Mutagenesis And Phenotypic Analysis Of The Type â…£ Secretion System In Xanthomonas Campestris

Many gram-negative bacteria secrete macromolecules across twomembranes by diverse macromolecular secretion systems that are capable ofexporting virulence factors across the membranes of the bacteria. Typeâ…£secretion system (T4SS) is the one of six major secretion systems of proteins.T4SS play fundamental role on disease development in several importantanimal and human pathogens. The role of T4SS on disease development is wellestablished at the case of crown gall tumors induced by Agrobacteriumtumefaciens. Xanthomonas campestris pv. campestris (Xcc) is the causal agent ofblack rot disease of worldwide cruciferous. Recent genome sequencing projectshave uncovered that T4SS was present in Xanthomonas campestris. WhetherT4SS is involved in pathogenicity of Xanthomonas campestris is unknown. Toconfirm the roll of T4SS on pathogenicity of X. campestris, deletion mutant ofT4SS of Xcc 8004 was constructed, and phenotype and pathogenicity of themutant were determinated.Construction of deletion mutant was conducted by molecular biologicaltechnique. Two DNA fragments (T4-L,1044bp; T4-R,938bp) at the left side orthe right side of T4SS gene were amplified respectively by PCR with twocouples of primers which were designed on the base of site and sequence ofVirB/D4 T4SS gene cluster in genome of Xcc 8004. One DNA fragment (Gm,826bp) was amplified by PCR with two primers of Gm-F/Gm-R and with atemplate from plasmid pPH1JI. All three fragments above (T4-L, T4-R, Gm)were cloned into the suicide plasmid pk18mobsacB. Recombinant plasmidpk18mobsacB::T4 was acquired by method of transformation and selection.The recombinant plasmid was transformed into Xcc 8004 by tri-parentalconjugation, and deletion mutant(8004â–³T4SS) of the VirB/D4 T4SS of Xcc8004 was acquired. The deletion mutant was confirmed by PCR.Phenotypic analysis of 8004â–³T4SS was conducted by common method. Results of phenotypic analysis of biochemical pathogenicity-related factorsindicated that no significant differences were found in the production ofexopolysaccharide, the activity of extracellular protease, the activity ofextracellular cellulose, the activity of extracellular amylase, between thedeletion mutant and the wild type strain. The mobility of the mutant did notchange obviously. Ability of growth and propagation of 8004â–³T4SS was thesame as that of 8004 grown at both NYGB and MMX. Virulence assay byinoculation method of clipping or spraying showed that the deletion of T4SS didnot affect the pathogenicity of Xcc significantly.