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Hydrogenases and Hydrogen Metabolism of Cyanobacteria

Department of Botany¹,¹ and Institute for Molecular and Cell Biology,² University of Porto, 4150-180 Porto, Portugal, and,² Department of Physiological Botany, EBC, Uppsala University, SE-752 36 Uppsala, Sweden³³

SUMMARY

INTRODUCTION

Cyanobacteria

Nitrogenases

Alternative nitrogenases.

(i) Mo-containing nitrogenases.

(ii) V-containing nitrogenase.

(iii) Fe-containing nitrogenase.

HYDROGENASES

Cyanobacterial Uptake Hydrogenases

hupSL and the deduced proteins.

Transcription of hupSL.

Cyanobacterial Bidirectional Hydrogenases

hox genes and the deduced proteins.

Transcription of hox genes.

Genetic Diversity of Cyanobacterial Hydrogenases

Accessory Genes

Organization and transcription of cyanobacterial hyp genes.

CYANOBACTERIAL BIOHYDROGEN

Rates of Cyanobacterial Hydrogen Production

Strategies for Improving Cyanobacterial Strains for Photobiological Hydrogen Production

Future Research and Development Directions and International Cooperation and Networks

ADDENDUM IN PROOF

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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At present, cyanobacteria are found in a wide range of habitats including aquatic (saltwater and freshwater), terrestrial, and extreme environments (like frigid lakes of the Antarctic or hot springs). Although called blue-green, cyanobacteria may display a variety of colors due to different combinations of the photosynthetic pigments chlorophyll a, carotenoids, and phycobiliproteins. All cyanobacteria combine the ability to perform an oxygenic photosynthesis (resembling that of chloroplasts) with typical prokaryotic features. The possession of chlorophyll a and the use of oxygenic photosynthesis distinguishes cyanobacteria from other photosynthetic bacteria, such as purple and green bacteria. Nevertheless, some cyanobacterial strains can perform anoxygenic photosynthesis by using hydrogen sulfide (H₂S) as the electron donor. Many cyanobacteria can fix atmospheric dinitrogen (N₂) (a capacity not possessed by any eukaryote) into ammonia (NH₃), a form in which the nitrogen is further available for biological reactions. Although quite uniform in nutritional and metabolic respects, cyanobacteria are a morphologically diverse group with unicellular, filamentous, and colonial forms. Among certain filamentous cyanobacteria, there is some degree of cellular differentiation. Within the filament, vegetative cells may develop into structurally modified and functionally specialized cells: the akinetes (resting cells) or the heterocysts (cells specialized in nitrogen fixation). For more detailed information on cyanobacteria, see reference 218.

Unicellular and filamentous cyanobacteria can form symbioses with a wide diversity of hosts. In symbiosis, some cyanobionts perform both photosynthesis and nitrogen fixation while others exhibit only one of these properties (3, 22, 133, 161, 184). It is believed that ancestors of cyanobacteria evolved to become plastids after a long period of endosymbiosis. In biochemical and structural detail, cyanobacteria are especially similar to the chloroplasts of red algae (47, 104).

The taxonomy of cyanobacteria is still a controversial subject, with two prevailing approaches, the botanical approach (8-10, 102, 103) and the bacterial approach (167, 168, 190). Despite the differences, both botanists and bacteriologists divide the cyanobacteria into four or five major subgroups. In fact, the five sections recognized by Rippka et al. (167) coincide broadly with orders of other classifications: Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales, and Stigonematales (for reviews on this subject, see references 218 and 219). At present, different molecular methods are being used and developed to infer phylogenetic relationships. However, the data available for cyanobacteria are still scarce (207, 219). Analysis of 16S rRNA sequences has given the most detailed hypothesis of the evolution of cyanobacteria (218, 219). A polyphasic taxonomy of cyanobacteria, integrating both phenotypic and genotypic characters is currently being assembled. A milestone in the study of cyanobacteria was the publication of the entire genome (3,573,470 bp) of the non-nitrogen-fixing unicellular cyanobacterium Synechocystis strain PCC 6803 (95, 144). A number of other cyanobacterial genome projects are being completed (see below).

Cyanobacteria may possess several enzymes directly involved in hydrogen metabolism: nitrogenase(s) catalyzing the production of hydrogen (H₂) concomitantly with the reduction of nitrogen to ammonia, an uptake hydrogenase catalyzing the consumption of hydrogen produced by the nitrogenase, and a bidirectional hydrogenase which has the capacity to both take up and produce hydrogen (Fig. 1) (13, 21, 58, 75, 87, 113, 115, 117).

