The clpX gene plays an important role in bacterial attachment, stress tolerance, and virulence in Xanthomonas campestris pv. campestris
Xanthomonas campestris pv. campestris is a bacterial pathogen and the causal agent of black rot in crucifers. In this study, a clpX mutant was obtained by EZ-Tn5 transposon mutagenesis of the X. campestris pv. campestris. The clpX gene was annotated to encode ClpX, the ATP-binding subunit of ATP-dependent Clp protease. The clpX mutant exhibited reduced bacterial attachment, extracellular enzyme production and virulence. Mutation of clpX also resulted in increased sensitivity to a myriad of stresses, including heat, puromycin, and sodium dodecyl sulfate. These altered phenotypes of the clpX mutant could be restored to wild-type levels by in trans expression of the intact clpX gene. Proteomic analysis revealed that the expression of 211 proteins differed not less than twofold between the wild-type and mutant strains. Cluster of orthologous group analysis revealed that these proteins are mainly involved in metabolism, cell wall biogenesis, chaperone, and signal transduction. The reverse transcription quantitative real-time polymerase chain reaction analysis demonstrated that the expression of genes encoding attachment-related proteins, extracellular enzymes, and virulence-associated proteins was reduced after clpX mutation. The results in this study contribute to the functional understanding of the role of clpX in Xanthomonas for the first time, and extend new insights into the function of clpX in bacteria.
KeywordsBiofilm formation Environmental adaptation Pathogenicity
This work was supported by Ministry of Science and Technology of Taiwan (Grants Nos. MOST104-2313-B-166-001-MY3 and MOST107-2313-B-166-001-MY3) to YMH, and Central Taiwan University of Science and Technology (grant No. CTU105-P-15) to HHL.
- Chen YY, Wu CH, Lin JW, Weng SF, Tseng YH (2010) Mutation of the gene encoding a major outer-membrane protein in Xanthomonas campestris pv. campestris causes pleiotropic effects, including loss of pathogenicity. Microbiology 156:2842–2854. https://doi.org/10.1099/mic.0.039420-0 CrossRefPubMedGoogle Scholar
- Gottesman S (2003) Proteolysis in bacterial regulatory circuits. Annu Rev Cell Dev Biol 19:565–587. https://doi.org/10.1146/annurev.cellbio.19.110701.153228 CrossRefPubMedGoogle Scholar
- Hsiao YM, Song WL, Liao CT, Lin IH, Pan MY, Lin CF (2012) Transcriptional analysis and functional characterization of XCC1294 gene encoding a GGDEF domain protein in Xanthomonas campestris pv. campestris. Arch Microbiol 194:293–304. https://doi.org/10.1007/s00203-011-0760-3 CrossRefPubMedGoogle Scholar
- Liao CT, Du SC, Lo HH, Hsiao YM (2014) The galU gene of Xanthomonas campestris pv. campestris is involved in bacterial attachment, cell motility, polysaccharide synthesis, virulence, and tolerance to various stresses. Arch Microbiol 196:729–738. https://doi.org/10.1007/s00203-014-1012-0 CrossRefPubMedGoogle Scholar
- Richard D et al (2017) Complete genome sequences of six copper-resistant Xanthomonas strains causing bacterial spot of solaneous plants, belonging to X. gardneri, X. euvesicatoria, and X. vesicatoria, using long-read technology. Genome Announc. https://doi.org/10.1128/genomeA.01693-16 CrossRefPubMedPubMedCentralGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Press, Cold Spring HarborGoogle Scholar
- Yang BY, Tseng YH (1988) Production of exopolysaccharide and levels of protease and pectinase activity in pathogenic and non-pathogenic strains of Xanthomonas campestris pv. campestris. Bot Bull Acad Sin 29:93–99Google Scholar