DevS/DosS sensor is bifunctional and its phosphatase activity precludes aerobic DevR/DosR regulon expression in Mycobacterium tuberculosis

Abstract

Two-component systems, comprising histidine kinases and response regulators, empower bacteria to sense and adapt to diverse environmental stresses. Some histidine kinases are bifunctional; their phosphorylation (kinase) and dephosphorylation (phosphatase) activities toward their cognate response regulators permit the rapid reversal of genetic responses to an environmental stimulus. DevR-DevS/DosR-DosS is one of the bestcharacterized two-component systems of Mycobacterium tuberculosis. The kinase function of DevS is activated by gaseous stress signals, including hypoxia, resulting in the induction of~48-genes DevR dormancy regulon. Regulon expression is tightly controlled and lack of expression in aerobic Mtb cultures is ascribed to the absence of phosphorylated DevR. Here we show that DevS is a bifunctional sensor and possesses a robust phosphatase activity toward DevR. We used site-specific mutagenesis to generate substitutions in conserved residues in the dimerization and histidine phosphotransfer domain of DevS and determined their role in kinase/phosphatase functions. In vitro and in vivo experiments, including a novel in vivo phosphatase assay, collectively establish that these conserved residues are critical for regulating kinase/phosphatase functions. Our findings establish DevS phosphatase function as an effective control mechanism to block aerobic expression of the DevR dormancy regulon. Asp-396 is essential for both kinase and phosphatase functions, whereas Gln-400 is critical for phosphatase function. The positive and negative functions perform opposing roles in DevS: the kinase function triggers regulon induction under hypoxia, whereas its phosphatase function prevents expression under aerobic conditions. A finely tuned balance in these opposing activities calibrates the dormancy regulon response output.

Target DNA stabilizes Mycobacterium tuberculosis DevR/DosR phosphorylation by the full‐length oxygen sensors DevS/DosS and DosT

“…DevS has paradoxically been proposed to serve, not only as a DevR kinase but also as a phospho‐DevR phosphatase . In the DevR‐DevS‐DosT system, however, there is no obvious need to respond rapidly to fast‐changing environmental conditions, as occurs in chemotaxis, or to reset the system enzymatically, as occurs after more stable phosphorylations (e.g., serine or threonine).…”

“…Normally, neither DKO nor wild‐type strains express genes from DevR‐regulated promoters in aerobic conditions; however, if Mtb is grown in media that force it to use acetate as a carbon source, certain DevR‐induced genes appear to be expressed in the DKO but not in the wild‐type strains. In media with adequate glucose as a carbon source, there was no gene expression attributable to an aerobic accumulation of phospho‐DevR . Kaur et al .…”

“…Compared to the full-length deoxy-DevS that we have used, the DevS 378-578 was an essentially inactive kinase (compare our Figs 1 and 2 to fig. 3e of Kaur et al [34]).…”

“…In vivo, such a dissociation and hydrolysis of phospho-DevR should correspond to a clearing of the DevR regulon upon a return to oxic conditions, and this should be more than fast enough to account for an Mtb response to environmental changes that takes place over hours to days. DevS has paradoxically been proposed to serve, not only as a DevR kinase but also as a phospho-DevR phosphatase [34]. In the DevR-DevS-DosT system, however, there is no obvious need to respond rapidly to fast-changing environmental conditions, as occurs in chemotaxis, or to reset the system enzymatically, as occurs after more stable phosphorylations (e.g., serine or threonine).…”

A temporal proteome dynamics study reveals the molecular basis of induced phenotypic resistance in Mycobacterium smegmatis at sub-lethal rifampicin concentrations

“…DevR-DevS/DosR-DosS is one of the best characterised two component systems of M. tuberculosis and responds rapidly to oxidative stress signals, including hypoxia and nitric oxide495051, inducing the DevR dormancy programme in mycobacteria by regulating the expression of an ~48 gene regulon4952. A recent proteomic study on M. tuberculosis Beijing B0/W148 exposed to high doses of rifampicin revealed the induction of DosR regulon proteins upon antibiotic exposure9.…”