Discovery of [NiFe] Hydrogenase Genes in Metagenomic DNA: Cloning and Heterologous Expression in Thiocapsa roseopersicina

Using a metagenomics approach, we have cloned a piece of environmental DNA from the Sargasso Sea that encodes an [NiFe] hydrogenase showing 60% identity to the large subunit and 64% to the small subunit of a Thiocapsa roseopersicina O₂-tolerant [NiFe] hydrogenase. The DNA sequence of the hydrogenase identified by the metagenomic approach was subsequently found to be 99% identical to the hyaA and hyaB genes of an Alteromonas macleodii hydrogenase, indicating that it belongs to the Alteromonas clade. We were able to express our new Alteromonas hydrogenase in T. roseopersicina. Expression was accomplished by coexpressing only two accessory genes, hyaD and hupH, without the need to express any of the hyp accessory genes (hypABCDEF). These results suggest that the native accessory proteins in T. roseopersicina could substitute for the Alteromonas counterparts that are absent in the host to facilitate the assembly of a functional Alteromonas hydrogenase. To further compare the complex assembly machineries of these two [NiFe] hydrogenases, we performed complementation experiments by introducing the new Alteromonas hyaD gene into the T. roseopersicina hynD mutant. Interestingly, Alteromonas endopeptidase HyaD could complement T. roseopersicina HynD to cleave endoproteolytically the C-terminal end of the T. roseopersicina HynL hydrogenase large subunit and activate the enzyme. This study refines our knowledge on the selectivity and pleiotropy of the elements of the [NiFe] hydrogenase assembly machineries. It also provides a model for functionally analyzing novel enzymes from environmental microbes in a culture-independent manner.Hydrogen is a promising energy carrier for the future (10). Photosynthetic microbes such as cyanobacteria have attracted considerable attention, because they can split water photolytically to produce H₂. However, one major drawback of the processes is that their H₂-evolving hydrogenases are extremely sensitive to O₂, which is an inherent by-product of oxygenic photosynthesis. Thus, transfer of O₂-tolerant [NiFe] hydrogenases into cyanobacteria might be one approach to overcome this O₂ sensitivity issue. A small number of O₂-tolerant hydrogenases has been identified (9, 21, 47). However, they tend to favor H₂ uptake over evolution. Searching for novel O₂-tolerant [NiFe] hydrogenases from environmental microbes therefore becomes an important part of the effort to construct such biophotolytic systems.The oceans harbor an abundance of microorganisms with H₂ production capability. Traditionally, new hydrogenases have been screened only from culturable organisms. However, since only a few microbes can be cultured (14), many of them have not been identified, and their functions remain unknown. Metagenomics is a rapidly growing field, which allows us to obtain information about uncultured microbes and to understand the true diversity of microbes in their natural environments. Metagenomics analysis provides a completely new approach for identifying novel [NiFe] hydrogenases from the oceans in a culture-independent manner. The Global Ocean Sampling (GOS) expedition has produced the largest metagenomic data set to date, providing a rich catalog of proteins and protein families, including those enzymes involved in hydrogen metabolism (45, 52, 56-58). Putative novel [NiFe] hydrogenase enzymes that were identified from marine microbial metagenomic data in these expeditions can be examined to find potentially important new hydrogenases. Because source organisms for metagenomic sequences are not typically known, these hydrogenases have to be heterologously expressed in culturable foreign hosts for protein and functional analyses.Unlike most proteins, hydrogenases have a complex architecture and must be assembled and matured through a multiple-step process (7, 11). Hydrogenases are divided into three distinct groups based on their metal contents (54): Fe-S cluster-free hydrogenases (22, 23, 48), [FeFe] hydrogenases (1, 12, 25), and [NiFe] hydrogenases (2, 3, 55). [NiFe] hydrogenases are heterodimers composed of a large subunit and a small subunit, and their NiFe catalytic centers are located in the large subunits (2, 15, 19, 40). A whole set of accessory proteins are required to properly assemble the catalytic centers (7). The accessory protein HypE first interacts with HypF to form a HypF-HypE complex, and the carbamyl group linked to HypF is then dehydrated by HypE in the presence of ATP to release the CN group that is transferred to iron through a HypC-HypD-HypE complex (6). The origin of the CO ligand that is also bound to the iron is not clear, and possibly it comes from formate, formyl-tetrahydrofolate, or acetate. The liganded Fe atom is inserted into the immature large subunit, in which HypC proteins function as chaperones to facilitate the metal insertion (5, 34, 36). Ni is delivered to the catalytic center by the zinc-metalloenzyme HypA that interacts with HypB, a nickel-binding and GTP-hydrolyzing protein. The final step in the maturation process is endoproteolytic cleavage. Once the nickel is transferred to the active site, the endopeptidase, such as HyaD or HynD, cleaves the C-terminal end of the large subunit (33, 43), which triggers a conformational change of the protein so that the Ni-Fe catalytic center can be internalized.Heterologous expression of functional [NiFe] hydrogenases has been demonstrated in several studies (4, 18, 31, 39, 44, 50), suggesting that it could be a feasible approach to express novel hydrogenases from the environment for functional analysis. In this study, we sought to prove the concept that metagenomically derived environmental DNA can give rise to a functional [NiFe] hydrogenase through expression in a foreign host and that novel [NiFe] hydrogenases from environmental microbes can be studied in a culture-independent manner. We cloned environmental DNA that harbors the genes of a putative novel hydrogenase that shows strong homology to a known O₂-tolerant hydrogenase, HynSL, from the phototrophic purple sulfur bacterium Thiocapsa roseopersicina (21, 28, 41, 59). We heterologously expressed the two structural genes (hyaA and hyaB) and two accessory genes (hupH and hyaD) of this novel environmental hydrogenase in T. roseopersicina, a foreign host that may already have the necessary machinery required to process the environmental hydrogenase since it carries the homologous hydrogenase HynSL. We analyzed the new hydrogenase protein and its functions. In addition, we compared the maturation mechanisms between the two homolog hydrogenases by performing complementation experiments.     