View larger version (45K): [in this window] [in a new window]   FIG. 1. Enzymes directly involved in hydrogen metabolism in cyanobacteria. While the uptake hydrogenase is present in all nitrogen-fixing strains tested so far, the bidirectional enzyme is distributed among both nitrogen-fixing and non-nitrogen-fixing cyanobacteria (although it is not a universal cyanobacterial enzyme) (192). The molecular masses indicated for the uptake hydrogenase subunits are mean values calculated from the deduced amino acid sequences of Anabaena strain PCC 7120 (39), Nostoc strain PCC 73102 (150), and A. variabilis ATCC 29413 (79), while the values for the subunits of the bidirectional enzyme are based on data exclusively from A. variabilis ATCC 29413 (173).

View larger version (92K): [in this window] [in a new window]   FIG. 2. Microphotographs of the three cyanobacteria from which genome sequence information is included in the present review. (A) Synechocystis strain PCC 6803; (B) Anabaena strain PCC 7120; (C) Nostoc punctiforme PCC 73102/ATCC 29133. Note the presence of heterocysts (arrows) in the nitrogen-fixing cultures of Anabaena strain PCC 7120 and Nostoc strain PCC 73102. Bar, 10 µm.

The activity of nitrogenase, the enzymatic multiprotein complex for nitrogen fixation, is essential for the maintenance of the nitrogen cycle, since this element often limits the growth of organisms (35, 65, 158, 187). The diversity of prokaryotic microorganisms with the ability to fix nitrogen is in contrast to the remarkable conservation of the nitrogenase itself (36, 58, 80).

The nitrogenase complex consists of two proteins: the dinitrogenase (MoFe protein or protein I) and the dinitrogenase reductase (Fe protein or protein II), (Fig. 1). The dinitrogenase is an ₂ß₂ heterotetramer of about 220 to 240 kDa, and the and ß subunits are encoded by nifD and nifK, respectively. The dinitrogenase reductase, encoded by nifH, is a homodimer of about 60 to 70 kDa and plays the specific role of mediating the transfer of electrons from the external electron donor (a ferredoxin or a flavodoxin) to the dinitrogenase (58, 127, 149). In addition to the three structural nif genes, many other genes are involved in the nitrogen fixation process and its regulation (for a review, see reference 26). Recently, following the completion of several cyanobacterial genome projects, it became evident that some strains contain multiple copies of certain nif genes; for example N. punctiforme (Nostoc strain PCC 73102) appears to have three copies of nifH (199; http://www.jgi.doe.gov/JGI_microbial/html/nostoc/nostoc_homepage.html).

The reduction of nitrogen to ammonia, catalyzed by nitrogenase, is a highly endergonic reaction requiring metabolic energy in the form of ATP. Moreover, this reaction is accompanied by an obligatory reduction of protons (H⁺) to hydrogen. Apparently, with a pure nitrogen atmosphere, at most 75% of the electrons (e^-) would be allocated for nitrogen reduction and at least 25% would be allocated for proton reduction. Since two ATP molecules are required for each electron transferred from dinitrogenase reductase to dinitrogenase, the reaction requires a minimum of 16 ATP molecules until the dinitrogenase has accumulated enough electrons to reduce nitrogen to ammonia. The overall reaction can be written as follows:

Nitrogenase is very oxygen labile; hence, all diazotrophs must protect the enzymatic complex from the deleterious effects of oxygen (O₂). Cyanobacteria and prochlorophytes are the only prokaryotic organisms known to perform oxygenic photosynthesis (58, 129). Cyanobacteria have evolved different mechanisms and strategies, ranging from fixing nitrogen only under anoxic conditions to temporal or even spatial separation of nitrogen fixation and oxygen evolution, to protect their nitrogen-fixing machinery not only from atmospheric oxygen but also from the intracellularly generated oxygen (for reviews, see references 22, 26, 56, 140, 188, 220, and 221). Temporal separation between photosynthetic oxygen evolution and nitrogen fixation seems to be the most common strategy adopted by nonheterocystous cyanobacteria (92, 137, 165). Spatial separation of the two processes is achieved in many filamentous cyanobacteria by differentiation of vegetative cells into cells specialized in nitrogen fixation, i.e., the heterocysts. In the marine filamentous nonheterocystous Trichodesmium, a spatial separation probably occurs between the two processes without any obvious cellular differentiation; only a small fraction of cells within a colony or along the filament are capable of nitrogen fixation. In contrast to the irreversible changes occurring during heterocyst differentiation, those occurring in nonheterocystous cyanobacteria can be reversed (21, 44, 61, 140).

