Acidobacteria are active and abundant members of diverse atmospheric H2-oxidizing communities detected in temperate soils

Significant rates of atmospheric dihydrogen (H₂) consumption have been observed in temperate soils due to the activity of high-affinity enzymes, such as the group 1h [NiFe]-hydrogenase. We designed broadly inclusive primers targeting the large subunit gene (hhyL) of group 1h [NiFe]-hydrogenases for long-read sequencing to explore its taxonomic distribution across soils. This approach revealed a diverse collection of microorganisms harboring hhyL, including previously unknown groups and taxonomically not assignable sequences. Acidobacterial group 1h [NiFe]-hydrogenase genes were abundant and expressed in temperate soils. To support the participation of acidobacteria in H₂ consumption, we studied two representative mesophilic soil acidobacteria, which expressed group 1h [NiFe]-hydrogenases and consumed atmospheric H₂ during carbon starvation. This is the first time mesophilic acidobacteria, which are abundant in ubiquitous temperate soils, have been shown to oxidize H₂ down to below atmospheric concentrations. As this physiology allows bacteria to survive periods of carbon starvation, it could explain the success of soil acidobacteria. With our long-read sequencing approach of group 1h [NiFe]-hydrogenase genes, we show that the ability to oxidize atmospheric levels of H₂ is more widely distributed among soil bacteria than previously recognized and could represent a common mechanism enabling bacteria to persist during periods of carbon deprivation.Subject terms: Soil microbiology, Biodiversity, Bacterial physiology     

A widely distributed hydrogenase oxidises atmospheric H2 during bacterial growth

Diverse aerobic bacteria persist by consuming atmospheric hydrogen (H₂) using group 1h [NiFe]-hydrogenases. However, other hydrogenase classes are also distributed in aerobes, including the group 2a [NiFe]-hydrogenase. Based on studies focused on Cyanobacteria, the reported physiological role of the group 2a [NiFe]-hydrogenase is to recycle H₂ produced by nitrogenase. However, given this hydrogenase is also present in various heterotrophs and lithoautotrophs lacking nitrogenases, it may play a wider role in bacterial metabolism. Here we investigated the role of this enzyme in three species from different phylogenetic lineages and ecological niches: Acidithiobacillus ferrooxidans (phylum Proteobacteria), Chloroflexus aggregans (phylum Chloroflexota), and Gemmatimonas aurantiaca (phylum Gemmatimonadota). qRT-PCR analysis revealed that the group 2a [NiFe]-hydrogenase of all three species is significantly upregulated during exponential growth compared to stationary phase, in contrast to the profile of the persistence-linked group 1h [NiFe]-hydrogenase. Whole-cell biochemical assays confirmed that all three strains aerobically respire H₂ to sub-atmospheric levels, and oxidation rates were much higher during growth. Moreover, the oxidation of H₂ supported mixotrophic growth of the carbon-fixing strains C. aggregans and A. ferrooxidans. Finally, we used phylogenomic analyses to show that this hydrogenase is widely distributed and is encoded by 13 bacterial phyla. These findings challenge the current persistence-centric model of the physiological role of atmospheric H₂ oxidation and extend this process to two more phyla, Proteobacteria and Gemmatimonadota. In turn, these findings have broader relevance for understanding how bacteria conserve energy in different environments and control the biogeochemical cycling of atmospheric trace gases.Subject terms: Environmental microbiology, Biogeochemistry     

Impact of alterations near the [NiFe] active site on the function of the H(2) sensor from Ralstonia eutropha

