Functional characterization of WalRK: A two-component signal transduction system from Bacillus anthracis

Abstract

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.

Keywords: 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.

Graphical abstract

Fig. 1

Domain organization of WalK & WalR of B. anthracis (SMART database). (A) WalK, histidine kinase, has two transmembrane domains (TM), a HAMP (a domain found in Histidine kinases, Adenylyl cyclases, Methyl binding proteins, Phosphatases) domain, two PAS (a domain common to Per-Arnt-Sim proteins) domains, a DHp (Dimerization and Histidine phosphotransfer, housing conserved histidine) domain and a CA (Catalytic and ATP-binding) domain. (B) WalR, response regulator, has a receiver (housing conserved aspartate) domain and an effector (DNA binding) domain.

Fig. 2

In vivo expression of walR and walK in B. anthracis at different growth stages – O.D._600nm ∼0.3 (early exponential phase), 0.6 (mid exponential phase), 0.9 (late exponential phase), 1.2 (onset of stationary phase). (A) qRT-PCR for walR and walK transcripts from B. anthracis total RNA. Transcripts for both the genes could be detected at all the tested growth stages. Normalized Ct values are plotted against different growth stages, using GraphPad Prism 5 software. Mean with SEM values, from two independent experiments carried out in triplicate are shown. (B) Immunoblotting for endogenous WalR protein expression in B. anthracis. WalR could be detected in B. anthracis lysates from all the tested growth stages using anti-WalR antisera. A single band could be observed at the same position as recombinant WalR monomer (+). Equal protein was loaded for all the samples as shown in the coomassie stained SDS–PAGE gel. No band could be detected in any of the B. anthracis fractions with anti-WalKc antisera. M – Molecular weight standards in kDa.

Fig. 3

Autophosphorylation of WalK and its variants, with its intensity analysis (ImageJ software). Autophosphorylation was observed within 5 min of γ-³²P-ATP addition and increased with time for each variant as shown. (A) Autophosphorylation of WalKc. (B) Autophosphorylation of WalKc(PH) with WalKc as a positive control. (C) Autophosphorylation of WalKc(H) with WalKc and WalKc(PH) as the positive controls. The reaction mixtures were analyzed by a 10% SDS–PAGE, followed by autoradiography. For each autoradiogram, intensity units (%) for phosphorylation are plotted against time in minutes.

Fig. 4

Phosphotransfer from WalK variants to WalR, with its intensity analysis (ImageJ software). Phosphotransfer was observed within 5 min of γ-³²P-ATP addition and increased with time as shown. (A) WalKc to WalR phosphotransfer. (B) WalKc(PH) to WalR phosphotransfer. (C) WalKc(H) to WalR phosphotransfer. The reaction mixtures were analyzed by a 10% SDS–PAGE, followed by autoradiography. For each autoradiogram, intensity units (%) for phosphotransfer are plotted against time in minutes.

Fig. 5

Phosphorylation of WalR by acetyl phosphate (AcP). (A) WalR could be phosphorylated with AcP which increased with time, reaching a maximum of 20–25% only. The phosphorylation of WalR was analyzed by 12% polyacrylamide native gel electrophoresis followed by coomassie staining and intensity analysis. Intensity units (%) of WalR phosphorylation are plotted against time in minutes. B. Acid/base treatment of WalR∼P. The stability of WalR∼P to acid/base treatment was tested in the presence of 0.5 N HCl and 0.5 N NaOH. The products were analyzed by 12% polyacrylamide native gel electrophoresis and coomassie staining. The phosphorylated form was base labile and acid stable as shown, which is characteristic of phospho-aspartate bonds in response regulator.

Fig. 6

B. anthracis WalK DHp domain (SMART database) with associated E/DxxT/N motif (Underlined). Residues highlighted in red form helix 1 of the DHp domain, which houses the conserved histidine (*) and interacts with the response regulator. Residues of helix 1 are marked on the basis of homology of WalK_Ban DHp with EnvZ_E.coli DHp domain which is also a HisKA domain (PDB database). The residues are numbered according to WalK_Ban amino acid sequence.

Fig. 7

Phosphatase activity of WalKc variants. WalR was phosphorylated with AcP for 60 min, following which the reaction was stopped with 40 mM EDTA. WalKc(H)/WalKc(PH) (5 μg each) was then added to assay for phosphatase activity at different time points. The reaction mixtures were analyzed on a 12% polyacrylamide native gel followed by coomassie staining and intensity analysis. (A) Both WalKc(H) and WalKc(PH) could mediate dephosphorylation of WalR∼P. (B) Comparative analysis of phosphatase activity of WalKc(H) and WalKc(PH). (C) WalR∼P levels remaining after 10 min of incubation with WalKc(H)/WalKc(PH) (5 μg each) in comparison with control. Intensity units (%) are plotted for both the variants.

Fig. 8

DNA binding ability of WalR. EMSA was used to check for WalR binding to DNA by incubating increasing amounts of purified WalR (2, 3 and 4 μg) with eag, ftsE and kinB3 probes containing the WalR binding site sequence. IS probe, lacking the WalR binding site sequence, was the negative control. Binding reactions were performed at room temperature and the reaction products were run on 8% polyacrylamide native gels in 0.5X TBE at 4 °C. Bands were detected after transfer to a positively charged nylon membrane using a chemiluminescent nucleic acid detection module. WalR could bind to eag, ftsE and kinB3 probes, but not IS probe as shown.