Some filamentous cyanobacteria are able to differentiate 5 to 10% of their vegetative cells into heterocysts (Fig 2). Heterocyst formation does not take place randomly within the filament. Recently, it was proposed that this process in Anabaena variabilis is not controlled by individual cells but by the entire filament (199). The filament senses the nitrogen status and responds by producing heterocyst differentiation signals. However, this does not exclude cell-to-cell signaling in the maintenance of heterocyst pattern (26, 199, 220), and it has been shown that the small diffusible peptide PatS and products of nitrogen fixation control heterocyst differentiation in Anabaena strain PCC 7120 (226, 227). The heterocyst provides a microanaerobic environment suitable for the functioning of nitrogenase since (i) it lacks photosystem II activity, (ii) it has a higher rate of respiratory oxygen consumption, and (iii) it is surrounded by a thick envelope that limits the diffusion of oxygen through the cell wall (56, 221). Heterocysts import carbohydrates and in return export glutamine to the vegetative cells. An interesting feature of heterocyst differentiation in cyanobacteria is the occurrence of developmentally regulated genome rearrangements (39-41, 67-69, 130, 134, 141, 142). These rearrangements occur late in heterocyst differentiation, at about the same time as the nitrogenase genes are transcribed. In vegetative cells of Anabaena strain PCC 7120, the fdxN, nifD, and hupL genes are interrupted by DNA elements that are excised, during heterocyst differentiation, by site-specific recombinases encoded by xisF, xisA, and xisC, respectively. In addition, the excision of the fdxN element requires the products of the two genes xisH and xisI (162).

Alternative nitrogenases. In the heterocystous cyanobacterium A. variabilis ATCC 29413 several different nitrogenases have been identified and characterized (for details, see references 98, 99, 195, and 197 to 199).

(i) Mo-containing nitrogenases. Two of the A. variabilis nitrogenases are molybdenum-dependent enzymes. The first one (the so-called conventional nitrogenase, encoded by the nif1 gene cluster) functions in heterocysts. The second enzyme (encoded by the nif2 gene cluster) functions strictly under anaerobic conditions in vegetative cells only (199).

(ii) V-containing nitrogenase. The occurrence of a vanadium-containing nitrogenase was first reported by Kentemich et al. (98) and subsequently confirmed by Thiel (195). This enzyme is encoded by the vnfDGK gene cluster, which is transcribed in the absence of molybdenum and in which vnfDG are fused into a single gene (195).

(iii) Fe-containing nitrogenase. A fourth nitrogenase is believed to be an iron enzyme similar to the one encoded by the anf gene cluster in Azotobacter vinelandii (23, 99).

Alternative nitrogenases have also been found in other cyanobacterial strains (23), and it is known that they differ from the conventional enzyme physically, chemically, and by their catalytic properties. One must bear in mind that all of the alternative enzymes investigated so far seem to allocate a higher proportion of electrons to the reduction of protons to hydrogen than does the conventional Mo enzyme complex. However, hydrogen produced by nitrogenase is generally taken up by an uptake hydrogenase, so that net hydrogen evolution by nitrogen-fixing cyanobacteria is barely observed, at least under aerobic conditions (7).

HYDROGENASES

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The capacity of certain microorganisms to metabolize molecular hydrogen was discovered at the end of the 19th century (85) and later identified to be catalyzed by a hydrogenase (191). Since then hydrogenases have been observed and characterized in many microorganisms, including some algae, trichomonads, anaerobic ciliates, and chytrid fungi (4, 5, 59, 77, 86, 94, 100, 176, 222, 223, 225). The enzyme catalyzes the simplest of chemical reactions, the reversible reductive formation of hydrogen from protons and electrons:

According to the metal composition of the active site, hydrogenases are classified into three major groups: NiFe, Fe, and metal-free hydrogenases (209).

The majority of hydrogenases are NiFe-containing enzymes, occurring in all bacterial classes (38, 159, 209). The first crystal structure of a NiFe hydrogenase, the representative heterodimeric soluble enzyme of Desulfovibrio gigas (6), was published in 1995 (212). This hydrogenase is involved in periplasmic H₂ oxidation and is phylogentically related to other bacterial uptake hydrogenases (209). The binuclear Ni-containing active site in the large subunit contains an additional Fe ion (37, 60, 212). The nickel ion is ligated to four conserved cysteine residues (RxCGxCxxxH and DPCxxCxxH) at the N- and C-terminal regions, respectively, two of which are bridging the Fe and Ni ions. The Fe is also coordinated by two cyanide ions and one carbon monoxide molecule, two very uncommon biological ligands (18, 78). The hydrogen molecule is believed to access the active site through an identified hydrophobic channel (139), while conserved histidine and glutamate residues spanning from the active site to the molecule surface are responsible for the proton transfer (212). The transfer of electrons between the active site and the redox partner involves the participation of [FeS] clusters in the small subunit of NiFe hydrogenases. Sequence comparisons demonstrated that they possess several conserved cysteines, shown to participate in the formation of the [FeS] clusters. The heterodimeric NiFe hydrogenases are often directly linked to a variable diaphorase part containing additional prosthetic groups, e.g., [FeS] clusters, flavins, and cytochromes (6).

Cyanobacteria possess two functionally different NiFe hydrogenases, an uptake enzyme and a bidirectional enzyme. In 1995, structural genes encoding the latter enzyme were sequenced and characterized (173). These authors, to avoid confusion with the tritium exchange 42-kDa enzyme previously cloned (51-54), chose to call it a bidirectional hydrogenase, a name that is used throughout this review. The 42-kDa enzyme is commonly accepted to be an aminotransferase (228).

