Functional characterization of WalRK: A two-component signal transduction system from Bacillus anthracis.
Two-component signal transduction systems (TCS), consisting of a sensor histidine protein kinase and its cognate response regulator, are an important mode of environmental sensing in bacteria. Additionally, they have been found to regulate virulence determinants in several pathogens. Bacillus anthracis, the causative agent of anthrax and a bioterrorism agent, harbours 41 pairs of TCS. However, their role in its pathogenicity has remained largely unexplored. Here, we show that WalRK of B. anthracis forms a functional TCS which exhibits some species-specific functions. Biochemical studies showed that domain variants of WalK, the histidine kinase, exhibit classical properties of autophosphorylation and phosphotransfer to its cognate response regulator WalR. Interestingly, these domain variants also show phosphatase activity towards phosphorylated WalR, thereby making WalK a bifunctional histidine kinase/phosphatase. An in silico regulon determination approach, using a consensus binding sequence from Bacillus subtilis, provided a list of 30 genes that could form a putative WalR regulon in B. anthracis. Further, electrophoretic mobility shift assay was used to show direct binding of purified WalR to the upstream regions of three putative regulon candidates, an S-layer protein EA1, a cell division ABC transporter FtsE and a sporulation histidine kinase KinB3. Our work lends insight into the species-specific functions and mode of action of B. anthracis WalRK.
Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
Alisha
A
Dhiman
Computational and Structural Biology Laboratory, Division of Biotechnology, Netaji Subhas Institute of Technology, Dwarka, New Delhi 110078, India.
Sonika
S
Bhatnagar
Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
Parul
P
Kulshreshtha
Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
Rakesh
R
Bhatnagar
AP, alkaline phosphatase; AcP, acetyl phosphate; Bacillus anthracis; ELISA, enzyme linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; HK, histidine kinase; HRP, horse radish peroxidase; Histidine kinase; RR, response regulator; Regulon; Response regulator; TCS, two-component signal transduction systems; Two-component signal transduction; VicRK; YycFG