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

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Author(s)

    • Kato Akinori
    • Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University
    • Ueda Shuhei
    • Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University
    • Oshima Taku
    • Graduate School of Biological Sciences, Nara Institute of Science and Technology
    • Inukai Yoichi
    • Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University
    • Eguchi Yoko
    • Department of Science and Technology on Food Safety, Faculty of Biology-Oriented Science and Technology, Kindai University
    • Utsumi Ryutaro
    • Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University

Abstract

<p>The WalK/WalR two-component system is essential for cell wall metabolism and thus for cell growth in <i>Bacillus subtilis</i>. Waldiomycin was previously isolated as an antibiotic that targeted WalK, the cognate histidine kinase (HK) of the response regulator, WalR, in <i>B. subtilis</i>. 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 (IC<sub>50</sub>s) 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 <i>B. subtilis</i> 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 <i>B. subtilis</i> cells with the H-box triple mutant alleles of the <i>walK</i> 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 <i>B. subtilis</i> by targeting the H-box region of WalK and other HKs.</p>

Journal

  • The Journal of General and Applied Microbiology

    The Journal of General and Applied Microbiology 63(4), 212-221, 2017

    Applied Microbiology, Molecular and Cellular Biosciences Research Foundation

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