Induction of the cydAB Operon Encoding the bd Quinol Oxidase Under Respiration-Inhibitory Conditions by the Major cAMP Receptor Protein MSMEG_6189 in Mycobacterium smegmatis

Abstract

The respiratory electron transport chain (ETC) of Mycobacterium smegmatis is terminated with two terminal oxidases, the aa3 cytochrome c oxidase and the cytochrome bd quinol oxidase. The bd quinol oxidase with a higher binding affinity for O2 than the aa3 oxidase is known to play an important role in aerobic respiration under oxygen-limiting conditions. Using relevant crp1 (MSMEG_6189) and crp2 (MSMEG_0539) mutant strains of M. smegmatis, we demonstrated that Crp1 plays a predominant role in induction of the cydAB operon under ETC-inhibitory conditions. Two Crp-binding sequences were identified upstream of the cydA gene, both of which are necessary for induction of cydAB expression under ETC-inhibitory conditions. The intracellular level of cAMP in M. smegmatis was found to be increased under ETC-inhibitory conditions. The crp2 gene was found to be negatively regulated by Crp1 and Crp2, which appears to lead to significantly low cellular abundance of Crp2 relative to Crp1 in M. smegmatis. Our RNA sequencing analyses suggest that in addition to the SigF partner switching system, Crp1 is involved in induction of gene expression in M. smegmatis exposed to ETC-inhibitory conditions.

Cyclic AMP is a critical mediator of intrinsic drug resistance and fatty acid metabolism in M. tuberculosis

“…Isolation of spontaneous resistant mutants to a series of novel inhibitors of the QcrB subunit of the cytochrome bc1-aa3 oxidase repeatedly identify mutations in rv1339 (Chandrasekera et al, 2017;O'Malley et al, 2018;Shelton et al, 2021). How loss of rv1339 and elevated cAMP promotes resistance to QcrB inhibitors remains to be defined, but in principle could be achieved by elevated cAMP levels increasing electron transport chain activity, for example by increasing expression of the alternative terminal oxidase cytochrome bd (Ko and Oh, 2020). Collectively, ours and published results lead to the following working model (Figure 5H).…”

“…Potential downstream cAMP effector proteins include transcription factors like CRP and Cmr, transporters, phospholipases, and others , highlighting the potentially pleiotropic consequences of modulating cAMP levels. Reduced activity of cAMP-responsive proteins, including Mt-Pat, may increase fatty acid uptake and catabolism (Nazarova et al, 2019) and oxidative TCA metabolism while simultaneously reducing electron transport chain activity (Ko and Oh, 2020). This derangement may ultimately lead to redox imbalance, reduced growth, and increased sensitivity to antibiotics through yet to be defined mechanisms.…”

Biosensor-integrated transposon mutagenesis reveals rv0158 as a coordinator of redox homeostasis in Mycobacterium tuberculosis

“…Also, the lack of Rv0158 may induce a physiological state in Mtb that is more tolerant to toxicity caused by fatty acids such as propionate. For example, cAMP homeostasis is associated with modulating redox balance, respiration, fatty acid uptake, and fatty acid toxicity in mycobacteria ( Ko and Oh, 2020 ; Nambi et al, 2013 ; Nazarova et al, 2019 ; Wong et al, 2023 ). Moreover, Myc obacterium bovis BCG elevates cAMP levels in response to propionate, and phosphodiesterases such as Rv0805 regulate cAMP homeostasis to modulate growth on propionate ( McDowell et al, 2023 ).…”

Deletion of rifampicin-inactivating mono-ADP-ribosyl transferase gene of Mycobacterium smegmatis globally altered gene expression profile that favoured increase in ROS levels and thereby antibiotic resister generation

“…In this regard, it could be speculated that the rifampicin surviving arr -KO cells might have entered anaerobiosis due to the increased expression of cydA (MSMEG_3233; see Section ETC genes arr -KO p < 0.05 in Supplementary Dataset S1). However, the upregulation of cydAB during hypoxia requires the activation of cAMP receptor protein ( cpr1 , MSMEG_6189) ( Aung et al., 2014 ; Ko and Oh, 2020 ). On the contrary, the expression cpr1 (MSMEG_6189) in the arr -KO MLP population was low (log2 -0.08) in comparison to its expression in the WT (Supplementary Dataset S6).…”