A soil actinobacterium scavenges atmospheric H2 using two membrane-associated, oxygen-dependent [NiFe] hydrogenases

Abstract

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 Δhyd23 (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 Δhyd13 (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.

Keywords: atmospheric chemistry; biogeochemical cycles; enzyme kinetics; mycobacteria.

Fig. 1.

H₂ oxidation during exponential growth of M. smegmatis mc²155 and derivatives in sealed serum vials. H₂ mixing ratios were measured upon inoculation of culture and throughout growth. All strains entered stationary phase at OD ∼ 2.5 (∼1.5 × 10⁸ cfu⋅mL⁻¹) after consuming the majority of the oxygen available in the headspace and hence becoming oxygen-limited. Blue circles represent OD₆₀₀ of the cultures. Red squares represent the H₂ mixing ratio in the headspace. Error bars are SDs from three biological replicates.

Fig. 2.

H₂ oxidation by carbon-limited cultures of M. smegmatis mc²155 and derivatives. All strains were inoculated to a density of 3 × 10⁷ cfu⋅mL⁻¹ (OD₆₀₀ = 0.4). H₂ mixing ratios were measured upon inoculation of culture and at regular intervals over 84 h. The mixing ratios are displayed on a logarithmic scale. Error bars represent SDs from three biological replicates. The dashed lines represent the average global mixing ratio of tropospheric H₂ (0.53 ppmv).

Fig. 3.

Determination of uptake hydrogenase localization. Protein samples of each cell fraction (5 µg) were separated on native polyacrylamide gels. (A) Total protein content was stained by using Coomassie brilliant blue. (B) Uptake hydrogenase activity was detected by using the artificial electron acceptor nitroblue tetrazolium chloride. (C) Cell fractions were added to a reaction chamber saturated with H₂ and O₂. A H₂ microsensor determined the rates of hydrogen consumption (negative values) or production (positive values). Error bars represent SDs from three technical replicates. C, cytosolic fraction; L, crude cell lysate; M, membrane fraction.

Fig. 4.

The wide spectrum of affinities for H₂ observed in hydrogen-oxidizing organisms. The types of [NiFe] hydrogenase encoded by each organism are shown in brackets. Pure cultures of the soil actinomycetes M. smegmatis (this work), R. equi (26), and certain Streptomyces species (12, 15, 17) have high affinities for H₂ (K_m = 30–130 nM). These activities depend on group 5 [NiFe] hydrogenases and correlate with the high-affinity portion of H₂ oxidation in whole soils (10, 16). By contrast, hydrogen-oxidizing soil Proteobacteria such as Bradyrhizobium japonicum (28), P. denitrificans (29), Xanthobacter autotrophicus (14), and R. eutropha (20) have low affinities for H₂ (K_m = 1–5 µM) similar to that observed for low-affinity portion of H₂ oxidation in whole soils (10). H₂ affinities of the cyanobacterium Anabaena sp. PCC 7120 (30), sulfate-reducing bacterium Desulfovibrio sp. G11, and methanogen Methanospirillum hungatei (31) are also shown for reference. Many organisms harboring group 5 [NiFe] hydrogenases (e.g., R. eutropha) and group 2a [NiFe] hydrogenases (e.g., Anabaena sp. PCC 7120) still have low affinities for H₂.