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

The Journal of General and Applied Microbiology

Ryutaro Utsumi¹  Shuhei Ueda¹  Yoichi Inukai¹  Akinori Kato¹  Yoko Eguchi²  Taku Oshima³  Masayuki Igarashi⁴  Toshihide Okajima⁵ 

[1] Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University;Department of Science and Technology on Food Safety, Faculty of Biology-Oriented Science and Technology, Kindai University;Graduate School of Biological Sciences, Nara Institute of Science and Technology;Institute of Microbial Chemistry, Tokyo;Institute of Scientific and Industrial Research, Osaka University

关键词: Antibiotics;    Bacillus subtilis (B. subtilis);    H-box region;    Histidine kinase (HK);    inhibitor;    two-component system (TCS);    Waldiomycin;    WalK/WalR;   

DOI  :  10.2323/jgam.2016.10.007

学科分类：微生物学和免疫学

来源: Applied Microbiology, Molecular and Cellulrar Biosciences Research Foundation

【 摘 要 】

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.

【 授权许可】

Unknown