available, A. variabilis and N. punctiforme (see also Oxelfelt et al., 1998; Happe et al., 2000), DNA hybridization studies showed that sequences similar to xisC were present in about half of the heterocystous strains tested (Tamagnini et al., 2000). These authors also showed that the presence of the bidirectional hydrogenase is not ubiquitous among heterocystous cyanobacteria, although they could not establish a correlation between the presence/absence of the bidirectional enzyme and hupL rearrangement.
hupSL intergenic region
The regions between hupS and hupL in cyanobacteria are longer than in other microorganisms, differ considerably in size (ranging from 43 to 689 bp; see Table 2) and are not particularly conserved (except for Nostoc sp. PCC 7120 and A. variabilis). A prominent feature within the hupSL intergenic region of heterocystous strains is the presence of Short Tandemly Repeated Repetive (STRR) sequences (with the exception of the relatively short 43-bp region of Nostoc sp. Mitsui 38901). STRR sequences have previously been shown to be frequent in heterocyst-forming cyanobacteria and relatively less frequent in unicellular strains (Asayama et al., 1996). Indeed, no STRR sequences could be discerned in the hupSL intergenic region from nonheterocystous cyanobacteria. However, in the filamentous nonheterocystous L. majuscula only about 10% of the intergenic region consists of nonrepetitive nucleotides, with two distinct sets of Long Repeated Repetitive (LRR) sequences clearly identified (for details see Leitão et al., 2005). Because the repetitive sequences within the hupSL intergenic region are highly variable or even absent (Table 2), it is unlikely that these repeats play a direct role in the regulation of gene expression. However, in all strains, a putative stem-loop structure, derived via 2D-computer modeling, might occur in the transcribed RNA (Lindberg et al., 2000; Tamagnini et al., 2002, 2005). The value of free energy (ΔG) was determined for each secondary structure and it was negative in all cases (ranging from −136.32 to −6.9 kcal mol−1), meaning that the formation of the hairpin is favored. It has been hypothesized that the occurrence of the hairpin may increase the stability of the transcript, and/or confer a translational coupling between hupS and hupL by sequestering the ribosome-binding site of hupL and thereby preventing the initiation of translation of this gene (Lindberg et al., 2000). However, although the sequestration of the hupL RBS may be effective in N. punctiforme in which the hairpin folds the entire hupSL intergenic region (Lindberg et al., 2000), it does not occur in all hupSL intergenic hairpin structures predicted. Only the construction of specific mutants will help to clarify the function of these intergenic regions.
|Crocosphaera watsonii WH 8501||67||No||NZ_AADV02000237|
|Cyanothece sp. ATCC 51142||126||No||DQ650318|
|Gloeothece sp. ATCC 27152||259||No||AY260103Oliveira et al. (2004)|
|Lyngbya aestuarii CCY 9616||118||No||DQ375444|
|Lyngbya majuscula CCAP 1446/4||643||LRR||AF368526Leitão et al. (2005)|
|Trichodesmium erythraeum IMS 101||689||No||NZ_AABK04000005|
|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||AF455565Oxelfelt (1998)|
|Nostoc punctiforme PCC 73102||192||STRR||AF030525; NZ_AAAY02000001 |
Oxelfelt et al. (1998)
hup promoter regions and transcriptional regulators
As mentioned previously, in all cyanobacteria studied so far the uptake hydrogenase structural genes are arranged in a contiguous manner with the gene encoding the smaller subunit located upstream of the gene of the larger one. The transcriptional start sites of the hup operons are localized 238, 59, 103 and 259 bp upstream from the hupS start codon for the unicellular Gloeothece sp. ATCC 27152, the filamentous L. majuscula and the filamentous heterocystous A. variabilis and N. punctiforme, respectively (Happe et al., 2000; Lindberg et al., 2000; Oliveira et al., 2004; Leitão et al., 2005) (Fig. 4). The analysis of the regions upstream the transcriptional start point (tsp) revealed the presence of a −10 and a −35 box in both L. majuscula and N. punctiforme, while in Gloeothece sp. ATCC 27152 and A. variabilis only a −10 box could be clearly discerned. A putative binding site for NtcA (a protein that operates global nitrogen control in cyanobacteria) could be found in Gloeothece sp. ATCC 27152, L. majuscula and N. punctiforme, although its relative position to the tsp varied depending on the strain. Moreover, in L. majuscula and N. punctiforme a possible