Characterization Of A Putative Two-Component Regulatory System Gene XC2229 In Xanthomonas Campestris Pv. Campestris

Two-component systems,consisting of a histidine protein kinase that senses a signal input and a response regulator that mediates the output,are involved in various signal transduction pathways in many prokaryotes,fungi,slime molds and plants.Xanthomonas campestris pv.campestris(Xcc)is a yellow pigmented gram-negative bacterium that belongs to theγ-subdivision of Proteobacteria.It is the causal agent of black-rot disease,one of the most destructive diseases of cruciferous plants worldwide.Xcc 8004 strian was subjected to Tn5 trasnposon mutagenesis to generate mutant defective in pathogenicity on the host plant Chinese radish(Raphanus sativus L.var.radiculus Pers.).A novel mutant 186B07 was decreased significantly in virulence but retained the ability to induce HR on the non host plant.The Tn5 insertional site in 186B07 was localized in open reading frame(ORF)XC2229 by TAIL-PCR and sequencing. By multiple sequence alignment,we found that the coding product of XC2229 is the sensor in a two-component regulatory system,its characteration can provide us a reference for the study of two-component regulatory system in pathogens.To investigte the function of XC2229,the deletion mutant of this gene,named DM2229,was constructed.The results of plant assay showed that on host plant,both DM2229 and 186B07 performed same virulence level which is significantly lower than that of the wild type Xcc 8004.By phenotypes investigation,we found that in DM2229,the production of exopolysaccharides (EPS)was surppressed,the ability to digest starch and the carboxylmethyl cellulose(CMC)was attenuated,and the cell motility was impaired.A pLALR6 carrying the full XC2229 gene with the original promoter was introduced into DM2229 to generate the complimentary strain CDM2229. The genetically complimention of XC2229 gene to DM2229 could restore fully the EPS production,the ability to digest CMC and starch,the cell mobility,and 95.4%of virulence to the wild type strain Xcc 8004.Because XC2229 located flanking the flagellar biosynthesis and control gene cluster,in order to investigate the relationships between XC2229 and the flagellar biosynthesis and control genes in this area,the semi-quantitative RT-PCR was used and the results showed that on the transcriptional level,XC2229 appeared to positively regulate the expression of some flagellar biosynthesisand control genes,such as fliL,fliJ and fliC;By the contrary,it was a negative regulator to flagellar gene flgA.And there weren't any significant regulative relations between XC2229 and other flagellar genes.This study demonstrated that XC2229 is an important gene which is invovled in many physical and chemical processes of Xcc,e.g.EPS production,cellulose digestion and flagellum biosynthesis,and the vilulenc of the pathogen.However,although there are two components in the system,the corresponding respose regulator gene of XC2229 is to be identified.