Since the small subunits of all known cyanobacterial uptake hydrogenases lack any N-terminal signal peptide (39, 79, 150) it has been suggested that the enzyme is localized on the cytoplasmic side of either the cytoplasmic or the thylakoid membrane (13, 209). In some filamentous strains, it is particularly expressed in the nitrogen-fixing heterocysts with little or no activity in the photosynthetic vegetative cells (see "Transcription of hupSL" below) (39, 87, 88, 152). The recycling of hydrogen produced by nitrogenase has been suggested to have at least three beneficial functions for the organism: (i) it provides the organism with ATP via the oxyhydrogen (Knallgas) reaction; (ii) it removes oxygen from nitrogenase, thereby protecting it from inactivation; and (iii) it supplies reducing equivalents (electrons) to nitrogenase and for other cell functions (32, 33, 90, 183, 216).

The uptake hydrogenase activity versus the bidirectional hydrogenase activity was characterized by Houchins and Burris (89) for Anabaena strain PCC 7120. In filaments, the uptake hydrogenase is resistant to atmospheric oxygen levels, but an inactivation that increased with the disruption of the filament was observed. The two hydrogenases differ in their thermal stability. Specifically, the uptake enzyme is more sensitive to high temperature, with a half-life of 12 min at 70°C, but less sensitive to competitive inhibition by carbon monoxide than is the bidirectional enzyme. Both enzymes have a low K_m for hydrogen, but only the uptake hydrogenase activity was shown to be elicited by addition of hydrogen to the gas phase (48, 87, 116, 151, 169).

Biochemical and molecular data concerning filamentous heterocystous cyanobacteria point to the existence of at least two dissimilar subunits of about 60 and 35 kDa (39, 79, 88, 89, 112, 116, 150, 193). However, the exact subunit composition of cyanobacterial uptake hydrogenases and the molecular mass of the active holoenzyme are still unknown. It is premature to exclude the presence of additional subunits in the active form of the enzyme, since no active cyanobacterial uptake hydrogenase has yet been purified.

The cellular and subcellular localization of hydrogenases in cyanobacteria is unclear. It has been suggested that the enzyme is located in the thylakoid membranes of heterocysts (48), while others reported its presence in both the vegetative cells and heterocysts and a particular association with the cytoplasmic membrane (88, 116, 160). There is a diversity in the number of nitrogenases in a single nitrogen-fixing cyanobacterial strain, ranging from one in, e.g., Anabaena strain PCC 7120 and Nostoc strain PCC 73102 to at least three different nitrogenases in A. variabilis (98, 99, 195, 197, 198). However, since no study has addressed the question of putatively several uptake hydrogenases in a single strain, the pattern(s) of uptake hydrogenase cellular and subcellular localization might be different for different strains under different environmental conditions.

A strong correlation between the activity of an uptake hydrogenase and nitrogen fixation has been demonstrated in filamentous cyanobacteria (87, 107, 151, 221). In A. variabilis, exogenous hydrogen produces only a slight stimulatory effect on the in vivo hydrogen uptake during the early stages of nitrogenase induction (203). The authors suggest that the level of hydrogen evolved by nitrogenase is enough for the induction of hydrogenase synthesis. The temporary stimulation of hydrogen uptake in Nostoc strain PCC 73102 cultures incubated in darkness (i.e., cells with a much lower in vivo nitrogenase activity [126]) implies a strong correlation between the hydrogen uptake and nitrogenase activities and indicates that the addition of exogenous hydrogen alone is not enough to maintain the hydrogen uptake activity (151).

The biosynthesis of NiFe hydrogenases is closely related to Ni metabolism, both to form an active enzyme and at the transcriptional level (101, 135, 211, 212). Cyanobacterial hydrogenases seem to be no exception. In several strains the hydrogen uptake activity is dependent on the concentration of nickel in the growth medium (45, 46, 151, 152, 182, 202, 224).

Addition of organic carbon to the growth medium demonstrated that cells of Nostoc strain PCC 73102, grown either photo- or chemoheterotrophically, reach both higher nitrogenase and hydrogen uptake activities than do photoautotrophically grown cells (151). The maximal in vivo light-dependent hydrogen uptake activity occurred in cells grown heterotrophically in darkness, a condition that mimics symbiosis. This finding is in agreement with the suggested coupling between hydrogen consumption and an efficient nitrogen fixation in Nostoc sp. strain Cc and other symbiotic microorganisms (122, 124, 202). In contrast, autotrophically grown cells of a cyanobacterium isolated from Macrozamia communis have a higher level of both nitrogenase and hydrogenase activity than do heterotrophically grown cells (46). A rapid decrease in hydrogen uptake activity in chemoheterotrophically grown cultures of Anabaena cycadeae has been reported (105). Moreover, an inhibition of the uptake hydrogenase activity by the addition of glucose, with a consequent increase in hydrogen production by nitrogenase, was observed in the Gunnera-Nostoc symbiosis (123). This set of conflicting data is not easily explained. As already pointed out (124), the omission of nickel from the growth medium in some of the experiments might have influenced their results. Moreover, symbiotic cyanobacteria within coralloid roots of the cycads Cycas revoluta and Zamia furfuracea showed a significant in vivo hydrogen uptake (124).