binding site for the integration host factor (IHF) – WATCAAN4TTR (Craig & Nash, 1984; Goodrich et al., 1990; Goodman et al., 1999) – could be recognized in the region between the NtcA motif and the tsp (Fig. 4). It has been postulated that the possible binding of the IHF to the promoter could bend the DNA (Friedman, 1988), and consequently allow the contact of the NtcA with the RNA polymerase complex, activating the hupSL transcription. In the unicellular Gloeothece sp. ATCC 27152, the potential NtcA-binding site is centered at −41.5 bp with respect to the tsp in place of the −35 box, like in the canonical NtcA-activated promoters with the consensus sequence signature GTAN8TAC (Herrero et al., 2001), a structure similar to that of class II bacterial promoters activated by catabolite activator protein (CAP). In L. majuscula and N. punctiforme, the NtcA-binding sites were found to be centered at positions −233.5 and −258.5, respectively, resembling class I CAP-dependent promoters (Busby & Ebright, 1999; Herrero et al., 2001, 2004). These data indicate that the type of the NtcA-activated promoter (class I vs II) is not correlated to the strategies used by heterocystous and nonheterocystous cyanobacteria to separate N2 fixation and photosynthesis. In the filamentous heterocystous A. variabilis, half of a sequence motif identical to the consensus Fnr-binding sequence was identified 144-bp upstream of the tsp (Happe et al., 2000) (Fig. 4). Fnr is a regulator of a fumarate nitrate reductase, which has been found to be involved in the regulation of the hyp operon in Escherichia coli (Lutz et al., 1991), and it is responsible for the induction of several operons in E. coli grown under anaerobic conditions (Spiro & Guest, 1990). In A. variabilis, although there is no rearrangement of the hupL gene, hupSL are expressed in heterocysts only. These differentiated cells have very low intracellular O2 pressures which led Happe et al. (2000) to suggest that the hupSL operon in A. variabilis could be regulated in a manner similar to that of the anaerobically induced operons in E. coli.
The possible interaction between NtcA and the hupSL(W) promoter regions in cyanobacteria was assessed by performing band shift assays. These experiments indicate a specific binding of NtcA to DNA sequences upstream of hupS in the three cyanobacterial strains tested (Gloeothece sp. ATCC 27152, L. majuscula and N. punctiforme), suggesting, indeed, the involvement of NtcA in the transcription regulation of the uptake hydrogenase gene cluster (Lindberg, 2003; Oliveira et al., 2004; Leitão et al., 2005). The fact that the transcription of the uptake hydrogenase structural genes is under the control of the transcriptional regulator that operates global nitrogen control in cyanobacteria reinforces the correlation observed between the activity of the uptake hydrogenase and N2 fixation, already demonstrated in several filamentous heterocystous cyanobacteria (Houchins, 1984; Wolk et al., 1994; Oxelfelt et al., 1995; Troshina et al., 1996).
Transcription and expression patterns of hup genes
The first transcriptional data on cyanobacterial uptake hydrogenases arose from RT-PCR experiments on Nostoc sp. PCC 7120, revealing that hupL is expressed only after a photosynthetic vegetative cell differentiates into a N2-fixing heterocyst (see above details about the DNA rearrangement occurring within this strain, Carrasco et al., 1995, 2005). Subsequent studies with other filamentous heterocystous strains have shown that hupSL is a transcriptional unit (Happe et al., 2000; Lindberg et al., 2000), present in cells grown under N2-fixing conditions (Axelsson et al., 1999; Happe et al., 2000; Hansel et al., 2001). Non-N2-fixing cultures of Nostoc muscorum, a strain without the hupL rearrangement, exhibit no in vivo H2-uptake activity (Axelsson et al., 1999). However, the transfer of N. muscorum cells from non-N2-fixing (ammonia) to N2-fixing conditions induced the appearance of a transcript (after c. 24 h), and the relative amounts of transcript increased in parallel with the H2-uptake activity (Axelsson et al., 1999). A similar pattern of transcription was observed for A. variabilis and N. punctiforme, two other strains with noninterrupted hupL genes (Happe et al., 2000; Hansel et al., 2001). These authors demonstrated that hupSL transcripts were missing in A. variabilis and in N. punctiforme cells grown with ammonia (and in A. variabilis cells grown with nitrate), but were present in both organisms grown under N2-fixing conditions.
