Cross talk between DevS sensor kinase homologue, Rv2027c, and DevR response regulator of Mycobacterium tuberculosis

Abstract

Rv2027c is a putative orphan histidine sensor kinase that bears strong homology to DevS of the hypoxia-responsive DevR-DevS two-component system in M. tuberculosis. The cytosolic C-terminal domain of Rv2027c protein (Rv2027c 194 ) was overexpressed in E. coli and biochemically characterized. Rv2027c 194 underwent autophosphorylation at a conserved His 392 residue and engaged in phosphotransfer with DevR response regulator. The rates of autophosphorylation and the stabilities of the phosphorylated species were broadly similar in Rv2027c and DevS. However, unlike DevS, Rv2027c utilized Ca 2þ as an alternative divalent ion during autophosphorylation. In contrast to DevS which completed phosphotransfer to DevR in 5-10 min, phosphotransfer from Rv2027c$P was only partial at 30 min. Unlike devS transcription that was hypoxia-responsive, Rv2027c transcript levels were not upregulated from basal levels during hypoxia. The differential regulation of devS and Rv2027c genes, the ability of Rv2027c to utilize Ca 2þ as a divalent cation in autophosphorylation at physiological concentrations and to engage in phosphotransfer with DevR suggests that the DevR regulon could be modulated by more than one environmental cue relayed through DevS and Rv2027c.

High-Throughput Microplate Phosphorylation Assays Based on DevR-DevS/Rv2027c 2-Component Signal Transduction Pathway to Screen for Novel Antitubercular Compounds

“…10,11,17 There is an inherent difficulty in using a coupled assay in a filter-based format owing to an inability to discriminate between the phosphorylated species of the histidine kinase and response regulator. Inhibitors of 2-component systems that inhibited the activity of AlgR1 and AlgR2 proteins regulating alginate biosynthesis in Pseudomonas aeruginosa were discovered using an algD-xylE reporter assay.…”

“…Thereafter, there was a time-dependent loss of net proteinbound radiolabeled phosphate, and dephosphorylation of both proteins was nearly complete within 10 min. 10,11 Moreover, as the concentration of DevR was increased in the phosphotransfer reaction, there was a proportionate increase in the extent of dephosphorylation of DevS~P within a 25-min period (Fig. 4).…”

Adaptation to Environmental Stimuli within the Host: Two-Component Signal Transduction Systems of Mycobacterium tuberculosis

“…Biochemical and genetic studies indicate that DosRDosS-DosT constitutes a functional TCSS. DosS and DosT autophosphorylate at conserved histidine residues (DosS at His-395 and DosT at His-392), and both proteins phosphorelay to Asp-54 of DosR (Tables 2 and 3) (125,(130)(131)(132).…”

“…While the differential regulation of dosR-dosS and genes from the DosR regulon is dependent on dosR (15,138), dosS is dispensable for this regulation (138). This observation led to the discovery of the second DosR kinase, DosT, which is encoded outside the DosR regulon (125,131). dosT appears to be constitutively expressed (60,125,131), and mutational analysis has suggested that this SK may mediate initial adaptation events to hypoxia (69).…”

“…This observation led to the discovery of the second DosR kinase, DosT, which is encoded outside the DosR regulon (125,131). dosT appears to be constitutively expressed (60,125,131), and mutational analysis has suggested that this SK may mediate initial adaptation events to hypoxia (69). Consistent with a role for DosT as a second DosR kinase, M. tuberculosis ⌬dosS ⌬dosT double mutants fail to increase the expression of DosR-regulated determinants during hypoxia (125).…”