Characterization of clpX and clpP Genes in Xanthomonas campestris pv. campestris

• Main Authors: LI,CHIH-EN, 李芷恩
• Other Authors: HSIAO,YI-MIN
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/uks677

碩士 === 中臺科技大學 === 生物科技暨醫學工程研究所 === 107 === The gram-negative Xanthomonas campestris pv. campestris (XCC) is the pathogenic bacterium that causes black rot disease in crucifers. The virulence determinants of this bacterium include extracellular enzymes, exopolysaccharides, and biofilm formation. Previously, a transposon mutant of XCC showing reduced production of bacterial attachment was isolated and subsequent analysis revealed this mutant carries the transposon insertion in clpX, a gene encoding ATPase subunit of the ClpXP protease. The Clp protease system is involved in the general stress response as well as the virulence of many pathogenic bacteria. In the fully sequenced genomes of XCC, several putative Clp protein encoding genes (such as clpA, clpX, clpY, clpP, and clpQ) have been annotated. However, none of them have been characterized. The aim of this study was to functionally characterize the clpX and clpP genes in XCC. Through genetic complementation and phenotypic evaluation, it was found that (i) mutation of clpX and clpP revealed decreased levels of extracellular enzymes and reductions in virulence on the host cabbage; (ii) the clpX mutant also exhibited impacted ability to attach to abiotic and biotic surfaces and showed reduced tolerance in the presence of a range of stresses, including temperature, puromycin and sodium dodecyl sulfate; and (iii) these affected phenotypes of the clpX and clpP mutants could be complemented to wild-type levels by the intact clpX and clpP genes. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) assays indicated that mutation in clpX diminished the expression of genes that encode the major extracellular enzymes and the biofilm-related proteins. The RT-qPCR assays also indicated that the expression of two clp genes (clpY and clpQ) that encode ClpYQ protease was increased after clpX mutation. Promoter activity assay indicated that clpX transcription exhibited a distinct expression profile under different culture conditions and was subject to catabolite repression. This is the first report to characterize the clpX and clpP genes in Xanthomonas.