Abstract
Members of the Mycobacterium genus are well known for their energetic flexibility, a trait which they exploit to grow and survive under a wide range of oxygen tensions, including hypoxia. Contributing to this flexibility is their ability to respire using two-terminal respiratory oxidases, a cytochrome bcc-aa3-type cytochrome c oxidase (qcrCAB operon – bcc complex) and a cytochrome bd-type menaquinol oxidase (cydAB operon). The former is typically thought to have a lower affinity for oxygen but a higher energetic efficiency than the latter. This body of work examines the role of terminal oxidases in Mycobacterium smegmatis.
Previously terminal oxidases have been typecast solely based on their affinities for oxygen. Through the creation of bcc complex (Delta-qcrCAB) and cytochrome bd (Delta-cydAB) mutants, we have been able to elucidate an additional role for terminal oxidases in redox balance and proton motive force (PMF) maintenance. We provide evidence for the presence of respiratory backpressure in mycobacteria and argue that it is sufficient to significantly impede proton-pumping complexes. We, therefore, suggest that instead of their affinities for oxygen controlling the response of the terminal oxidases, it is instead their differences in proton-pumping abilities that dictates their role.
Furthermore, we characterise a putative respiratory complex III (QcrB) inhibitor, TB47: a pyrazolo[1,5-a]pyridine-3-carboxamide and use it to understand the transcriptional response of M. smegmatis wild type to bcc-aa3 inhibition. RNA sequencing undertaken on TB47-treated cells showed significant upregulation of genes associated with the Dos regulon, which is involved in the ability of mycobacteria to adapt to low oxygen environments (hypoxia). These data provide the first evidence for a link between terminal oxidases and an oxygen-sensing two-component system. Upregulation of alternative electron donors and acceptors involved in recycling reducing equivalents eludes to a possible mechanism by which inhibiting a terminal oxidase leads to dysregulation of NADH/NAD+ homeostasis. In conclusion, we show that terminal oxidase shut down leads to respiratory dysregulation, which has multiple effects, including a more reduced NADH/NAD+ pool, respiratory backpressure and metabolic redox stress.