Target DNA stabilizes Mycobacterium tuberculosis DevR/DosR phosphorylation by the full-length oxygen sensors...

Mycobacterium tuberculosis strongly relies on a latency, or nonreplicating persistence, to escape a human host's immune system. The DevR (DosR), DevS (DosS), and DosT proteins are key components of this process. Like the rhizobial FixL oxygen sensor, DevS and DosT are histidine protein kinases with a heme-binding domain. Like the FixJ partner and substrate of FixL, DevR is a classical response regulator of the two-component class. When activated by DevS or DosT during hypoxia in vivo, DevR induces a dormancy regulon of more than 40 genes. To investigate the contributions of DevS, DosT, and target DNA to the phosphorylation of DevR, we developed an in vitro assay in which the full-length, sensing, DevS and DosT proteins were used to phosphorylate DevR with ATP, in the presence of target DNAs that were introduced as oligonucleotides linked to magnetic nanoparticles. We found that the DevR phosphorylations proceeded only for the deoxy states of the sensors. The reaction was strongly inhibited by O₂ , but not CO or NO. The production of phospho-DevR was enhanced sixfold by target consensus DNA or acr-DNA. The phospho-DevR bound tightly to that DNA (K_d ~ 0.8 nm toward acr-DNA), and it was only slightly displaced by a 200-fold excess of unphosphorylated DevR or of a truncated DevR with only a DNA-binding domain. To our knowledge, this represents the first in vitro study of the ligand regulation of DevR phosphorylation by full-length DevS and DosT, and demonstration of a positive effect of DNA on this reaction.