The thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV oxidizes subatmospheric H2 with a high-affinity, membrane-associated [NiFe] hydrogenase.
ABSTRACT: The trace amounts (0.53?ppmv) of atmospheric hydrogen gas (H2) can be utilized by microorganisms to persist during dormancy. This process is catalyzed by certain Actinobacteria, Acidobacteria, and Chloroflexi, and is estimated to convert 75?×?1012?g H2 annually, which is half of the total atmospheric H2. This rapid atmospheric H2 turnover is hypothesized to be catalyzed by high-affinity [NiFe] hydrogenases. However, apparent high-affinity H2 oxidation has only been shown in whole cells, rather than for the purified enzyme. Here, we show that the membrane-associated hydrogenase from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV possesses a high apparent affinity (Km(app)?=?140?nM) for H2 and that methanotrophs can oxidize subatmospheric H2. Our findings add to the evidence that the group 1h [NiFe] hydrogenase is accountable for atmospheric H2 oxidation and that it therefore could be a strong controlling factor in the global H2 cycle. We show that the isolated enzyme possesses a lower affinity (Km?=?300?nM) for H2 than the membrane-associated enzyme. Hence, the membrane association seems essential for a high affinity for H2. The enzyme is extremely thermostable and remains folded up to 95?°C. Strain SolV is the only known organism in which the group 1h [NiFe] hydrogenase is responsible for rapid growth on H2 as sole energy source as well as oxidation of subatmospheric H2. The ability to conserve energy from H2 could increase fitness of verrucomicrobial methanotrophs in geothermal ecosystems with varying CH4 fluxes. We propose that H2 oxidation can enhance growth of methanotrophs in aerated methane-driven ecosystems. Group 1h [NiFe] hydrogenases could therefore contribute to mitigation of global warming, since CH4 is an important and extremely potent greenhouse gas.
Project description: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 ?mol of H2 per min and mg of protein. However, an unexpectedly high Michaelis constant (Km) for H2 of 3.6 ± 0.5 ?M was determined, which is in contrast to the previously proposed low Km 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.
Project description:Diverse aerobic bacteria persist by consuming atmospheric hydrogen (H<sub>2</sub>) 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 H<sub>2</sub> 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 H<sub>2</sub> to sub-atmospheric levels, and oxidation rates were much higher during growth. Moreover, the oxidation of H<sub>2</sub> 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 H<sub>2</sub> 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.
Project description:Emissions of the strong greenhouse gas methane (CH4) to the atmosphere are mitigated by methanotrophic microorganisms. Methanotrophs found in extremely acidic geothermal systems belong to the phylum Verrucomicrobia. Thermophilic verrucomicrobial methanotrophs from the genus Methylacidiphilum can grow autotrophically on hydrogen gas (H2), but it is unknown whether this also holds for their mesophilic counterparts from the genus Methylacidimicrobium. To determine this, we examined H2 consumption and CO2 fixation by the mesophilic verrucomicrobial methanotroph Methylacidimicrobium tartarophylax 4AC. We found that strain 4AC grows autotrophically on H2 with a maximum growth rate of 0.0048 h-1 and a yield of 2.1 g dry weight?mol H2 -1, which is about 12 and 41% compared to the growth rate and yield on methane, respectively. The genome of strain 4AC only encodes for an oxygen-sensitive group 1b [NiFe] hydrogenase and H2 is respired only when oxygen concentrations are below 40 ?M. Phylogenetic analysis and genomic comparison of methanotrophs revealed diverse [NiFe] hydrogenases, presumably with varying oxygen sensitivity and affinity for H2, which could drive niche differentiation. Our results show that both thermophilic and mesophilic verrucomicrobial methanotrophs can grow as autotrophs on H2 as a sole energy source. Our results suggest that verrucomicrobial methanotrophs are particularly well-equipped to thrive in hostile volcanic ecosystems, since they can consume H2 as additional energy source.