In proteobacteria capable of H(2) oxidation under (micro)aerobic conditions, hydrogenase gene expression is often controlled in response to the availability of H(2). The H(2)-sensing signal transduction pathway consists of a heterodimeric regulatory [NiFe]-hydrogenase (RH), a histidine protein kinase and a response regulator. To gain insights into the signal transmission from the Ni-Fe active site in the RH to the histidine protein kinase, conserved amino acid residues in the L0 motif near the active site of the RH large subunit of Ralstonia eutropha H16 were exchanged. Replacement of the strictly conserved Glu13 (E13N, E13L) resulted in loss of the regulatory, H(2)-oxidizing and D(2)/H(+) exchange activities of the RH. According to EPR and FTIR analysis, these RH derivatives contained fully assembled [NiFe] active sites, and para-/ortho-H(2) conversion activity showed that these centres were still able to bind H(2). This indicates that H(2) binding at the active site is not sufficient for the regulatory function of H(2) sensors. Replacement of His15, a residue unique in RHs, by Asp restored the consensus of energy-linked [NiFe]-hydrogenases. The respective RH mutant protein showed only traces of H(2)-oxidizing activity, whereas its D(2)/H(+)-exchange activity and H(2)-sensing function were almost unaffected. H(2)-dependent signal transduction in this mutant was less sensitive to oxygen than in the wild-type strain. These results suggest that H(2) turnover is not crucial for H(2) sensing. It may even be detrimental for the function of the H(2) sensor under high O(2) concentrations.     

Production of biohydrogen by heterologous expression of oxygen-tolerant Hydrogenovibrio marinus [NiFe]-hydrogenase in Escherichia coli

Oxygen sensitivity of hydrogenase is a critical issue in efficient biological hydrogen production. In the present study, oxygen-tolerant [NiFe]-hydrogenase from the marine bacterium, Hydrogenovibrio marinus, was heterologously expressed in Escherichia coli, for the first time. Recombinant E. coli BL21 expressing H. marinus [NiFe]-hydrogenase actively produced hydrogen, but the parent strain did not. Recombinant H. marinus hydrogenase required both nickel and iron for biological activity. Compared to the recombinant E. coli [NiFe]-hydrogenase 1 described in our previous report, recombinant H. marinus [NiFe]-hydrogenase displayed 1.6- to 1.7-fold higher hydrogen production activity in vitro. Importantly, H. marinus [NiFe]-hydrogenase exhibited relatively good oxygen tolerance in analyses involving changes of surface aeration and oxygen proportion within a gas mixture. Specifically, recombinant H. marinus [NiFe]-hydrogenase produced ∼7- to 9-fold more hydrogen than did E. coli [NiFe]-hydrogenase 1 in a gaseous environment containing 5-10% (v/v) oxygen. In addition, purified H. marinus [NiFe]-hydrogenase displayed a hydrogen evolution activity of ∼28.8 nmol H₂/(min mg protein) under normal aerobic purification conditions. Based on these results, we suggest that oxygen-tolerant H. marinus [NiFe]-hydrogenase can be employed for in vivo and in vitro biohydrogen production without requirement for strictly anaerobic facilities.     

Functional analysis by site-directed mutagenesis of the NAD(+)-reducing hydrogenase from Ralstonia eutropha

The tetrameric cytoplasmic [NiFe] hydrogenase (SH) of Ralstonia eutropha couples the oxidation of hydrogen to the reduction of NAD(+) under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site-directed mutagenesis in order to dissect the molecular mechanism of hydrogen activation. Based on phenotypic characterizations, 27 mutants were grouped into four different classes. Mutants of the major class, class I, failed to grow on hydrogen and were devoid of H(2)-oxidizing activity. In one of these isolates (HoxH I64A), H(2) binding was impaired. Class II mutants revealed a high D(2)/H(+) exchange rate relative to a low H(2)-oxidizing activity. A representative (HoxH H16L) displayed D(2)/H(+) exchange but had lost electron acceptor-reducing activity. Both activities were equally affected in class III mutants. Mutants forming class IV showed a particularly interesting phenotype. They displayed O(2)-sensitive growth on hydrogen due to an O(2)-sensitive SH protein.     

