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.

NMDS using the Bray-Curtis dissimilarity index (Fig. S5c,d), as observed in the long-read amplicon libraries ( Fig. 1d (rarefied data), S4b (unrarefied data)). The diversity and richness were typically higher in the temperate soils (beech forest and managed grassland soils) relative to the biological soil crusts (Fig. S5e,f). These diversity and richness patterns were similar to the long-read amplicon libraries when data were rarefied and unrarefied (Fig. S4c,d).
Although similar alpha-and beta-diversity patterns amongst soils were observed across the 3 investigated primer pair sets, we observed clear differences in the phylogenetic affiliation of the OTU representatives using RAxML EPA trees (Fig. S6 -comparison of the grey shaded clusters and dots).
This newly designed primer pair also covered an additional group of actinobacterial sequences (Fig.   S6c, top-most Actinobacteria cluster), as well as an additional deep-branching cluster of sequences containing group 1h [NiFe]-hydrogenases of diverse origin, such as members of the Actinobacteria, Chloroflexi, Bacteriodetes, Acidobacteria, Proteobacteria and Euryarchaeota (Fig. 6c, "Distant group 1h cluster"). Although these sequences were all classified as a group 1h [NiFe]-hydrogenase based on the HydDB (8), they appear to be distantly related to the other, main branch of the tree (Fig. 2b).
Furthermore, we detected numerous sequences that were placed in the tree without reference sequences (Fig. S6c, indicated by grey dots) in the long-read amplicon libraries, in comparison to 1 ([NiFe]-1129f/1640R) or 5 ([NiFe]-244f/568R) OTU representatives without reference sequences in the short-read libraries (Fig. S6a- (Fig. S9a). Unlike the DNA-based libraries stemming from the [NiFe]-244F/568R primer pair as well as the long-read amplicon libraries, the group 1h [NiFe]-hydrogenase communities did not separate based on soil type (Fig. S9b). This suggests that a diverse collection of microorganisms were expressing their hhyL, which was not (directly) influenced by the edaphic properties of the investigated soils. The diversity and richness were typically higher in the managed grassland soils relative to the beech forest soil (Fig. S9c). The rhizosphere soil diversity was lower than of the bulk soil from the managed grassland soil (Fig. S9c). This could suggest that bacteria with low-affinity hydrogenases might be more active in the rhizosphere, as these bacteria are believed to grow on the high concentrations of H2 produced in microniches, such as N2-fixing root nodules (9), which are found in rhizosphere soil. The OTU representatives of the cDNA libraries stemming from the [NiFe]-244F/568R primer pair spanned numerous phylogenetic phyla based on RAxML EPA, such as Actinobacteria, Proteobacteria, Verrucomicrobia, Acidobacteria, Planctomycetes and Chloroflexi (Fig. S10).

Summary
Although similar patterns in diversity and community composition were observed across the   Figure S1. Establishment of the acidobacterial strain-specific group 1h [NiFe]-hydrogenase hhyL (panels a-d) and hhyS (panels e-h) qPCR assays. qPCR amplification (panels a,e), standards (panels b,f), melting curves (panels c,g) are depicted for a serially diluted DNA standard. Neighbor-joining phylogenetic tree of amplified products illustrating specificity of the reaction (panels d, h). Data from Acidobacteriaceae bacterium KBS 83 strain specific primers are shown for qPCR amplification, standards and melting curves. Similar patterns were observed for E. aggregans.      Analysis of variance with a Tukey's HSD mean separation was performed across the soil types for the diversity statistics; similar letters indicate that no significant difference was observed (P > 0.05), 'nsd' = no significant difference. Libraries were rarified to 2,300 sequences. To determine carbon-limiting conditions, strains were grown in the VSB-6 defined medium (see Materials and Methods for more details on the medium composition) in differing glucose concentrations (5 mM and 10 mM glucose). Carbon-limiting conditions were defined when the cellular yield (optical density 600 nm) was proportional to the amount of carbon provided. Growth was monitored by measuring the optical density at 600 nm.   Figure S13. H 2 consumption assays of cells in exponential phase. Cells were harvested for both strains in exponential phase (panel a); arrows depict the time points in which the cells were harvested from each respective strain. Controls for this assay can be found in Figure S3. H 2 consumption of the exponential phase cells of both strains was monitored over time (panel b  Figure S3. These were classified manually as orginating from "temperate soils", "thermophilic environments (such as soils, bagasse and volcanic deposits, all with temperatures >40˚C), "acid mine drainage", "aquatic" (such as deep-sea octacoral samples), "insect-associated" (such as the termite hindgut and gut of New Zealand's endemic Huhubeetle larvae), "microbial mats" "various" (such as "hot water", "leaves", "skin" and "waste pile") and "unknown" (no description was given in the sequence entries at NCBI, along with no associated manuscript). The percent of these sequences found in each respective environment is depicted. 16S rRNA gene ATT CCA CKC ACC TCT CCC AYC AY (Steger, 2010)