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.

Main Figure. Overview and highlight of biomolecular protein-DNA interactions between the PrrA^Mtb WT protein and the prrA^Mtb, dosR^Mtb, and cydA^Mtb promoters established by surface plasmon resonance. The PrrA^Mtb WT protein rapidly and strongly binds the dosR^Mtb and prrA^Mtb promoters with binding affinity values in the low nanomolar range.