Expression of a functional NAD-reducing [NiFe] hydrogenase from the gram-positive Rhodococcus opacus in the gram-negative Ralstonia eutropha

The actinomycete Rhodococcus opacus MR11 harbors a bidirectional NAD-reducing [NiFe] hydrogenase (SH). This cytoplasmic enzyme is composed of two heterodimeric modules which catalyze distinct enzymatic activities. The hydrogenase moiety mediates H(2):benzyl viologen oxidoreductase activity and the FMN-containing diaphorase module displays NADH:benzyl viologen oxidoreductase activity. The SH of Rh. opacus resembles [NiFe] hydrogenases present in strains of the proteobacterium Ralstonia eutropha and in species of cyanobacteria. Heterologous expression of active [NiFe] hydrogenases failed in most cases due to protein-assisted maturation processes implicated in the assembly of the NiFe bimetallic site. This study reports on the construction of a recombinant plasmid harboring the four SH subunit genes hoxFUYH and the associated endopeptidase gene hoxW from Rh. opacus under the regime of the SH promoter from R. eutropha H16. The resulting recombinant plasmid restored lithoautotrophic growth in a R. eutropha mutant impaired in H(2)-oxidizing ability. The SH of Rh. opacus was functionally active in R. eutropha and displayed the typical features described for its natural host. It readily dissociated in vitro into two active subforms. Dissociation was accompanied by the loss of the H(2)-dependent NAD-reducing activity, which was partially reconstituted by addition of 5 mM MgSO(4) and 0.5 mM NiCl(2). Activity and stability of the SH from Rh. opacus were enhanced almost three-fold by co-overexpression of the SH-associated metal insertion genes hypA2B2F2 of R. eutropha. Under optimal conditions the heterologously expressed Rh. opacus SH catalyzed NAD-reduction at a specific activity of 1.7 units per mg protein, which is approximately 30% of the yield obtained for the R. eutropha SH. The results indicate that, despite an enormous phylogenetic distance of the two bacterial species, their SH proteins are highly related.     

Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Hydrogenases catalyze reversible reaction between hydrogen (H₂) and proton. Inactivation of hydrogenase by exposure to oxygen is a critical limitation in biohydrogen production since strict anaerobic conditions are required. While [FeFe]-hydrogenases are irreversibly inactivated by oxygen, it was known that [NiFe]-hydrogenases are generally more tolerant to oxygen. The physiological function of [NiFe]-hydrogenase 1 is still ambiguous. We herein investigated the H₂ production potential of [NiFe]-hydrogenase 1 of Escherichia coli in vivo and in vitro. The hya A and hya B genes corresponding to the small and large subunits of [NiFe]-hydrogenase 1 core enzyme, respectively, were expressed in BL21, an E. coli strain without H₂ producing ability.     

A trimeric supercomplex of the oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Ralstonia eutropha H16

The oxygen-tolerant membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha H16 consists of three subunits. The large subunit HoxG carries the [NiFe] active site, and the small subunit HoxK contains three [FeS] clusters. Both subunits form the so-called hydrogenase module, which is oriented toward the periplasm. Membrane association is established by a membrane-integral cytochrome b subunit (HoxZ) that transfers the electrons from the hydrogenase module to the respiratory chain. So far, it was not possible to isolate the MBH in its native heterotrimeric state due to the loss of HoxZ during the process of protein solubilization. By using the very mild detergent digitonin, we were successful in isolating the MBH hydrogenase module in complex with the cytochrome b. H(2)-dependent reduction of the two HoxZ-stemming heme centers demonstrated that the hydrogenase module is productively connected to the cytochrome b. Further investigation provided evidence that the MBH exists in the membrane as a high molecular mass complex consisting of three heterotrimeric units. The lipids phosphatidylethanolamine and phosphatidylglycerol were identified to play a role in the interaction of the hydrogenase module with the cytochrome b subunit.     

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.     