While the heterocyst provides a microaerobic environment protecting the oxygen-sensitive nitrogenases and uptake hydrogenases from the atmospheric and intracellulary generated oxygen, the nonheterocystous cyanobacteria developed different approaches. The temporal separation between photosynthesis (light) and nitrogen-fixation/hydrogen uptake (dark) seems to be the most common strategy adopted by the later cyanobacteria (Bergman et al., 1997; Böhme, 1998; Berman-Frank et al., 2003). In fact, in the nonheterocystous Gloeothece sp. ATCC 27152 (unicellular) and L. majuscula (filamentous), grown under nitrogen-fixing conditions and 12 h light/12 h dark cycles, there is an evident light/dark regulation with the highest levels of hupSL(W) transcripts detected during the light phase or in the transition between the light and dark phase, respectively (Oliveira et al., 2004; Leitão et al., 2005). It has also been demonstrated that both organisms exhibit higher hydrogen-uptake activities during the dark period (in agreement with the nitrogen fixation rates; see Reade et al., 1999; Lundgren et al., 2003). In L. majuscula, the increase of the HupL protein levels coincides with the increase of hydrogenase uptake activity during the dark phase. In the beginning of the light phase, no hupSL transcription is detectable, and the levels of both polypeptides and H2 uptake activity begin to decline (Leitão et al., 2005). These results suggest that in L. majuscula, a protein turnover occurs, with degradation taking place during the light period and de novo synthesis taking place during the dark phase. The time difference between the hupSL transcription and the hydrogen uptake activity, both in Gloeothece sp. ATCC 27152 and L. majuscula, might be due to the complexity of the maturation process of the uptake hydrogenase. Thus, it is possible that the translation occurs as soon as the transcript is available, while the enzyme becomes active only after the maturation process is completed. The temporal separation between the photosynthesis and nitrogen fixation/hydrogen uptake activity may also influence the time lag between transcription and activity.
In the presence of combined nitrogen, hupSLW transcription is totally repressed in Gloeothece sp. ATCC 27152, while in L. majuscula the levels of hupSL transcription and expression are significantly reduced but it is possible to discern a pattern similar to the one observed in cells grown under N2-fixing conditions (Oliveira et al., 2004; Leitão et al., 2005, Ferreira et al., 2007). The results obtained for L. majuscula under non-N2-fixing conditions could be explained by the mode of growth of this cyanobacterium, in which the inner cells are probably not in the same conditions notably in terms of access to the combined nitrogen.
Besides the source of nitrogen, other factors were proven to influence the transcription/expression of the cyanobacterial uptake hydrogenases. Similar to any NiFe hydrogenase, the activity of the cyanobacterial uptake enzyme was shown to be dependent on nickel availability, and the addition of external nickel to the growth medium (up to a certain concentration) increased the uptake hydrogenase activity in several strains (Xiankong et al., 1984; Daday et al., 1985; Kumar & Polasa, 1991; Oxelfelt et al., 1995; Axelsson & Lindblad, 2002). Furthermore, the addition of exogenous hydrogen was shown to induce hupSL transcription and hydrogen uptake activity in N. muscorum and N. punctiforme (Oxelfelt et al., 1995; Axelsson & Lindblad, 2002), as well as hydrogen uptake activity in Nostoc sp. PCC 7120 (Houchins & Burris, 1981b). Both cyanobacterial hydrogenases are affected by the oxygen partial pressure. Nostoc muscorum and N. punctiforme cultures transferred from aerobic to anaerobic conditions showed an increase in both the transcription of hupL and hydrogen uptake activity (Axelsson & Lindblad, 2002). Similarly, the uptake hydrogenase activity could be elicited by removing oxygen from the sparging gas of a culture of Nostoc sp. PCC 7120 (Houchins & Burris, 1981b). The addition of organic carbon to the culture medium can also influence the hydrogen uptake activity. Cells of N. punctiforme grown either photo- or chemoheterotrophically reach both higher nitrogenase and hydrogen uptake activities than photoautotrophically grown cells (Oxelfelt et al., 1995). However, the effect of carbon substrates on the cyanobacterial uptake hydrogenase activity is difficult to assess, and apparently contradictory results are reported in the literature (Houchins, 1984; Kumar et al., 1986; Chen et al., 1989; Margheri et al., 1991).