AbstractThe influence of carbon metabolism on oxidative phosphorylation is poorly understood in mycobacteria.M.tuberculosisexpresses two respiratory terminal oxidases, the cytochromebc1:aa3and the cytochromebdoxidase, which are jointly required for oxidative phosphorylation and mycobacterial viability. The essentiality of the cytochromebc1:aa3for optimum growth is illustrated by its vulnerability to chemical inhibition by the clinical drug candidate Q203 and several other chemical series. The cytochromebdoxidase is not strictly essential for growth but is required to maintain bioenergetics when the function of the cytochromebc1:aa3is compromised. In this study, we observed that the potency of drugs targeting the cytochromebc1:aa3is influenced by carbon metabolism. The efficacy of Q203 and related derivatives was alleviated by glycerol supplementation. The negative effect of glycerol supplementation on Q203 potency correlated with an upregulation of the cytochromebdoxidase-encodingcydABDCoperon. Upon deletion ofcydAB, the detrimental effect of glycerol on the potency of Q203 was abrogated. The same phenomenon was also observed in recent clinical isolates, but to a lesser extent compared to the laboratory-adapted strain H37Rv. This study reinforces the importance of optimizingin vitroculture conditions for drug evaluation in mycobacteria, a factor which appeared to be particularly essential for drugs targeting the cytochromebc1:aa3terminal oxidase.