Addition of the protein biosynthesis inhibitor chloramphenicol to Nostoc strain PCC 73102 cultures (151) inhibited the stimulation observed by nickel and organic carbon, suggesting a regulation at the synthesis and transcriptional level and not regulation of the activity of preexisting protein(s). A similar phenomenon has been reported for the filamentous nonheterocystous cyanobacterium Oscillatoria subbrevis (182). Based on midpoint potentials and inhibitor studies, the probable candidate for the initial electron acceptor of the uptake hydrogenase is plastoquinone (87). In A. variabilis, in vitro the uptake hydrogenase is activated by a reduced thioredoxin, leading to the proposal that in vivo the enzyme may be subjected to a form of redox control that also involves a thioredoxin (152, 185).

hupSL and the deduced proteins. The first molecular data concerning cyanobacterial hydrogenases appeared in 1995. Carrasco et al. (39) described a novel developmental genome rearrangement for Anabaena strain PCC 7120, occurring in addition to the known nifD and fdxN rearrangements (see above), during the differentiation of a vegetative cell into a heterocyst. This third rearrangement occurs within a gene (hupL) encoding the large subunit of the uptake hydrogenase. The excision of a 10.5-kb DNA element occurs late during the heterocyst differentiation process, indicating that HupL in Anabaena strain PCC 7120 is expressed in heterocysts only. The excision occurs by a site-specific recombination, and the recombinase gene, xisC, was found 115 bp inside the right border of the excised element (Fig. 3A). Subsequently, the structural genes encoding both the small (hupS) and the large (hupL) subunits of the uptake hydrogenase have been sequenced and characterized in Anabaena strain PCC 7120 (www.kazusa.or.jp./cyano/anabaena), Nostoc strain PCC 73102 (150), and A. variabilis (79) (Fig. 3A).

View larger version (15K): [in this window] [in a new window]   FIG.3. hupSL in the cyanobacteria Nostoc strain PCC 73102 (N. PCC 73102) (150), A. variabilis ATCC 29413 (79), and Anabaena strain PCC 7120 (A. PCC 7120) (39). (A) Thin vertical lines correspond to the nucleotide triplets encoding the conserved cysteine residues compared to HupSL in other organisms. Arrows show the positions of transcriptional start in Nostoc strain PCC 73102 and A. variabilis ATCC 29413. Grey boxes (a, b, c, and d) indicate the positions of putative promoter elements; a, NtcA-binding site; b, IHF-binding site; c, -10 promoter sequence; d, part of an FNR-binding site. Intergenic-sequence hairpin structures are also indicated (for details, see panel B), as well as positions and directions of putative ORFs, labeled 1 to 3, downstream of hupL. Note the presence of xisC and the subsequent rearrangement within hupL in Anabaena strain PCC 7120. (B) hupSL intergenic sequence hairpin structures. The stop codon of hupS and the start codon of hupL are boxed, and calculated G values for each hairpin structure are shown below the respective structures (112).

Generally, the structural genes encoding NiFe uptake hydrogenases are clustered in a similar physical organization forming a transcript unit, with hupS being located upstream of hupL. In several microorganisms, a third ORF, hupC, has been identified and is located directly downstream of the hupSL genes (83, 208, 211). It has been proposed that HupC, a b-type cytochrome, could play a role in mediating the electron transport to the terminal acceptor oxygen (43, 211). The cyanobacterial hupSL genes follow the general pattern (Fig. 3A) and are transcribed as a single transcript in A. variabilis ATCC 29413 (79) and in Nostoc strain PCC 73102 (112). In both organisms, as well as in Anabaena strain PCC 7120, ORFs have been detected downstream of the respective hupL genes, but none of them show any similarity to hupC and they are different in the three strains examined (Fig. 3A).

