Surviving within terrestrial and marine environments alike can pose considerable challenges to microbial life. Nutrient scarcity and fierce intermicrobial competition often place severe restrictions on growth and necessitate a transition to a dormant state until conditions improve. While the cessation of growth and replication are key characteristics of dormancy, minimal metabolic activity is required to sustain cellular integrity and maintenance. Many members of the bacterial genus Mycobacterium leverage their metabolic flexibility during dormancy to prolong survival when confronted with resource deprivation. As obligate aerobes, mycobacteria depend on aerobic respiration to generate the energy required for growth and replication and transition to dormancy when exposed to hypoxia (oxygen deprivation). Without oxygen available as the terminal respiratory electron acceptor, mycobacteria must rely on anaerobic processes to satisfy the energetic requirements of dormancy. Here we investigate two of these processes in M. smegmatis; anaerobic respiration, where the function of the electron transport chain is maintained by the use of alternative terminal electron acceptors, and fermentation, where glycolytic pathways become the primary source of ATP and NAD(P)+ is regenerated by dispensing of reducing equivalents through fermentative end products.
Previous studies have shown that M. smegmatis produces hydrogen as a fermentative end product during hypoxia and recycles it when electron acceptors become available. Here we show that hypoxic M. smegmatis cultures produce substantially more hydrogen than previously observed, suggesting that hydrogen fermentation plays a major role in hypoxic energy metabolism. We have demonstrated that M. smegmatis uses nitrate as an anaerobic electron acceptor to drive hydrogen oxidation, and that this is mediated by the nitrate reductase NarGHI and the uptake hydrogenase Huc.
We have employed a differential isotope labelling approach to directly demonstrate that glucose is incompletely oxidised during the onset of hypoxia, a key criterion of fermentation, and that this is a result of increased flux through the pentose phosphate pathway (PPP). The PPP generates NADPH, providing reductive power to anabolic pathways such as fatty acid synthesis. This is consistent with existing evidence that mycobacteria, including M. tuberculosis, produce large energy stores in the form of triacylglycerides (TAGs) during hypoxia. We are expanding on these findings by investigating the broader changes to metabolism during the transition to hypoxia and onset of fermentation, by conducting paired proteomic and metabolomics, alongside cryo-EM tomography to assess any changes to cellular morphology. This will provide important insight into the adaptations of environmental and pathogenic mycobacteria to hypoxia.