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Functional characterization and proteomic analysis of lolA in Xanthomonas campestris pv. campestris.

ABSTRACT: BACKGROUND:The gram-negative Xanthomonas campestris pv. campestris is the pathogenic bacterium that causes black rot disease in crucifers. The virulence determinants of this bacterium include extracellular enzymes, exopolysaccharides, and biofilm formation. Here, one transposon mutant of X. campestris pv. campestris strain 17 that affects biofilm formation was isolated, and subsequent analyses led to the identification of the lolA gene, which encodes an outer membrane lipoprotein chaperone. RESULTS:The lolA mutant exhibited significant reductions in bacterial attachment, extracellular enzyme production, virulence, and tolerance in the presence of myriad membrane-perturbing agents. These phenotypic changes of the mutant could be complemented to the wild-type level through the intact lolA gene. Proteomic analysis revealed that 109 proteins were differentially expressed after lolA mutation. These differentially expressed proteins were categorized in various functional groups and were mainly associated with the membrane component, were involved in transport, and contained receptor activity. Through reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis, deletion of lolA was determined to have caused significantly reduced expression of genes that encode the major extracellular enzymes, the biofilm-related proteins, and the virulence-related proteins. The RT-qPCR analysis also indicated that the expression of several genes that encode putative outer membrane lipoproteins and TonB-dependent receptors was reduced after lolA mutation. CONCLUSIONS:This is the first report to define the lolA gene as a virulence factor and to contribute to the functional understanding of, and provide new information concerning, the role of lolA in Xanthomonas. Furthermore, the results of this study provide and extend new insights into the function of lolA in bacteria.

SUBMITTER: Liao CT

PROVIDER: S-EPMC6341742 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Functional characterization and proteomic analysis of lolA in Xanthomonas campestris pv. campestris.

Liao Chao-Tsai CT Chiang Ying-Chuan YC Hsiao Yi-Min YM

BMC microbiology 20190121 1

<h4>Background</h4>The gram-negative Xanthomonas campestris pv. campestris is the pathogenic bacterium that causes black rot disease in crucifers. The virulence determinants of this bacterium include extracellular enzymes, exopolysaccharides, and biofilm formation. Here, one transposon mutant of X. campestris pv. campestris strain 17 that affects biofilm formation was isolated, and subsequent analyses led to the identification of the lolA gene, which encodes an outer membrane lipoprotein chapero ...[more]

PMID: 30665348

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Project description:UnlabelledN-Acetylglucosamine (GlcNAc), the main component of chitin and a major constituent of bacterial peptidoglycan, is present only in trace amounts in plants, in contrast to the huge amount of various sugars that compose the polysaccharides of the plant cell wall. Thus, GlcNAc has not previously been considered a substrate exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, expresses a carbohydrate utilization system devoted to GlcNAc exploitation. In addition to genes involved in GlcNAc catabolism, this system codes for four TonB-dependent outer membrane transporters (TBDTs) and eight glycoside hydrolases. Expression of all these genes is under the control of GlcNAc. In vitro experiments showed that X. campestris pv. campestris exploits chitooligosaccharides, and there is indirect evidence that during the early stationary phase, X. campestris pv. campestris recycles bacterium-derived peptidoglycan/muropeptides. Results obtained also suggest that during plant infection and during growth in cabbage xylem sap, X. campestris pv. campestris encounters and metabolizes plant-derived GlcNAc-containing molecules. Specific TBDTs seem to be preferentially involved in the consumption of all these plant-, fungus- and bacterium-derived GlcNAc-containing molecules. This is the first evidence of GlcNAc consumption during infection by a phytopathogenic bacterium. Interestingly, N-glycans from plant N-glycosylated proteins are proposed to be substrates for glycoside hydrolases belonging to the X. campestris pv. campestris GlcNAc exploitation system. This observation extends the range of sources of GlcNAc metabolized by phytopathogenic bacteria during their life cycle.ImportanceDespite the central role of N-acetylglucosamine (GlcNAc) in nature, there is no evidence that phytopathogenic bacteria metabolize this compound during plant infection. Results obtained here suggest that Xanthomonas campestris pv. campestris, the causal agent of black rot disease on Brassica, encounters and metabolizes GlcNAc in planta and in vitro. Active and specific outer membrane transporters belonging to the TonB-dependent transporters family are proposed to import GlcNAc-containing complex molecules from the host, from the bacterium, and/or from the environment, and bacterial glycoside hydrolases induced by GlcNAc participate in their degradation. Our results extend the range of sources of GlcNAc metabolized by this phytopathogenic bacterium during its life cycle to include chitooligosaccharides that could originate from fungi or insects present in the plant environment, muropeptides leached during peptidoglycan recycling and bacterial lysis, and N-glycans from plant N-glycosylated proteins present in the plant cell wall as well as in xylem sap.

Dataset Information

Functional characterization and proteomic analysis of lolA in Xanthomonas campestris pv. campestris.

Publications

Functional characterization and proteomic analysis of lolA in Xanthomonas campestris pv. campestris.

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