Project description:Aerobic soil bacteria persist by scavenging molecular hydrogen (H2) from the atmosphere. This key process is the primary sink in the biogeochemical hydrogen cycle and supports the productivity of oligotrophic ecosystems. In Mycobacterium smegmatis, atmospheric H2 oxidation is catalyzed by two phylogenetically distinct [NiFe]-hydrogenases, Huc (group 2a) and Hhy (group 1h). However, it is currently unresolved how these enzymes transfer electrons derived from H2 oxidation into the aerobic respiratory chain. In this work, we used genetic approaches to confirm that two putative iron-sulfur cluster proteins encoded on the hydrogenase structural operons, HucE and HhyE, are required for H2 consumption in M. smegmatis. Sequence analysis show that these proteins, while homologous, fall into distinct phylogenetic clades and have distinct metal-binding motifs. H2 oxidation was reduced when the genes encoding these proteins were deleted individually and was eliminated when they were deleted in combination. In turn, the growth yield and long-term survival of these deletion strains was modestly but significantly reduced compared to the parent strain. In both biochemical and phenotypic assays, the mutant strains lacking the putative iron-sulfur proteins phenocopied those of hydrogenase structural subunit mutants. We hypothesize that these proteins mediate electron transfer between the catalytic subunits of the hydrogenases and the menaquinone pool of the M. smegmatis respiratory chain; however, other roles (e.g., in maturation) are also plausible and further work is required to resolve their role. The conserved nature of these proteins within most Hhy- or Huc-encoding organisms suggests that these proteins are important determinants of atmospheric H2 oxidation.
Project description:Methanotrophs play a key role in balancing the atmospheric methane concentration. Recently, the microbial methanotrophic diversity was extended by the discovery of thermoacidophilic methanotrophs belonging to the Verrucomicrobia phylum in geothermal areas. Here we show that a representative of this new group, Methylacidiphilum fumariolicum SolV, is able to grow as a real 'Knallgas' bacterium on hydrogen/carbon dioxide, without addition of methane. The full genome of strain SolV revealed the presence of two hydrogen uptake hydrogenases genes, encoding an oxygen-sensitive (hup-type) and an oxygen-insensitive enzyme (hhy-type). The hhy-type hydrogenase was constitutively expressed and active and supported growth on hydrogen alone up to a growth rate of 0.03?h<sup>-1</sup>, at O<sub>2</sub> concentrations below 1.5%. The oxygen-sensitive hup-type hydrogenase was expressed when oxygen was reduced to below 0.2%. This resulted in an increase of the growth rate to a maximum of 0.047?h<sup>-1</sup>, that is 60% of the rate on methane. The results indicate that under natural conditions where both hydrogen and methane might be limiting strain SolV may operate primarily as a methanotrophic 'Knallgas' bacterium. These findings argue for a revision of the role of hydrogen in methanotrophic ecosystems, especially in soil and related to consumption of atmospheric methane.
Project description:Streptomyces soil isolates exhibiting the unique ability to oxidize atmospheric H(2) possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase hhyS and hhyL, respectively. Genome data mining revealed that the hhyL gene is unevenly distributed in the phyla Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria. The hhyL gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H(2)-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H(2) oxidation activity and the hhyL gene are ubiquitous. A quantitative PCR assay revealed that soil contained 10(6) to 10(8) hhyL gene copies g (dry weight)(-1). Assuming one hhyL gene copy per genome, the abundance of presumptive high-affinity H(2)-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H(2) oxidation activity measured in soil. Our data indicate that the abundance of the hhyL gene should not be taken as a reliable proxy for the uptake of atmospheric H(2) by soil, because high-affinity H(2) oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur.
Project description:In the Earth's lower atmosphere, H2 is maintained at trace concentrations (0.53 ppmv/0.40 nM) and rapidly turned over (lifetime ? 2.1 y(-1)). It is thought that soil microbes, likely actinomycetes, serve as the main global sink for tropospheric H2. However, no study has ever unambiguously proven that a hydrogenase can oxidize this trace gas. In this work, we demonstrate, by using genetic dissection and sensitive GC measurements, that the soil actinomycete Mycobacterium smegmatis mc(2)155 constitutively oxidizes subtropospheric concentrations of H2. We show that two membrane-associated, oxygen-dependent [NiFe] hydrogenases mediate this process. Hydrogenase-1 (Hyd1) (MSMEG_2262-2263) is well-adapted to rapidly oxidize H2 at a range of concentrations [Vmax(app) = 12 nmol?g?dw(-1)?min(-1); Km(app) = 180 nM; threshold = 130 pM in the ?hyd23 (Hyd1 only) strain], whereas Hyd2 (MSMEG_2719-2720) catalyzes a slower-acting, higher-affinity process [Vmax(app) = 2.5 nmol?g?dw(-1)?min(-1); Km(app) = 50 nM; threshold = 50 pM in the ?hyd13 (Hyd2 only) strain]. These observations strongly support previous studies that have linked group 5 [NiFe] hydrogenases (e.g., Hyd2) to the oxidation of tropospheric H2 in soil ecosystems. We further reveal that group 2a [NiFe] hydrogenases (e.g., Hyd1) can contribute to this process. Hydrogenase expression and activity increases in carbon-limited cells, suggesting that scavenging of trace H2 helps to sustain dormancy. Distinct physiological roles for Hyd1 and Hyd2 during the adaptation to this condition are proposed. Soil organisms harboring high-affinity hydrogenases may be especially competitive, given that they harness a highly dependable fuel source in otherwise unstable environments.
