Characterization of H-box region mutants of WalK inert to the action of waldiomycin in <i>Bacillus subtilis</i>
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- Kato Akinori
- Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University
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- Ueda Shuhei
- Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University
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- Oshima Taku
- Graduate School of Biological Sciences, Nara Institute of Science and Technology
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- Inukai Yoichi
- Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University
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- Okajima Toshihide
- Institute of Scientific and Industrial Research, Osaka University
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- Igarashi Masayuki
- Institute of Microbial Chemistry, Tokyo
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- Eguchi Yoko
- Department of Science and Technology on Food Safety, Faculty of Biology-Oriented Science and Technology, Kindai University
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- Utsumi Ryutaro
- Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University
Bibliographic Information
- Other Title
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- Characterization of H-box region mutants of WalK inert to the action of waldiomycin in Bacillus subtilis
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Abstract
<p>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.</p>
Journal
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- The Journal of General and Applied Microbiology
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The Journal of General and Applied Microbiology 63 (4), 212-221, 2017
Applied Microbiology, Molecular and Cellular Biosciences Research Foundation