Characterization of hns Gene of Xanthomonas campestris pv. campestris

• Main Authors: Ching-Hua Wang, 王清華
• Other Authors: 翁淑芬
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/56591956311878025919

碩士 === 國立中興大學 === 分子生物學研究所 === 95 === The gram-negative plant pathogenic Xanthomonas campestris pv. campestris causes black rot in crucifers. The virulence of this bacterium is mainly depending on its ability to produce large mounts of an exopolysaccharide (EPS, also called xanthan gum) and several extracellular enzymes, the virulence factors. It is known that the global transcription factor Clp (cAMP receptor protein-like protein) is involved in the production and secretion of these virulence factors. Using the consensus Clp-binding sequences (CBS) predicted in our laboratory for genome-wide search, 46 genes with at least one CBS located in the upstream region, suggesting possible regulation by Clp, were found in X. campestris pv. campestris strain 17. One of them was XCC0637, which was predicted to be a DNA-binding protein genes belonging to functional category Ⅲ possibly involved in macromolecule metabolism. It was designated as an X. campestris pv. campestris H-NS, because it possesses an H-NS conserved domain and shares 72, 62, 46 and 35% identities with the global regulator H-NS from Stenotrophomonas maltophilia, Xylella fastidiosa, Rhodopseudomonas palustris and Escherichia coli, respectively. In enterobacteria, H-NS regulates gene expression primarily as a transcriptional repressor, with only a few examples required for a direct positive control. In addition, H-NS and CRP in E. coli regulate gene expression independently. In this study, gel retardation and Phns-lacZ transcriptional fusion assays show that Clp positively regulates expression of hns by direct binding to the promoter. An hns mutan was constructed by insertional mutation. Testing with this mutant indicated that, relative to the wild-type, i) production of EPS and extracellular enzymes was slightly reduced, ii) appearance of the black rot symptom was slightly delayed, by about one day slower, iii) swarming motility was drastically reduced. Finally, transcriptional fusion assays also demonstrated that the X. campestris pv. campestris H-NS exerts negative control on the expression of hns, pelB and xpsE and positive control on the expression of engA.