Transcription of hupSL. The first transcriptional study concerning cyanobacterial hupSL genes was performed with Anabaena strain PCC 7120, where non-nitrogen-fixing filaments of vegetative cells were transferred to nitrogen-fixing conditions. Reverse transcription-PCR (RT-PCR) demonstrated that hupL transcription coincides with heterocyst formation (39). Subsequent studies of Nostoc muscorum and A. variabilis ATCC 29413 (cyanobacteria without rearrangement of hupL; see "Genetic diversity of cyanobacterial hydrogenases" below) using RT-PCR and Northern blot analysis, respectively, confirmed the induction of a hupL transcript under nitrogen-fixing conditions only (17, 79). In N. muscorum, the induction of the transcript was followed by the appearance of an in vivo hydrogen uptake activity (Fig. 4) (17). In contrast to these results, a low level of hupL expression (detected by RT-PCR) has been found in A. variabilis ATCC 29413 vegetative cells grown with the addition of ammonia (28). Interestingly, when exposed to anaerobic conditions A. variabilis synthesizes a second Mo nitrogenase cellularly localized in the vegetative cells (199). The concomitant induction of the already identified uptake hydrogenase (79) or a possible alternative uptake hydrogenase deserves further studies. It was previously shown that ammonia-grown cells of A. variabilis have low but consistent in vivo hydrogen uptake, an activity that was attributed to the bidirectional enzyme (203). Moreover, some uptake hydrogenase activity could be detected in vegetative cells of Anabaena spp. grown microaerobically or anaerobically (88, 157).

View larger version (27K): [in this window] [in a new window]   FIG. 4. Induction of an in vivo light-dependent hydrogen uptake in Nostoc muscorum cells during a shift from non-nitrogen-fixing to nitrogen-fixing conditions. Ammonia-grown cells were transferred to nitrogen-fixing conditions (time = 0) and analyzed with a Clark-type electrode for the appearance of an in vivo light-dependent hydrogen uptake (A) and by RT-PCR for the presence of a hupL transcript (B) after 14, 24, 41, and 61 h (17). M, marker (100-bp DNA ladder). Controls (right panel) include only a PCR (-RT; all RNA samples, only t = 0 h shown, negative control), replacement of RNA with water (H2O, negative control), and use of genomic DNA instead of RNA (DNA, positive control).

The cyanobacterial hupSL genes differ from those of other microorganisms in being separated by longer intergenic regions (79, 150). These regions consist largely of 7-bp repeated sequences belonging to different families of short tandemly repeated repetitive sequences commonly found in heterocystous cyanobacteria (112, 131). Even though the specific short tandemly repeated repetitive sequences are not conserved, sequence analyses revealed that the possibility of a hairpin formation is a common feature, indicating a conserved two-dimensional structure rather than a specific sequence of the repeat itself (Fig. 3B) (112). The specific function(s) and origin of the repeats are not known (93, 131). One possible function of hairpins could be to increase the stability of the transcript. Another suggestion is that the hairpin structure may confer a translational coupling between the two structural genes (112).

The physiological role of the bidirectional hydrogenase has been a matter of speculation and is still unclear. One possibility is that it functions as a mediator of the release of excess of reducing power in anaerobic environments. Kentemich et al. (97) and Schmitz et al. (173) reported that the low K_m for hydrogen indicates that the enzyme generally operates in the direction of hydrogen uptake. Since the proton gradient in cyanobacteria is directed outward and the bidirectional hydrogenase has high affinity for hydrogen, they suggested that there may be a function in the oxidation of hydrogen at the periplasmic side and that the enzyme may allocate electrons to the respiratory chain (97, 173). Furthermore, it has been proposed that the enzyme functions as a valve for low-potential electrons generated during the light reaction of the photosynthesis, thus preventing the slowing of the electron transport chain under stress conditions (11). An incomplete version of respiratory complex I (11 subunits out of 14 strictly conserved in other prokaryotes like E. coli) was identified in cyanobacteria, and the bidirectional hydrogenase was suggested to fulfill the functions of the missing subunits (11, 12, 30, 174). However, other data do not support this theory (27, 64, 91, 189), including the fact that the bidirectional enzyme is not a universal cyanobacterial enzyme (192, 194). Since the bidirectional hydrogenase is absent in a significant set of strains, it seems unlikely that it plays a common, central role in cyanobacterial respiratory complex I, unless different cyanobacteria have adopted different strategies. Some of the conserved sequence motifs of the bidirectional hydrogenase are similar in the two corresponding complex I subunits, but apart from this there are only low sequence similarities (64). These authors proposed that the cyanobacterial and chloroplastidial complex, which both have 11 subunits in common with respiratory complex I, might work as a NADPH:plastoquinone oxidoreductase, possibly involved in the cyclic photosynthetic electron transport, and that the sequence similarities observed between the NADH dehydrogenase part of complex I and the bidirectional hydrogenase are due to a common ancestor. It should be pointed out that, at present, the existence of a respiratory 14-subunit complex I in cyanobacteria cannot be ruled out. Nostoc strain PCC 73102, a strain lacking the bidirectional hydrogenase (27, 194), respires at rates comparable to those of other cyanobacteria (27), and bidirectional hydrogenase-minus mutants of Synechocystis strain PCC 6803 exhibited growth and respiration rates comparable to those of the wild type (91). It is obvious that more data are necessary to clarify the function of the bidirectional hydrogenase in cyanobacteria.

