Inactivation Of Two-component Signaling Genes And Analysis Of A Polysaccharide Biosynthesis Gene Of Bacterial Phytopathogen Xanthomonas Campestris Pv. Campestris

Xanthomonas campestris pv. campestris (Xcc) , a gram-negative bacterial pathogen, is the causative agent of black rot disease of cruciferous plant. Two-component signal transduction system is the dominant signaling mechanism in prokaryotes. It is also important for regulating bacterial virulence. In order to systematically study two-component signal transduction system of Xcc, we obtained corresponding gene mutants by genetic inactivation based on homologous recombination approach. We previously predicted all response regulator (RR) genes of Xcc ATCC 33913 by bioinformatics analysis, the present study selected 10 genes (rr01-rr10) from the result. The PCR fragments of RR genes rr01-rr10 were individually cloned into suicide vectors of pKnockout-? or pK18mob which can not replicates in Xcc. After confirmation, these recombinant plasmids were transformed into Xcc ATCC 33913 competent cells. Suicide vectors subsequently integrated into the targeted genes that located at bacterial chromosome. By this way we got all of the ten insertional inactivation gene mutants. For each gene, at least six candidate clones were analyzed by Southern blotting. The results confirmed the gene knockouts and these mutants were subjected into phenotypic characterization.In our previous functional genomic study, Xcc 8004 was randomly inserted with a transposon EZ::TN Tnp. A pathogenicity-deficient mutant with insertional site in the CDS (protein coding sequence) XC3814 was identified. Southern blotting analysis revealed that the transposon was a single-copy insertional one. This mutant was virulence deficient in cabbage (Brassica oleracae cultivar Jingfeng NO.1), and became small and dry when growing on NYGB agar plate. Our analysis showed that the yield of extracellular polysaccharide(EPS) accounted for only 26% of wild-type of Xcc 8004. We restored these phenotypes by constructing overexpression and genetical complement bacterial strains. Our result genetically confirmed that the XC3814 gene was involved in pathogenicity by affecting EPS biosynthesis. Because in planta growth assay failed to discriminate the difference between XC3814 mutant and wild-type Xcc 8004 in terms of population dynamics, XC3814 gene was likely to play a role in early stage of pathogenesis. In Xcc ATCC 33913 there was a same CDS with XC3814. Blast search based on homology and protein domain prediction showed that this gene putatively encoded a...