Atmospheric hydrogen consumption is regulated by glycerol-mediated catabolite repression in mycobacteria

Consumption of atmospheric hydrogen (H₂) enables diverse aerobic microorganisms to grow and persist in resource-deprived environments. In the aerobic saprophyte <i>Mycobacterium smegmatis</i>, hydrogen oxidation is catalyzed by two differentially expressed, high-affinity, oxygen-insensitive uptake hydrogenases, Huc and Hhy. Huc enables mixotrophic growth and facilitates the transition from growth to dormancy. Although the <i>huc</i> operon is known to be upregulated in response to organic carbon deprivation, the specific signals and regulators modulating its expression remain unresolved. Here, we show that GylR, a glycerol-3-phosphate-sensing regulator of glycerol metabolism, plays a role in the repression of <i>huc</i> expression in response to the availability of glycerol but not other carbon sources. Based on proteomic analyses and activity assays, mutation or knockdown of <i>gylR</i> leads to enhanced Huc production and activity. GylR and other key catabolite repressor proteins (Crp1 and Crp2) do not directly bind to the <i>huc</i> operon, indicating that repression is mediated by unidentified transcription factors, with GylR acting as an upstream sensor. Here, we present data that suggest atmospheric H₂ oxidation is regulated in response to organic carbon source availability through the process of catabolite repression. By identifying a key signal that prompts atmospheric H₂ oxidation, these findings advance understanding of how aerobic bacteria adapt to changing environmental conditions and suggest that organic carbon levels are a key factor regulating the main sink of atmospheric H₂ in soils globally.IMPORTANCESoil microorganisms collectively consume 70 million tonnes of atmospheric hydrogen (H₂) a year, regulating atmospheric composition and climate change. In turn, consuming this dependable trace gas enables these microorganisms to survive even when their preferred organic energy sources are exhausted. Despite the importance of H₂ consumption for soil biodiversity and atmospheric regulation, the signals and sensors that regulate this process remain to be understood. Here, we demonstrate that a model soil bacterium turns on the machinery required for atmospheric H₂ consumption in direct response to being limited by organic carbon availability, through the process of catabolite repression. Specifically, in the absence of a sensor of the organic carbon source glycerol, a H₂-consuming hydrogenase is highly expressed and active. These findings suggest that organic carbon levels have a major role in regulating trace gas oxidation, with implications for predicting how trace gas consumption and soil biodiversity respond to environmental change.