Flagellar Basal Body Structural Proteins FlhB, FliM, and FliY Are Required for Flagellar-Associated Protein Expression in Listeria monocytogenes

Abstract

<i>Listeria monocytogenes</i> is a food-associated bacterium that is responsible for food-related illnesses worldwide. In the <i>L. monocytogenes</i> EGD-e genome, FlhB, FliM, and FliY <i>(encoded</i> by <i>lmo0679, lmo0699</i>, and <i>lmo0700</i>, respectively) are annotated as putative flagella biosynthesis factors, but their functions remain unknown. To explore whether FlhB, FliM, and FliY are involved in <i>Listeria</i> flagella synthesis, we constructed <i>flhB, fliM, fliY</i>, and other flagellar-related gene deletion mutants using a homologous recombination strategy. Then, we analyzed the motility, flagella synthesis, and protein expression of these mutant strains. Motility and flagella synthesis were completely abolished in the absence of <i>flhB, fliM</i>, or <i>fliY</i>. These impaired phenotypes were fully restored in the complemented strains CΔ<i>flhB</i>, CΔ<i>fliM</i>, and CΔ<i>fliY</i>. The transcriptional levels of flagellar-related genes, including <i>flaA, fliM, fliY, lmo0695, lmo0698, fliI</i>, and <i>fliS</i>, were downregulated markedly in the absence of <i>flhB, fliM</i>, or <i>fliY</i>. Deletion of <i>flhB</i> resulted in the complete abolishment of FlaA expression, while it decreased FliM and FliY expression. The expression of FlaA was abolished completely in the absence of <i>fliM</i> or <i>fliY</i>. No significant changes were found in the expression of FlhF and two flagella synthesis regulatory factors, MogR and GmaR. We demonstrate for the first time that FlhB, FliM, and FliY not only mediate <i>Listeria</i> motility, but also are involved in regulating flagella synthesis. This study provides novel insights that increase our understanding of the roles played by FlhB, FliM, and FliY in the flagellar type III secretion system in <i>L. monocytogenes</i>.