A widely distributed hydrogenase oxidises atmospheric H2 during bacterial growth

Abstract

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.

Fig. 1. Expression of the group 2a [NiFe]-hydrogenase in three bacterial strains during growth and survival.

The normalised transcript copy number of the large subunit gene (hucL) are plotted for (a) Gemmatimonas aurantiaca (locus GAU_0412), (b) Acidithiobacillus ferrooxidans (locus AFE_0702), and (c) Chloroflexus aggregans (locus CAGG_0471). Copy number was analysed by qRT-PCR in cultures harvested during exponential phase and stationary phase, in the presence of either ambient H₂ or 10% H₂. Error bars show standard deviations of three biological replicates (averaged from two technical duplicates) per condition. Values denoted by different letters were determined to be statistically significant based on a one-way ANOVA with post-hoc Tukey’s multiple comparison (p < 0.05).

Fig. 2. Hydrogenase activity in three bacterial strains during growth and survival.

H₂ oxidation by cultures of (a) Gemmatimonas aurantiaca, (b) Acidithiobacillus ferrooxidans, and (c) Chloroflexus aggregans. Error bars show the standard deviation of three biological replicates, with media-only vials monitored as negative controls. Dotted lines show the atmospheric concentration of hydrogen (0.53 ppmv). d Biomass-normalised first-order rate constants based on H₂ oxidation observed in exponential and stationary phase cultures. Error bars show standard deviations of three biological replicates and statistical significance was tested using a two-way ANOVA with post-hoc Tukey’s multiple comparison (**= p < 0.01; ****= p < 0.0001).

Fig. 3. Effects of H₂ supplementation on growth of three bacterial strains.

The final growth yield (OD₆₀₀) of (a) Gemmatimonas aurantiaca, (b) Acidithiobacillus ferrooxidans, and (c) Chloroflexus aggregans is shown in ambient air vials containing H₂ at either ambient, 1%, or 10% concentrations. Error bars show the standard deviation of three biological replicates and statistical significance was tested using a one-way ANOVA with post-hoc Tukey’s multiple comparison (*= p < 0.05; **= p < 0.01; ***= p < 0.001).

Fig. 4. Radial phylogenetic tree showing the distribution and evolutionary history of the group 2a [NiFe]-hydrogenase.

Amino acid sequences of the catalytic subunit of the group 2a [NiFe]-hydrogenase (hucL) are shown for 171 bacterial genera. The taxon names of the three study species, G. aurantiaca, A. ferrooxidans, and C. aggregans, are coloured in blue. The tree was constructed using the maximum-likelihood method (gaps treated with partial deletion), bootstrapped with 500 replicates, and rooted at the mid-point. Accession numbers and amino acid sequences used to construct the tree are listed in Table S2. The total number of genomes identified per phylum are as follows: Acidobacteriota (1), Actinobacteriota (27), Aquificota (4), Bacteroidota (34), Chloroflexota (5), Cyanobacteria (61), Deinococcota (2), Firmicutes (19), Gemmatimonadota (1), Myxococcota (1), Nitrospirota (2), Planctomycetota (1), Proteobacteria (49).