Characterization of H-box region mutants of WalK inert to the action of waldiomycin in Bacillus subtilis

• Published in: Journal of general and applied microbiology Vol. 63; no. 4; pp. 212 - 221
• Main Authors: Kato, Akinori, Ueda, Shuhei, Oshima, Taku, Inukai, Yoichi, Okajima, Toshihide, Igarashi, Masayuki, Eguchi, Yoko, Utsumi, Ryutaro
• Format: Journal Article
• Language: English
• Published: Japan Applied Microbiology, Molecular and Cellular Biosciences Research Foundation 01-01-2017; Japan Science and Technology Agency
• Subjects: Anti-Bacterial Agents - pharmacology; Antibiotics; Bacillus subtilis (B. subtilis); Bacillus subtilis - drug effects; Bacillus subtilis - enzymology; Bacillus subtilis - genetics; Bacteria; Bacterial Proteins - drug effects; Bacterial Proteins - genetics; Cell growth; Cell morphology; Cell Wall - metabolism; Cell walls; Conserved sequence; Cytology; Drug Resistance, Bacterial - genetics; Gram-positive bacteria; H-box region; Histidine; Histidine kinase; Histidine kinase (HK); Histidine Kinase - drug effects; Histidine Kinase - genetics; inhibitor; Inhibitory Concentration 50; Kinases; Lymphocytes B; Metabolism; Minimum inhibitory concentration; Mutants; Mutation; Protein Kinases - drug effects; Protein Kinases - genetics; Protein Kinases - metabolism; Quinones - pharmacology; Transcription; two-component system (TCS); Waldiomycin; WalK/WalR; Histidine kinase (HK); H-box region; Antibiotics; inhibitor; Waldiomycin; WalK/WalR; Bacillus subtilis (B. subtilis); two-component system (TCS)
• ISSN: 0022-1260; 1349-8037
• DOI: 10.2323/jgam.2016.10.007

The WalK/WalR two-component system is essential for cell wall metabolism and thus for cell growth in Bacillus subtilis. Waldiomycin was previously isolated as an antibiotic that targeted WalK, the cognate histidine kinase (HK) of the response regulator, WalR, in B. subtilis. To gain further insights into the action of waldiomycin on WalK and narrow down its site of action, mutations were introduced in the H-box region, a well-conserved motif of the bacterial HKs of WalK. The half-maximal inhibitory concentrations (IC50s) of waldiomycin against purified WalK protein with triple substitutions in the H-box region, R377M/R378M/S385A and R377M/R378M/R389M, were 26.4 and 55.1 times higher than that of the wild-type protein, respectively, indicating that these residues of WalK are crucial for the inhibitory effect of waldiomycin on its kinase activity. Surprisingly, this antibiotic severely affected cell growth in a minimum inhibitory concentration (MIC) assay, but not transcription of WalR-regulated genes or cell morphology in B. subtilis strains that harbored the H-box triple substitutions on the bacterial chromosome. We hypothesized that waldiomycin targets other HKs as well, which may, in turn, sensitize B. subtilis cells with the H-box triple mutant alleles of the walK gene to waldiomycin. Waldiomycin inhibited other HKs such as PhoR and ResE, and, to a lesser extent, CitS, whose H-box region is less conserved. These results suggest that waldiomycin perturbs multiple cellular processes in B. subtilis by targeting the H-box region of WalK and other HKs.