A soil actinobacterium scavenges atmospheric H ₂ using two membrane-associated, oxygen-dependent [NiFe] hydrogenases

Abstract

In the Earth's lower atmosphere, H 2 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 H 2 . 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 H 2 . We show that two membraneassociated, oxygen-dependent [NiFe] hydrogenases mediate this process. Hydrogenase-1 (Hyd1) (MSMEG_2262-2263) is well-adapted to rapidly oxidize H 2 at a range of concentrations [V max(app) = 12 nmol·g·dw −1 ·min −1 ; K m(app) = 180 nM; threshold = 130 pM in the Δhyd23 (Hyd1 only) strain], whereas Hyd2 (MSMEG_2719-2720) catalyzes a slower-acting, higher-affinity process [V max(app) = 2.5 nmol·g·dw −1 ·min −1 ; K m(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 H 2 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 H 2 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.atmospheric chemistry | biogeochemical cycles | enzyme kinetics | mycobacteria

An obligately aerobic soil bacterium activates fermentative hydrogen production to survive reductive stress during hypoxia

“…In contrast to this system, the tight regulation and sustained activity of Hyd3 under oxygen limitation in M. smegmatis is consistent with Hyd3 being specifically selected to catalyze the fermentative production of H 2 during dormancy. The conservation of Hyd3 homologs across other actinobacteria, e.g., mycobacteria, streptomycetes, and rhodococci (38), suggests that hydrogen fermentation may be a widespread, but previously unanticipated, mechanism for long-term survival among obligate aerobic soil bacteria.…”

“…Our data also surprisingly show that Hyd2, a group 5 [NiFe]-hydrogenase (actinobacteria-type uptake hydrogenase), is activated by DosR from a previously unidentified promoter. In contrast to Hyd3, this enzyme acts as a high-affinity, oxygen-dependent uptake hydrogenase during carbon limitation (15,38). Neighboring operons encoding a putative sulfate ABC importer (CysTWA) (MSMEG_4530-4532) and a sulfite reductase (SirA), phosphoadenosine phosphosulfate reductase, and siroheme chelatase (Che1) (MSMEG_4527-4529) also exhibited a fivefold decrease in expression in ΔdosR.…”

“…2A). We have previously shown that Hyd1 is a rapid-acting, highaffinity group 2a [NiFe]-hydrogenase (38) that is up-regulated during oxygen limitation in a manner independent of DosR (15). H 2 oxidation could not be observed at later time points, likely because the remaining O 2 is depleted to levels insufficient to sustain respiratory activity.…”

“…Although group 3b [NiFe]-hydrogenases are widespread in actinobacteria and proteobacteria (38), until now, they have only been characterized in thermophilic archaea. Hyd3 shares >30% sequence identity with the cytosolic hydrogenases of Pyrococcus furiosus (39,40) and Thermococcus kodakaraensis (41), including motifs predicted to bind a FAD moiety, a [NiFe] center, and a relay of six [FeS] clusters.…”

The Redox Cofactor F 420 Protects Mycobacteria from Diverse Antimicrobial Compounds and Mediates a Reductive Detoxification System

“…No CFU or MIC differences were observed for DMSO-only controls. To test the capacity of strains to grow on compounds as a sole source of carbon, exponential-phase cultures were inoculated to a starting OD 600 of 0.005 into HBTP medium (25) supplemented with either 1 mM or 10 mM the compounds of interest, and growth was determined by the OD 600 following incubation (200 rpm, 37°C, 24 h). Eight compounds were tested: coumarin, methoxsalen, imperatorin, menadione, azure B, malachite green, crystal violet, and phenol red.…”