hox genes and the deduced proteins. In 1995, Schmitz et al. (173) sequenced a set of structural genes (hox genes) encoding a bidirectional hydrogenase in A. variabilis ATCC 29413. These authors suggested that the bidirectional enzyme is a heterotetrameric enzyme consisting of a hydrogenase part (encoded by hoxYH) and a diaphorase part (encoded by hoxFU) (Fig. 1 and 5). In subsequent works, hox genes have been sequenced and characterized in the unicellular Synechocystis strain PCC 6803, Synechococcus strain PCC 6301 (12, 28-30, 95, 144, 174), and (partially) C. thermalis CALU 758 (178), as well as in the filamentous strains Anabaena strain PCC 7120 (www.kazusa.or.jp./cyano/anabaena) and (partially) A. variabilis IAM M58 (GenBank accession no. AB057405). The physical organizations of the structural genes encoding the bidirectional enzyme are similar (Fig. 5). In A. variabilis, Anabaena strain PCC 7120, Synechococcus strain PCC 6301, and Synechocystis strain PCC 6803, one or several additional ORFs have been identified between some of the structural genes. In A. variabilis IAM M58, an additional ORF is localized between hoxF and hoxU (Fig. 5). The intergenic distances between hoxF and hoxU in both Synechococcus strain PCC 6301 and Anabaena strain PCC 7120 are longer (at least 16 and approximately 9 kb, respectively) than in other strains (Fig. 5). An ORF positioned upstream of hoxF and termed hoxE has been identified and sequenced in Synechococcus strain PCC 6301 and Synechocystis strain PCC 6803, and is also present in Anabaena strain PCC 7120 (http://www.kazusa.or.jp./cyano/anabaena). It has been suggested to encode a third diaphorase subunit (28). Moreover, in Synechococcus strain PCC 6301, an ORF termed hoxW is localized downstream of hoxH, putatively encoding a protease (Fig. 5) (30).

View larger version (29K): [in this window] [in a new window]   FIG. 5. Physical map of the genes encoding the bidirectional hydrogenase (hox) and additional ORFs (labeled 1 to 7, with identical numbers indicating homologous ORFs) in the cyanobacteria A. variabilis (two strains; A. ATCC 29413 [173] and A. IAM M58 [Gen. Bank accession no. AB057405]), Anabaena strain PCC 7120 (A. PCC 7120) (http://www.kazusa.or.jpn/cyano/anabaena), Synechococcus PCC 6301 (S. PCC 6301) (29, 30), and Synechocystis PCC 6803 (S. PCC 6803) (12, 95, 144). Vertical lines correspond to the positions of triplets encoding conserved cysteine residues compared to other microorganisms (for more details, see Table 4). and represent the NAD- and flavin mononucleotide-binding regions, respectively.

The cellular and subcellular localization of the bidirectional hydrogenase is a topic that needs further investigation. Earlier work indicated that the enzyme is present in both the vegetative cells and heterocysts and that through gentle cell disruption procedures it was easily solubilized and thus located in the cytoplasm (73, 88, 89). However, other works suggest an association of the bidirectional hydrogenase with cell membranes. The enzyme of the unicellular Synechococcus strain PCC 6301 seems to be loosely associated with the cytoplasmic membrane (96, 97), whereas associations with the thylakoid membranes have been demonstrated in A. variabilis and Synechocystis strain PCC 6803 (11, 177).

Nucleotide sequence comparisons have shown that there is a reasonably high degree of homology between the hox genes of cyanobacteria and genes encoding the NAD⁺-reducing hydrogenase from the chemolithotrophic hydrogen-metabolizing bacterium R. eutropha as well as those encoding methyl viologen-reducing hydrogenases from species of the archaeal genera Methanobacterium, Methanococcus, and Methanothermus (173). Sequence comparisons between the bidirectional hydrogenase genes or deduced proteins of A. variabilis ATCC 29413, A. variabilis IAM M58, Anabaena strain PCC 7120, Synechococcus strain PCC 6301, and Synechocystis strain PCC 6803 reveal that they are highly homologous, with A. variabilis ATCC 29413 and Anabaena strain PCC 7120 being at least 95% identical when the deduced amino acid sequences of proteins are compared (Table 3). The two A. variabilis strains are less similar (78 to 88% identical), and all other comparisons are in the range of 58 to 71% identical amino acids of the deduced proteins, with the exception of 80 to 88% identical amino acids when comparing Anabaena strain PCC 7120 and A. variabilis IAM M58 (Table 3).

View larger version (47K): [in this window] [in a new window]   FIG. 6. Visualization of a hoxH transcript from N. muscorum during a shift from non-nitrogen-fixing to nitrogen-fixing conditions (the same experiment as described in the legend to Fig. 4). Transcripts from t = 0, 14, 24, 41, and 61 h after the transfer to nitrogen-fixing conditions are shown (17). M, marker (100-bp DNA ladder).