Hydrogenase activity in Thiocapsa roseopersicina according to the D2--H20 metabolic reaction]

Extracts of Thiocapsa roseopersicina cells show hydrogenase activity, measured by evolution of H2 from reduced methylviologene (MV) and by D2-H2O exchange reaction. According to these reactions the most part of hydrogenases is found to be in the soluble fraction. Hydrogenase activity measured in the exchange reaction is completely inhibited by p-chloromercurybenzoate (5-10- minus 3 M), iodacetate (1-10- minus 2 M) and 26% inhibited by KCN and o-phenanthroline (5-10- minus 3 M). Evolution of H2 from reduced MV was not inhibited by o-phenanthroline, KCN and iodacetate and was inhibited by 66% only with p-chloromercurybenzoate. Light and ATP stimulated hydrogenase activity of chromatophores did not affect on its activity in the soluble fraction. The results obtained show that there are certain differences in hydrogenase systems responsible for the exchange reaction and evolution of H2.     

Unusual Organization of the Genes Coding for HydSL, the Stable [NiFe]Hydrogenase in the Photosynthetic Bacterium Thiocapsa roseopersicina BBS

The characterization of a hyd gene cluster encoding the stable, bidirectional [NiFe]hydrogenase 1 enzyme in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium belonging to the family Chromatiaceae, is presented. The heterodimeric hydrogenase 1 had been purified to homogeneity and thoroughly characterized (K. L. Kovacs et al., J. Biol. Chem. 266:947–951, 1991; C. Bagyinka et al., J. Am. Chem. Soc. 115:3567–3585, 1993). As an unusual feature, a 1,979-bp intergenic sequence (IS) separates the structural genes hydS and hydL, which encode the small and the large subunits, respectively. This IS harbors two sequential open reading frames (ORFs) which may code for electron transfer proteins ISP1 and ISP2. ISP1 and ISP2 are homologous to ORF5 and ORF6 in the hmc operon, coding for a transmembrane electron transfer complex in Desulfovibrio vulgaris. Other accessory proteins are not found immediately downstream or upstream of hydSL. A hup gene cluster coding for a typical hydrogen uptake [NiFe]hydrogenase in T. roseopersicina was reported earlier (A. Colbeau et al. Gene 140:25–31, 1994). The deduced amino acid sequences of the two small (hupS and hydS) and large subunit (hupL and hydL) sequences share 46 and 58% identity, respectively. The hup and hyd genes differ in the arrangement of accessory genes, and the genes encoding the two enzymes are located at least 15 kb apart on the chromosome. Both hydrogenases are associated with the photosynthetic membrane. A stable and an unstable hydrogenase activity can be detected in cells grown under nitrogen-fixing conditions; the latter activity is missing in cells supplied with ammonia as the nitrogen source. The apparently constitutive and stable activity corresponds to hydrogenase 1, coded by hydSL, and the inducible and unstable second hydrogenase may be the product of the hup gene cluster.     

Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Synechococcus elongatus

Oxygen-tolerant [NiFe] hydrogenases may be used in future photobiological hydrogen production systems once the enzymes can be heterologously expressed in host organisms of interest. To achieve heterologous expression of [NiFe] hydrogenases in cyanobacteria, the two hydrogenase structural genes from Alteromonas macleodii Deep ecotype (AltDE), hynS and hynL, along with the surrounding genes in the gene operon of HynSL were cloned in a vector with an IPTG-inducible promoter and introduced into Synechococcus elongatus PCC7942. The hydrogenase protein was expressed at the correct size upon induction with IPTG. The heterologously-expressed HynSL hydrogenase was active when tested by in vitro H(2) evolution assay, indicating the correct assembly of the catalytic center in the cyanobacterial host. Using a similar expression system, the hydrogenase structural genes from Thiocapsa roseopersicina (hynSL) and the entire set of known accessory genes were transferred to S. elongatus. A protein of the correct size was expressed but had no activity. However, when the 11 accessory genes from AltDE were co-expressed with hynSL, the T. roseopersicina hydrogenase was found to be active by in vitro assay. This is the first report of active, heterologously-expressed [NiFe] hydrogenases in cyanobacteria.     

Accessory proteins functioning selectively and pleiotropically in the biosynthesis of [NiFe] hydrogenases in Thiocapsa roseopersicina.

There are at least two membrane-bound (HynSL and HupSL) and one soluble (HoxEFUYH) [NiFe] hydrogenases in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium. Genes coding for accessory proteins that participate in the biosynthesis and maturation of hydrogenases seem to be scattered along the chromosome. Transposon-based mutagenesis was used to locate the hydrogenase accessory genes. Molecular analysis of strains showing mutant phenotypes led to the identification of hupK (hoxV ), hypC1, hypC2, hypD, hypE, and hynD genes. The roles of hynD, hupK and the two hypC genes were investigated in detail. The putative HynD was found to be a hydrogenase-specific endoprotease type protein, participating in the maturation of the HynSL enzyme. HupK plays an important role in the formation of the functionally active membrane-bound [NiFe] hydrogenases, but not in the biosynthesis of the soluble enzyme. In-frame deletion mutagenesis showed that HypC proteins were not specific for the maturation of either hydrogenase enzyme. The lack of either HypC protein drastically reduced the activity of every hydrogenase. Hence both HypCs might participate in the maturation of [NiFe] hydrogenases. Homologous complementation with the appropriate genes substantiated the physiological roles of the corresponding gene products in the H2 metabolism of T. roseopersicina.     

Interaction of HydSL hydrogenase from the purple sulfur bacterium Thiocapsa roseopersicina BBS with methyl viologen and positively charged polypeptides

The effect of polypeptides having different charge on the activity of Thiocapsa roseopersicina HydSL hydrogenase was studied. Strong inhibition was shown for poly-L-lysine bearing positive charge. The inhibition was reversible and competitive to methyl viologen, an electron acceptor, in the reaction of hydrogen oxidation catalyzed by the hydrogenase. Peptides carrying less positive charge had weaker inhibiting effect, while neutral and negatively charged peptides did not inhibit the hydrogenase. Molecular docking of poly-L-lysine to T. roseopersicina hydrogenase showed strong affinity of this polypeptide to the acceptor-binding site of the enzyme. The calculated binding constant is close to the experimentally measured value (K _i = 2.1 μM).     

Localization of hydrogenase in Thiocapsa roseopersicina photosynthetic membrane

The photosynthetic cell membrane is impermeable to the oxidized redox dyes Methyl Viologen and Benzyl Viologen, whereas the reduced forms easily penetrate into the cells. By exploiting this permeability difference, the orientation of the membrane-bound hydrogenase has been determined.     

Localization of hydrogenase in the purple sulphur bacterium Thiocapsa roseopersicina

The localization of hydrogenase in the phototrophic bacterium Thiocapsa roseopersicina was investigated by subcellular fractionations, and transmission electron microscopic immunocytochemistry. By using sonicated cells and measuring in vitro hydrogenase activities in soluble and membrane fractions, respectively, a weak hydrophobic interaction between the hydrogenase enzyme and the T. roseopersicina membranes was observed. Polyclonal antisera directed against the purified hydrogenase were raised in rabbits and exhibited one band in native-PAGE/Western immunoblot analysis. Native-PAGE/activity stain confirmed the identity of this band as being hydrogenase. Immunocytolocalization experiments using ultrathin sections showed an internal localization of the hydrogenase enzyme. A higher specific labeling was associated with chromatophores, indicating a possible coupling of hydrogenase with the photosynthetic membranes in the T. roseopersicina cells.     

