In this work, we provide insight into the physiological role of hydrogen scavenging by observing the effect of deleting Hyd2 throughout BAY 73-4506 755037-03-7 exponential growth, upon entry into stationary phase, and during long-term survival. Using a combinatorial approach, we show that hydrogen scavenging is required for the efficient metabolism of certain carbon sources and infer that atmospheric H2 is a source of reductant for mycobacterial metabolism. There were particularly extensive changes in central intermediary metabolism. MSMEG_3706, a bifunctional enzyme that catalyses key reactions in the glyoxylate shunt and methylcitrate cycle , was significantly downregulated; this enzyme is usually upregulated during slow growth of M. smegmatis and M. bovis BCG. In compensation, several predicted glycolytic and tricarboxylic acid cycle enzymes were upregulated, i.e. pyruvate dehydrogenase, isocitrate dehydrogenase, ketoglutarate-ferredoxin oxidoreductase, and lactate 2-monooxygenase. All of these upregulated enzymes catalyse oxidative decarboxylation reactions that yield reduced cofactors concomitant with the loss of CO2. To compensate for downregulation of the methylisocitrate cycle, the strain also increased expression of the enzymes of the methylmalonyl-CoA pathway that converts propionate to succinate in an ATP-dependent manner. These changes suggest that M. smegmatis compensates for loss of hydrogen oxidation by re-routing carbon flux from anabolic to catabolic pathways. In amino acid metabolism, the operon encoding the determinants of the urea cycle was upregulated. These ATP-consuming enzymes convert the carbon components of amino acids into the tricarboxylic acid intermediate fumarate, while removing excess nitrogen as urea. Transcripts encoding the predicted NAD-dependent glutamate synthase MSMEG_64586459 were also significantly more abundant. We also observed that the putative operons encoding six ABC transporters were upregulated, including those predicted to transport trehalose, methionine, branched-chain amino acids, and alkane sulfonates. Some of these compounds may be scavenged from the cell envelope; it has previously been observed that trehalose is produced by mycobacteria as a byproduct of mycolic acid cell envelope biosynthesis, and the recycling of this compound by a homologous ABC transporter is essential for virulence in Mycobacterium tuberculosis. In conclusion, it is clear that hydrogen scavenging enhances the growth and survival of Mycobacterium smegmatis under a range of conditions. Single and double markerless deletions of the hydrogen-scavenging enzymes Hyd1 or Hyd2 grew to lower yields than the wild-type strain. Mutant strains were defective when cultured on minimal medium at low carbon concentrations, acidic pH, and, most significantly, on short-chain fatty acids. Reduced growth yields of the Dhyd2 strain have also been observed during growth on rich media, e.g. LBT . All defects were observed when strains were grown in flasks aerated with ambient air, i.e. when H2 is available at trace concentrations.