View larger version (21K): [in this window] [in a new window]   FIG. 7. Physical organization and conserved regions (vertical lines; for further explanations, see Table 6) of hitherto identified cyanobacterial hyp genes. The histidine-rich N terminals of hypB are indicated (). Anabaena strain PCC 7120 (A. PCC 7120) (70, 71; http://www.kazusa.or.jpn/cyano/anabaena), Nostoc PCC 73102 (N. PCC 73102) (74; http://www.jgi.doe.gov/JGI_microbial/html/nostoc/nostoc_homepage/html); Synechococcus strain PCC 7002 (S. PCC 7002) (171), Synechococcus strain PCC 6301 (S. PCC 6301) (29), and Synechocystis strain PCC 6803 (S. PCC 6803) (95, 144).

CYANOBACTERIAL BIOHYDROGEN

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As discussed above, two cyanobacterial enzymes are capable of hydrogen production: the nitrogenase(s) and the bidirectional hydrogenase. Individual strains may harbor both a bidirectional hydrogenase (although this is not a universal cyanobacterial enzyme [Table 5] [192]) and none to several nitrogenases, encoded by different structural genes. There is no report describing the existence of either several uptake hydrogenases or several bidirectional hydrogenases in a single cyanobacterial strain. An efficient photoconversion of water to hydrogen by cyanobacteria is certainly influenced by many other factors, and only an extensive knowledge of this field can lead to improvement of the rates of cyanobacterial hydrogen production. For example, it is known that immobilized cells produce more hydrogen than do free-living cultures (125) and that non-Mo-containing nitrogenases allocate more electrons to the production of hydrogen (206). The gas phase (214), the age and density of the culture, and the composition, pH, and temperature of the growth medium are also crucial to the final result. Most of the research on cyanobacterial hydrogen production has been carried out with nitrogen-fixing strains (31, 55, 106, 111, 166, 205, 206). In these organisms, the net hydrogen production is the result of hydrogen evolution catalyzed by nitrogenase and of hydrogen consumption catalyzed mainly by an uptake hydrogenase. Consequently, the production and selection of mutants deficient in hydrogen uptake activity is of great interest (76, 79, 115, 136). Moreover, the nitrogenase has a high ATP requirement and this lowers the potential solar energy conversion efficiencies considerably. On the other hand, the bidirectional hydrogenase requires much less metabolic energy but is extremely sensitive to oxygen (14, 15). For reviews on the potential, problems and prospects of hydrogen production by cyanobacteria, see references (19, 20, 72, 75, 113, 117, 120, 125, and 164).

Several strategies are available for improving existing cyanobacterial strains for the biotechnological production of hydrogen. Inactivation of a gene encoding the uptake hydrogenase leads to a mutant that is not able to recycle the hydrogen evolved by the nitrogenase under nitrogen-fixing conditions. Consequently, hydrogen produced through the action of a nitrogenase will either be oxidized by the bidirectional hydrogenase or, if this enzyme is not present, be evolved from the cells. Recent experiments demonstrated that a nitrogen-fixing culture of an uptake-deficient mutant (a hup mutant generated by introducing a cassette of foreign DNA into hupL) of A. variabilis ATCC 29413 (a strain containing both an uptake hydrogenase and a bidirectional enzyme [Table 5]) produces molecular hydrogen (79). The absence of a bidirectional enzyme in cyanobacteria such as Nostoc strain PCC 73102 (Table 5) makes these strains interesting candidates for inactivation experiments. Identification and engineering of an oxygen-stable hydrogen-evolving hydrogenase might result in a photosynthesizing microorganism that evolves significant amounts of hydrogen. Interestingly, replacing Azotobacter vinelandii hydrogenase small-subunit cysteines with serines can create insensitivity to inhibition by oxygen and preferentially damage hydrogen oxidation over hydrogen evolution (132). Moreover, overproducing mutants might be obtained by providing genes encoding a selected hydrogenase on an expression vector. Coupling the genes to a strong promoter might lead to an increased amount of the hydrogenase and thus to increased levels of hydrogen produced by the organism. Similarly, overexpression might also be used to increase the nitrogenase activity. A thorough examination of the genes involved in the regulation of hydrogenase expression, e.g., hyp genes, might generate knowledge leading to further strategies for improving hydrogen production rates in cyanobacteria.

Genetic engineering has become increasingly important with the establishment of molecular biological tools and techniques for cyanobacteria. A few unicellular strains, including Synechococcus strains PCC 6301 and PCC 7942 and Synechocystis strain PCC 6803, are naturally transformable. Protocols and vector systems useful for the transfer of DNA into different cyanobacteria are available for nontransformable strains (196). These methods have been used with success in filamentous genera such as Anabaena and Nostoc, which are interesting candidates for future photobiotechnological applications (75, 76, 79). The introduction of the structural genes encoding a clostridial Fe hydrogenase into the unicellular cyanobacterium Synechococcus elongatus PCC 7942 and their subsequent transcription and translation into a functional hydrogen-evolving enzyme in a photosynthetic microorganism (16, 138) is interesting and deserves further studies.