Project description:The majority of microbial cells in global soils exist in a spectrum of dormant states. However, the metabolic processes that enable them to survive environmental challenges, such as nutrient-limitation, remain to be elucidated. In this work, we demonstrate that energy-starved cultures of Pyrinomonas methylaliphatogenes, an aerobic heterotrophic acidobacterium isolated from New Zealand volcanic soils, persist by scavenging the picomolar concentrations of H2 distributed throughout the atmosphere. Following the transition from exponential to stationary phase due to glucose limitation, the bacterium up-regulates by fourfold the expression of an eight-gene operon encoding an actinobacteria-type H2-uptake [NiFe]-hydrogenase. Whole-cells of the organism consume atmospheric H2 in a first-order kinetic process. Hydrogen oxidation occurred most rapidly under oxic conditions and was weakly associated with the cell membrane. We propose that atmospheric H2 scavenging serves as a mechanism to sustain the respiratory chain of P. methylaliphatogenes when organic electron donors are scarce. As the first observation of H2 oxidation to our knowledge in the Acidobacteria, the second most dominant soil phylum, this work identifies new sinks in the biogeochemical H2 cycle and suggests that trace gas oxidation may be a general mechanism for microbial persistence.
Project description:To persist when nutrient sources are limited, aerobic soil bacteria metabolize atmospheric hydrogen (H2). This process is the primary sink in the global H2 cycle and supports the productivity of microbes in oligotrophic environments. H2-metabolizing bacteria possess [NiFe] hydrogenases that oxidize H2 to subatmospheric concentrations. The soil saprophyte Mycobacterium smegmatis has two such [NiFe] hydrogenases, designated Huc and Hhy, that belong to different phylogenetic subgroups. Both Huc and Hhy are oxygen-tolerant, oxidize H2 to subatmospheric concentrations, and enhance bacterial survival during hypoxia and carbon limitation. Why does M. smegmatis require two hydrogenases with a seemingly similar function? In this work, we resolved this question by showing that Huc and Hhy are differentially expressed, localized, and integrated into the respiratory chain. Huc is active in late exponential and early stationary phases, supporting energy conservation during mixotrophic growth and transition into dormancy. In contrast, Hhy is most active during long-term persistence, providing energy for maintenance processes following carbon exhaustion. We also show that Huc and Hhy are obligately linked to the aerobic respiratory chain via the menaquinone pool and are differentially affected by respiratory uncouplers. Consistently, these two enzymes interacted differentially with the respiratory terminal oxidases. Huc exclusively donated electrons to, and possibly physically associated with, the proton-pumping cytochrome bcc-aa 3 supercomplex. In contrast the more promiscuous Hhy also provided electrons to the cytochrome bd oxidase complex. These results indicate that, despite their similar characteristics, Huc and Hhy perform distinct functions during mycobacterial growth and survival.
Project description:An oxygen-tolerant respiratory [NiFe]-hydrogenase is proven to be a four-electron hydrogen/oxygen oxidoreductase, catalyzing the reaction 2 H2 + O2 = 2 H2O, equivalent to hydrogen combustion, over a sustained period without inactivating. At least 86% of the H2O produced by Escherichia coli hydrogenase-1 exposed to a mixture of 90% H2 and 10% O2 is accounted for by a direct four-electron pathway, whereas up to 14% arises from slower side reactions proceeding via superoxide and hydrogen peroxide. The direct pathway is assigned to O2 reduction at the [NiFe] active site, whereas the side reactions are an unavoidable consequence of the presence of low-potential relay centers that release electrons derived from H2 oxidation. The oxidase activity is too slow to be useful in removing O2 from the bacterial periplasm; instead, the four-electron reduction of molecular oxygen to harmless water ensures that the active site survives to catalyze sustained hydrogen oxidation.