M. tuberculosis PrrA binds the dosR promoter and regulates mycobacterial adaptation to hypoxia.

The PrrAB two-component system (TCS) is essential for Mycobacterium tuberculosis viability. Previously, it was demonstrated that PrrA binds DNA in the absence of PrrB-mediated transphosphorylation and that non-cognate serine/threonine-kinases phosphorylate PrrA threonine-6 (T6). Therefore, we investigated the differential binding affinity and regulatory properties of the M. tuberculosis-derived wild-type PrrA, PrrA phosphomimetic (D58E, T6E), and PrrA phosphoablative (D58A, T6A) proteins with the prrA Mtb , dosR Mtb , and cydA Mtb genes. While we hypothesized greater DNA binding affinity and more pronounced regulation by PrrA phosphomimetic variants, recombinant, wild-type PrrA Mtb bound DNA with greatest affinity. Collectively, wild-type PrrA Mtb recombinant protein displayed the highest binding affinity to the dosR Mtb promoter (K D 3.46 ± 2.09 nM), followed by the prrA Mtb promoter (K D 9.00 ± 2.66 nM). To establish PrrA Mtb regulatory activity, we constructed M. smegmatis ΔprrAB Msmeg ::prrA Mtb strains with each of the PrrA Mtb variants and extrachromosomal prrA Mtb , dosR Mtb , and cydA Mtb promoter-mCherry reporter fusions. Our findings showed that PrrA Mtb is autoregulatory and induces dosR Mtb expression only during in vitro, hypoxic growth. Combined expression of prrAB Mtb in M. smegmatis ΔprrAB significantly induced cydA Mtb promoter-mCherry expression. Our studies advanced the understanding of PrrA function and PrrAB phosphorylation-mediated regulatory mechanisms and control of mycobacterial dosR and cydA hypoxic and low-oxygen responsive genes.