The Role of the Huc Hydrogenase on the Growth of Mycobacterium Smegmatis under Ambient and Elevated Hydrogen Gas Conditions

Abstract

Hydrogen (H2) gas exists ubiquitously in the atmosphere. Microorganisms in soil use H2 oxidation to support microbial processes related to growth and survival. One model organism for understanding H2 oxidation is Mycobacterium smegmatis MC2 155, which possesses both a high-affinity (Hhy) hydrogenase and mid-affinity (Huc) hydrogenase. While both hydrogenases oxidize H2 at atmospheric concentrations, only Huc is upregulated in response to H2 supplementation. A knockout mutant of Huc (Δhuc) was compared with wildtype (WT) to test whether Huc contributes to growth under ambient (0.53 ppm) or elevated (50 ppm) H2. Soil was sterilized and different bacterial assemblages (WT, Δhuc, WT:Δhuc (50:50)) were distributed to mesocosms, then exposed to elevated or ambient H2 for 8 days. After the experiment was complete, there was an increase in H2 uptake for all assemblages at both H2 concentrations. High affinity oxidation was suppressed in all assemblages exposed to the elevated concentration of H2. Colony forming unit enumeration showed the most growth in the WT assemblage, but this growth was independent of H2 supplementation. Based on previous research, it was assumed when both microbes are present, WT would outgrow Δhuc, but our qPCR results did not support that theory. The ratio of copy number stayed relatively the same, with the data indicating the possibility that Δhuc outgrew WT. Based on these methods our results showed that Huc could contribute to growth, but elevated H2 did not have a measurable impact. These results also demonstrated that both growth and H2 oxidation from this microorganism can be measured in sterilized soil.