Respiratory backpressure determines normoxic distribution of terminal oxygen reduction pathways

Cytochrome bd (CydAB), a high-affinity oxidase that performs terminal oxygen reduction in cellular respiration, is used by various bacteria to grow in hypoxic environments, form nonreplicating persister cells, or resist antimicrobial killing. It is thought that oxygen concentration solely determines the usage of CydAB in bacteria, with the tradeoff being that CydAB-containing respiratory chains generate less ATP than those containing low-affinity proton-pumping cytochrome oxidases, as CydAB does not pump protons when generating a proton motive force (PMF). However, CydAB has roles in acid, nitrosative and oxidative stress that suggest the existence of oxygen-independent CydAB usage. Here, we show that an obligate aerobe, the soil actinomycete Mycobacterium smegmatis, uses CydAB to normoxically maintain a homeostatic balance of the PMF, involving an oxygen-independent interaction with the classical hypoxia regulator (DosR). We show that overpotentials of the PMF apply feedback inhibition on the proton-pumping, low-affinity, cytochrome c oxidase supercomplex bcc-aa3, and that CydAB can immediately escape this inhibition and promote a PMF-responsive programme. Overall design: RNA Sequencing on Mycobacterium smegmatis wild-type or cytochrome bd deletion mutant, either treated with DMSO or treated with 50 nM TB47. Sequencing was performed across two lanes.