| In pathogenic bacteria, sensory systems composed of kinases and phosphatases promote reversible protein phosphorylation in response to changes in environmental stimuli and regulate highly diverse processes ranging from cell wall biogenesis to virulence. With this observation, we hypothesized that a similar kinase-phosphatase system, essential for growth and virulence, exists in B. anthracis. We therefore examined the genome of B. anthracis for candidate systems that regulate reversible phosphorylation, and identified a Serine/Threonine (Ser/Thr) kinase-phosphatase pair for further study.;B. anthracis STPK101, a null mutant lacking a putative phosphatase (BA-Stp1) and a putative kinase (BA-Stk1), was impaired in its ability to survive within macrophages, and this correlated with an observed reduction in virulence in a mouse model of pulmonary anthrax. Biochemical analyses confirmed that BA-Stp1 is a PP2C phosphatase and dephosphorylates phosphoserine and phosphothreonine residues. Moreover, mutagenesis of any one of four conserved aspartic acid residues, predicted to coordinate Mn 2+ or Mg2 ions, abolished phosphatase activity of BA-Stp1. Analyses of the regulatory interactions between BA-Stp1 and BA-Stk1 demonstrated that BA-Stp1 dephosphorylates autophosphorylated BA-Stk1. Importantly; this dephosphorylation influenced the enzymatic function of BA-Stk1. Using a combination of mass-spectrometry and mutagenesis approaches, three phosphorylated residues T165, S173, and S214 in BA-Stk1 were identified as putative targets of BA-Stp1. Further analysis demonstrated that phosphorylation of T165 and S173 was important for regulating BAStk1 autophosphorylation, while phosphorylation S214 was important for complete ATP-hydrolysis, autophosphorylation, and substrate phosphorylation. These findings provide a mechanistic insight into a previously undescribed Stp/Stk pair in B. anthracis. |