产甲烷古菌铁氢酶研究进展

根据活性中心金属原子的不同,氢酶主要分为镍铁、铁铁、铁氢酶三大类。铁氢酶是发现较晚、存在物种单一且结构较为特殊的一类氢酶。目前,铁氢酶仅发现于氢营养型产甲烷古菌中。该酶直接催化氢气异裂,还原产甲烷代谢途径中一碳载体四氢蝶呤的次甲基转化为亚甲基。与其他两类氢酶相比,铁氢酶不含传递电子的铁硫簇和双金属活性中心,在结构组成上有较大的差异。此外,铁氢酶活性中心的吡啶环被高度取代,活性中心铁原子直接与酰基碳成键,这些奇特的活性分子结构预示着氢酶全新的催化机制,以及古菌细胞在合成特殊结构大分子方面的特殊功能。本文总结了从1990年发现这类新型氢酶以来的相关研究,分别从氢酶的生理功能、结构特征、催化机制、成熟过程及应用研究等方面阐述铁氢酶的研究进展。     

The three classes of hydrogenases from sulfate-reducing bacteria of the genus Desulfovibrio

Three types of hydrogenases have been isolated from the sulfate-reducing bacteria of the genus Desulfovibrio. They differ in their subunit and metal compositions, physico-chemical characteristics, amino acid sequences, immunological reactivities, gene structures and their catalytic properties. Broadly, the hydrogenases can be considered as 'iron only' hydrogenases and nickel-containing hydrogenases. The iron-sulfur-containing hydrogenase ([Fe] hydrogenase) contains two ferredoxin-type (4Fe-4S) clusters and an atypical iron-sulfur center believed to be involved in the activation of H2. The [Fe] hydrogenase has the highest specific activity in the evolution and consumption of hydrogen and in the proton-deuterium exchange reaction and this enzyme is the most sensitive to CO and NO2-. It is not present in all species of Desulfovibrio. The nickel-(iron-sulfur)-containing hydrogenases [( NiFe] hydrogenases) possess two (4Fe-4S) centers and one (3Fe-xS) cluster in addition to nickel and have been found in all species of Desulfovibrio so far investigated. The redox active nickel is ligated by at least two cysteinyl thiolate residues and the [NiFe] hydrogenases are particularly resistant to inhibitors such as CO and NO2-. The genes encoding the large and small subunits of a periplasmic and a membrane-bound species of the [NiFe] hydrogenase have been cloned in Escherichia (E.) coli and sequenced. Their derived amino acid sequences exhibit a high degree of homology (70%); however, they show no obvious metal-binding sites or homology with the derived amino acid sequence of the [Fe] hydrogenase. The third class is represented by the nickel-(iron-sulfur)-selenium-containing hydrogenases [( NiFe-Se] hydrogenases) which contain nickel and selenium in equimolecular amounts plus (4Fe-4S) centers and are only found in some species of Desulfovibrio. The genes encoding the large and small subunits of the periplasmic hydrogenase from Desulfovibrio (D.) baculatus (DSM 1743) have been cloned in E. coli and sequenced. The derived amino acid sequence exhibits homology (40%) with the sequence of the [NiFe] hydrogenase and the carboxy-terminus of the gene for the large subunit contains a codon (TGA) for selenocysteine in a position homologous to a codon (TGC) for cysteine in the large subunit of the [NiFe] hydrogenase. EXAFS and EPR studies with the 77Se-enriched D. baculatus hydrogenase indicate that selenium is a ligand to nickel and suggest that the redox active nickel is ligated by at least two cysteinyl thiolate and one selenocysteine selenolate residues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of the [NiFe]-hydrogenase of Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H(2)ase) that has been implicated in H(2) production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H(2)ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H(2)ase were cloned and then expressed in an MR-1 mutant without hyaB and hydA genes. Expression of recombinant MR-1 [NiFe]-H(2)ase in trans restored the mutant's ability to produce H(2) at 37% of that for the wild type. Following purification, MR-1 [NiFe]-H(2)ase coupled H(2) oxidation to reduction of Tc(VII)O(4)(-) and methyl viologen. Change of the buffers used affected MR-1 [NiFe]-H(2)ase-mediated reduction of Tc(VII)O(4)(-) but not methyl viologen. Under the conditions tested, all Tc(VII)O(4)(-) used was reduced in Tris buffer, while in HEPES buffer, only 20% of Tc(VII)O(4)(-) was reduced. The reduced products were soluble in Tris buffer but insoluble in HEPES buffer. Transmission electron microscopy analysis revealed that Tc precipitates reduced in HEPES buffer were aggregates of crystallites with diameters of ～5 nm. Measurements with X-ray absorption near-edge spectroscopy revealed that the reduction products were a mixture of Tc(IV) and Tc(V) in Tris buffer but only Tc(IV) in HEPES buffer. Measurements with extended X-ray adsorption fine structure showed that while the Tc bonding environment in Tris buffer could not be determined, the Tc(IV) product in HEPES buffer was very similar to Tc(IV)O(2)·nH(2)O, which was also the product of Tc(VII)O(4)(-) reduction by MR-1 cells. These results shows for the first time that MR-1 [NiFe]-H(2)ase catalyzes Tc(VII)O(4)(-) reduction directly by coupling to H(2) oxidation.     

