Persistence of the dominant soil phylum Acidobacteria by trace gas scavenging

Abstract

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.

Keywords: dormancy; extremophile; hydrogen; hydrogenase; rare biosphere.

Fig. 1.

Hydrogenase determinants in P. methylaliphatogenes strain K22^T. (A) Phylogenetic tree showing the phylogeny of the P. methylaliphatogenes hydrogenase large subunit sequence compared with those of Group 5 [NiFe]-hydrogenases. The tree was constructed using the neighbor-joining method, bootstrapped with 500 replicates, and rooted using representatives of the Group 1 and Group 2 [NiFe]-hydrogenases. (B) RT-PCR analysis determining the structure of the operon encoding the hydrogenase structural components. The structure was determined by detecting the presence of intergenic PCR products for cDNA samples against positive controls (gDNA) and negative controls (mRNA). Primers were designed to amplify the start, end, and intergenic regions of adjacent genes (loci numbers shown above the lane). (C) To-scale structure of the elucidated operon showing locus numbers and functional annotations of the genes.

Fig. S1.

Multiple sequence alignments (Clustal) of the genes encoding the large subunits of the Group 5 [NiFe]-hydrogenases in P. methylaliphatogenes (PYK22_03065), Solibacter usitatus (Acid_6556), Mycobacterium smegmatis (MSMEG_2719), Streptomyces avermitilis (SAV_7367), and Ralstonia eutropha (PHG065). The four cysteine residues highlighted in yellow ligate the [NiFe] center that serves as the catalytic center. Sequences in red are predicted to be cleaved by the HupD endopeptidase during hydrogenase maturation.

Fig. S2.

Multiple sequence alignments (Clustal) of the genes encoding the small subunits of the Group 5 [NiFe]-hydrogenases in P. methylaliphatogenes (PYK22_03064), Solibacter usitatus (Acid_6555), Mycobacterium smegmatis (MSMEG_2720), Streptomyces avermitilis (SAV_7367), and Ralstonia eutropha (PHG064). The subunit ligates a 3Cys1Asp[4Fe4S]_proximal (ligands highlighted in green), 4Cys[4Fe4S]_medial cluster (ligands highlighted in blue), and a 3Cys1His[4Fe4S]_distal cluster (ligands highlighted in pink).

Fig. 2.

Hydrogenase expression in P. methylaliphatogenes strain K22^T. (A) Growth of P. methylaliphatogenes in FS1V minimal medium supplemented with 2.5 mM glucose. Cells were harvested for RNA extraction during exponential phase (OD₆₀₀ = 0.1) and stationary phase (t = 120 h). (B) Glucose concentration of the external medium during cell growth. (C) Expression ratios of the genes encoding the hydrogenase small and large subunit during stationary vs. exponential phase as determined by qRT-PCR and normalized to 16S rRNA gene expression. In both cases, error bars represent SDs from three biological replicates.

Fig. 3.

Hydrogenase activity in carbon-limited stationary-phase cultures of P. methylaliphatogenes strain K22^T. (A) Density-dependent whole-cell oxidation of H₂. (B) Whole-cell oxidation of H₂ following nitrogen-sparging (anoxic), cyanide-treatment, or heat-killing of the cells. For both A and B, H₂ concentration was measured using a H₂ microsensor. Positive values infer net H₂ evolution, whereas negative values infer net H₂ oxidation. Error bars represent SDs from three biological replicates; *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test). (C) Zymographic detection of hydrogenase activity in concentration-normalized cell lysates (L), cytosols (C), and membranes (M) separated by native PAGE and anaerobically stained with the artificial electron acceptor nitroblue tetrazolium. Acidobacterial samples (labeled acido in subscript) were run against the M. smegmatis mc²155 as a positive control (+ve) and its hydrogenase triple mutant derivative as a negative control (−ve).

Fig. 4.

H₂ uptake in carbon-limited stationary-phase cultures of P. methylaliphatogenes strain K22^T. The depletion of a headspace of H₂ was measured as a function of time in cultures inoculated with the bacterium (blue circles) against no-culture controls (red triangles). Mixing ratios are displayed on a logarithmic scale. Error bars represent SDs from three biological replicates. The gray dotted line shows the global mixing ratio of H₂ in the lower atmosphere (0.53 ppmv).

Fig. S3.

Hydrogen oxidation by stationary-phase cultures of P. methylaliphatogenes strain K22^T at H₂ partial pressures capable of saturating the [NiFe]-hydrogenase. The depletion of a headspace of H₂ was measured as a function of time in cultures inoculated with the bacterium. Mixing ratios are displayed on a linear scale. Error bars show SDs from three biological replicates.

Fig. 5.

Model of the respiratory chain of P. methylaliphatogenes in carbon-replete (Upper) and carbon-limiting (Lower) conditions. The genome of P. methylaliphatogenes suggests its respiratory chain is highly minimalistic. It comprises three primary dehydrogenases (nuo: NADH dehydrogenase type I; sdh: succinate dehydrogenase; hhy: high-affinity [NiFe]-hydrogenase), a single terminal oxidase (cyd: cytochrome bd oxidase), and an F₁F_o-ATP synthase. In carbon-replete conditions, import and oxidation of heterotrophic carbon sources such as glucose yields NADH and succinate that serve as an input into the respiratory chain via nuo and sdh. A large proton-motive force is generated, primarily through the action of the proton-translocating nuo, generating sufficient ATP for growth. In carbon-limiting conditions, the high-affinity hydrogenase hhy oxidizes the atmospheric H₂ that diffuses into the cell. This may generate a small proton-motive force via a redox-loop mechanism dependent on menaquinone protonation and menaquinol deprotonation, providing sufficient ATP for maintenance.

Fig. S4.

Distribution of Group 5 [NiFe]-hydrogenases. The phylogenetic tree shows the phylogeny of the P. methylaliphatogenes hydrogenase large subunit sequence compared with those of Group 5 [NiFe]-hydrogenases. The tree was constructed using the neighbor-joining method, bootstrapped with 500 replicates, and rooted using representatives of the Group 1 and Group 2 [NiFe]-hydrogenases. Over 100 Group 5 [NiFe]-hydrogenases have been identified in the genomes of the dominant soil phyla Actinobacteria, Acidobacteria, Chloroflexi, Verrucomicrobia, Planctomycetes, and Proteobacteria.