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

首页  ꄲ  发表论文  ꄲ  2018年  ꄲ  Flagellar Basal Body Structural Proteins FlhB, FliM, and FliY Are Required for Flagellar-Associated Protein Expression in Listeria monocytogenes - PubMed

doi: 10.3389/fmicb.2018.00208. eCollection 2018.

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

Changyong Cheng et al. Front Microbiol. .

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Abstract

Listeria monocytogenes is a food-associated bacterium that is responsible for food-related illnesses worldwide. In the L. monocytogenes EGD-e genome, FlhB, FliM, and FliY (encoded by lmo0679, lmo0699, and lmo0700, respectively) are annotated as putative flagella biosynthesis factors, but their functions remain unknown. To explore whether FlhB, FliM, and FliY are involved in Listeria flagella synthesis, we constructed flhB, fliM, fliY, 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 flhB, fliM, or fliY. These impaired phenotypes were fully restored in the complemented strains CΔflhB, CΔfliM, and CΔfliY. The transcriptional levels of flagellar-related genes, including flaA, fliM, fliY, lmo0695, lmo0698, fliI, and fliS, were downregulated markedly in the absence of flhB, fliM, or fliY. Deletion of flhB 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 fliM or fliY. 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 Listeria 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 L. monocytogenes.

Keywords: Listeria monocytogenes; flagella; motility; protein expression; type III secretion system.

Figures

FIGURE 1
FIGURE 1

Growth of L. monocytogenes mutants in BHI medium at 37°C (A) and 30°C (B). Overnight cultures were resuspended in fresh BHI medium and then incubated at 37°C (A) and 30°C (B) for 12 h. Then, the OD600 nm was measured at 1-h intervals. The data are expressed as the mean ± standard deviation (SD) of three independent experiments.

FIGURE 2
FIGURE 2

FlhB, FliM, and FliY contribute to L. monocytogenes motility and flagellar formation. A motility assay (A,B) and TEM (C) were performed by growing the L. monocytogenes wild-type strain EGD-e, the mutants ΔflhB, ΔfliM and ΔfliY, and the complemented strains CΔflhB, CΔfliM, and CΔfliY on soft agar (0.25%) at 30°C or 37°C for 16 h. Scale: 1 μm.

FIGURE 3
FIGURE 3

The flagellar-motility gene cluster in L. monocytogenes EGD-e (A) and the changes of the transcriptional levels of flagellar-related genes in the absence of flhB (B), fliM (C), or fliY (D). Relative quantification of flagellar-related gene mRNA levels in the L. monocytogenes wild-type strain EGD-e, the mutants ΔflhB, ΔfliM, and ΔfliY, and the complemented strains CΔflhB, CΔfliM, and CΔfliY at 30°C. Values are expressed as the mean ± SD. Solid lines indicate a 1.5-fold change in transcription of the genes of interest.

FIGURE 4
FIGURE 4

Changes in the levels of flagellar-related proteins in the absence of flhB (A), fliM (B), or fliY (C). Bacterial overnight cultures of the L. monocytogenes wild-type strain EGD-e, the ΔflhB, ΔfliM, ΔfliY mutants, and the complemented strains CΔflhB, CΔfliM, and CΔfliY were diluted 1:100 into 100 ml of fresh BHI broth and statically grown to stationary phase. Bacterial sediments and culture supernatants were collected to obtain the different cell fractions. Proteins were separated by 12% SDS-PAGE and immunoblotted with α-FlgG, α-MogR, α-FlhF, α-FliM, α-FliY, α-FlaA, α-FliS, or α-GmaR antisera. GAPDH was used as an internal control. The predicted molecular weight of each protein is indicated on the right.

FIGURE 5
FIGURE 5

Model for the roles of FlhB, FliY, and FliM in flagellar formation and bacterial motility. FlhB as an export switch factor is responsible for transporting intracellular proteins to the extracellular, including FlgG and FlaA. Besides its role in the flagellar switch complex, the C-ring is also involved in the export process, FliM and FliY are components of the C ring, their defects may lead to a variety of specific substrate proteins that can not be transported. FliS chaperone binds to filament (FlaA) in the cytoplasm and efficiently transfers FlaA to a sorting platform of the flagellar export apparatus during the flagellar filament assembly. The flhB, fliM, and fliY mutant strains are incapable of synthesizing filament, thus causing the bacteria to lose power and non-motile. FlhF is a GTPase associated with flagellin localization and flagellar transcription is still subject to temperature-dependent regulation of MogR and GmaR strictly, all these three regulators are not disturbed by FlhB, FliM, and FliY.

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