Novel,Oxygen-Insensitive Group 5 [NiFe]-Hydrogenase in Ralstonia eutropha

Recently, a novel group of [NiFe]-hydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. This so-called group 5 [NiFe]-hydrogenase is widespread in soil-living actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward H₂. Here, we provide a biochemical characterization of a group 5 hydrogenase from the betaproteobacterium Ralstonia eutropha H16. The hydrogenase was designated an actinobacterial hydrogenase (AH) and is catalytically active, as shown by the in vivo H₂ uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H₂. The AH was purified to homogeneity by affinity chromatography and consists of two subunits with molecular masses of 65 and 37 kDa. Among the electron acceptors tested, nitroblue tetrazolium chloride was reduced by the AH at highest rates. At 30°C and pH 8, the specific activity of the enzyme was 0.3 μmol of H₂ per min and mg of protein. However, an unexpectedly high Michaelis constant (K_m) for H₂ of 3.6 ± 0.5 μM was determined, which is in contrast to the previously proposed low K_m of group 5 hydrogenases and makes atmospheric H₂ uptake by R. eutropha most unlikely. Amperometric activity measurements revealed that the AH maintains full H₂ oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O₂.     

Genetic Analysis of the Hox Hydrogenase in the Cyanobacterium Synechocystis sp. PCC 6803 Reveals Subunit Roles in Association,Assembly, Maturation,and Function

Hydrogenases are metalloenzymes that catalyze 2H⁺ + 2e⁻ ↔ H₂. A multisubunit, bidirectional [NiFe]-hydrogenase has been identified and characterized in a number of bacteria, including cyanobacteria, where it is hypothesized to function as an electron valve, balancing reductant in the cell. In cyanobacteria, this Hox hydrogenase consists of five proteins in two functional moieties: a hydrogenase moiety (HoxYH) with homology to heterodimeric [NiFe]-hydrogenases and a diaphorase moiety (HoxEFU) with homology to NuoEFG of respiratory Complex I, linking NAD(P)H ↔ NAD(P)⁺ as a source/sink for electrons. Here, we present an extensive study of Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. We identify the presence of HoxEFUYH, HoxFUYH, HoxEFU, HoxFU, and HoxYH subcomplexes as well as association of the immature, unprocessed large subunit (HoxH) with other Hox subunits and unidentified factors, providing a basis for understanding Hox maturation and assembly. The analysis of mutants containing individual and combined hox gene deletions in a common parental strain reveals apparent alterations in subunit abundance and highlights an essential role for HoxF and HoxU in complex/subcomplex association. In addition, analysis of individual and combined hox mutant phenotypes in a single strain background provides a clear view of the function of each subunit in hydrogenase activity and presents evidence that its physiological function is more complicated than previously reported, with no outward defects apparent in growth or photosynthesis under various growth conditions.     

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.