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

Abstract

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.

Fig. 1. Activity and diversity of bacteria mediating atmospheric H₂ oxidation in three soil ecosystems.

a Hydrogen consumption by soils collected from a beech forest, managed grassland and desert biological soil crust. Dotted lines represent atmospheric concentrations of H₂ (~0.53 ppmv). Data points depict the mean ± standard deviation. b Michaelis–Menten kinetics of H₂ oxidation by the beech forest soil. Best-fit curve was determined using a Michaelis–Menten non-linear regression model. c Alpha diversity (Shannon index) and d beta diversity (Bray–Curtis dissimilatory) of the group 1h [NiFe]-hydrogenase large subunit (hhyL) genes in the soils detected by long-read amplicon sequencing. Analysis of variance with a Tukey’s HSD mean separation was performed across the soil types for the Shannon index; similar letters indicate that no significant difference was observed (p value > 0.05). Data were rarefied; unrarefied comparisons can be found in Fig. S4.

Fig. 2. Taxonomic and phylogenetic analysis of the group 1h [NiFe]-hydrogenase large subunit (hhyL) genes in three soil ecosystems.

a Predicted taxonomic distribution of amplified hhyL sequences across the investigated soils based on their closest hits to sequences in the NCBI-nr database. Triplicate samples are depicted for each soil. Colors represent different taxonomic groups. b RAxML-EPA tree of amino acid sequences of the group 1h [NiFe]-hydrogenase large subunit (hhyL) from long-read amplicon sequencing and reference sequences. The phylogenetic placements of OTU representatives stemming from nearly full-length sequences are depicted in gray: collapsed clusters containing OTU representative are shaded gray and non-clustered OTU representatives are colored gray. The number of the placed, nearly full-length hhyL sequences and the total number of sequences in each cluster are depicted. The proportions of sequences within each soil are depicted to the right of the clusters managed grassland (red), rhizosphere (orange), beech forest (blue) and biological soil crust (purple). Sequences from group 1g [NiFe]-hydrogenases were used as an outgroup. The scale bar indicates the number of substitutions per site.

Fig. 3. Phylogenetic tree of the group 1h [NiFe]-hydrogenase large subunit (hhyL) sequences stemming from reference genomes and metagenome-assembled genomes (MAGs) based on amino acid sequences (n = 1650 sequences, ca. 570 amino acid positions).

The tree was calculated using FastTree using the JTT + CAT model; FastTree confidence values of >95% (black circles) and >80% (gray circle) are depicted. The scale bar indicates the number of substitutions per site. The acidobacterial cluster is depicted in blue. The environmental source of the sequences (if available) is depicted in the name of each MAG. Additional information on publicly available MAG sequences depicted in the tree can be found in Table S4. Group 1g [NiFe]-hydrogenase sequences were used as an outgroup.

Fig. 4. Expression, activity, and kinetics of the enzymes mediating atmospheric H₂ oxidation in two acidobacterial strains isolated from temperate soils, Acidobacteriaceae bacterium KBS 83 and Edaphobacter aggregans.

a, d Growth curves of the strains over time (days) (x-axis) and expression levels (inset) of the group 1h [NiFe]-hydrogenase structural subunit genes (large, hhyL; small, hhyS) during exponential and stationary phase. Arrows depict the growth phases in which cells were harvested for gene expression investigations (gray arrow, exponential phase; black arrow, stationary phase). During this experiment, H₂ consumption was measured on stationary phase cells (black arrows) and in a parallel experiment on exponential phase cells (Fig. S13), as H₂ consumption assays required the entire biomass of such an experiment. b, e H₂ consumption of stationary phase stage cells of each respective strain; x-axis depicts the start of measurements for H₂ consumption after harvesting cells from growth curves of panels a, d. Dashed lines represent atmospheric H₂ concentrations (~0.53 ppmv), whereas red points depict the heat-killed controls for each respective strain. The final sub-atmospheric H₂ measurement for each strain was performed on a gas chromatograph with a pulsed discharge helium ionization detector (model TGA-6791-W-4U-2, Valco Instruments Company Inc.); this measurement is indicated by an asterisk. Over the course of the experiment, we observed a slight decrease in H₂ for our medium control (8%). Even when this loss is accounted for in the final sub-atmospheric measurement, the concentration is still below atmospheric levels of H₂, (0.27 ppmv for Acidobacteriaceae bacterium KBS 83 and 0.39 ppmv for E. aggregans). Additional controls can be found in Fig. S3. c, f Apparent kinetic parameters of H₂ oxidation for the strains based on whole-cell assays. Best-fit curves were determined using the Michaelis–Menten non-linear regression model; similar values were observed using Hanes–Woolf plots (Table S5).

Fig. 5. Comparison of apparent substrate affinity (K_m[app], in nM) for H₂-oxidizing bacteria and archaea spanning different taxonomic groups.

Figure was amended from [17]; data were extracted from Greening et al. [17] and Islam et al. [22]. Asterisks depict microorganisms harboring a group 1h [NiFe]-hydrogenase. Acidobacteria estimates derived in this study are depicted in blue, and data from the investigated beech forest soil are shown in green.