Three-dimensional structure of the nickel-containing hydrogenase from Thiocapsa roseopersicina.

The three-dimensional structure of the nickel-containing hydrogenase from Thiocapsa roseopersicina has been determined at a resolution of 2 nm in the plane and 4 nm in the vertical direction by electron microscopy and computerized image processing on microcrystals of the enzyme. The enzyme forms a large ring-shaped complex containing six each of the large (62-kDa) and small (26-kDa) subunits. The complex is very open, with six well-separated dumbbell-shaped masses surrounding a large cylindrical hole. Each dumbbell is interpreted as consisting of one large and one small subunit.     

Induced mutagenesis by bleomycin in the purple sulfur bacterium Thiocapsa roseopersicina

Cell death and mutagenesis in bleomycin-treated cells of Thiocapsa roseopersicina (a purple sulfur bacterium) was studied by cultivation in a semisolid medium (agar-shake technique). This technique has also proven useful in assessing the frequency of antibiotic mutations by detecting and counting individual colonies of Thiocapsa roseopersicina. The frequencies of spontaneous mutants resistant to ampicillin, rifampicin, cloramphenicol, tetracycline, kanamycin, streptomycin, and neomycin were also studied: they ranged between 2×10^-9 and 9×10^-8. Bleomycin (4 g/ml) sharply increased the frequency of ampicillin-resistant mutants, from 10^-8 (spontaneous) to 4×10^-4 (induced), in 17 h. An inducible, error-prone mechanisms of DNA synthesis seems to be responsible for this enhancement of the mutagenic effect. This is the first report on the sensitivity to several antibiotics, and capacity of lethality and mutagenesis by bleomycin has been studied in a purple sulfur bacterium.     

Growth and oxidation of sulfur compounds by Thiocapsa roseopersicina in darkness]

The purple sulphur bacterium Thiocapsa roseopersicina, strain BBS, grown in the darkness in aerobic autotrophic conditions, oxidized sulphides to free sulphur and then to sulphates. This was accompanied with the fixation of carbon dioxide by the cells. Addition of glucose to the mineral medium increased the biomass yield; the cells oxidized thiosulphate still at a high rate. These results prove the possibility of switching T. roseopersicina from photosynthesis to a dark chemolithautotrophic way of life.     

The role of Hox hydrogenase in the H2 metabolism of Thiocapsa roseopersicina

The purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS synthesizes at least three NiFe hydrogenases (Hox, Hup, Hyn). We characterized the physiological H(2) consumption/evolution reactions in mutants having deletions of the structural genes of two hydrogenases in various combinations. This made possible the separation of the functionally distinct roles of the three hydrogenases. Data showed that Hox hydrogenase (unlike the Hup and Hyn hydrogenases) catalyzed the dark fermentative H(2) evolution and the light-dependent H(2) production in the presence of thiosulfate. Both Hox(+) and Hup(+) mutants demonstrated light-dependent H(2) uptake stimulated by CO(2) but only the Hup(+) mutant was able to mediate O(2)-dependent H(2) consumption in the dark. The ability of the Hox(+) mutant to evolve or consume hydrogen was found to depend on a number of interplaying factors including both growth and reaction conditions (availability of glucose, sulfur compounds, CO(2), H(2), light). The study of the redox properties of Hox hydrogenase supported the reversibility of its action. Based on the results a scheme is suggested to describe the role of Hox hydrogenase in light-dependent and dark hydrogen metabolism in T. roseopersicina BBS.     

Growth of Thiocapsa roseopersicina purple sulfur bacteria in darkness under anaerobic conditions]

The phototrophic sulphur bacterium. Thiocapsa roseopersicina, strain BBS, was grown under anaerobic conditions in the darkness on the medium containing glucose and thiosulphate or molecular sulphur. The assimilation of glucose is accompanied by the accumulation of small amounts of pyruvate in the medium, and the uptake of thiosulphate or molecular sulphur leads to the formation of sulphates and hydrogen sulphide.