[Identification of a gene involved in the expression of the pathogenicity-related gene XC3814 in Xanthomonas campestris].

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of the black rot disease of cruciferous plants. Our previous work had demonstrated that XC3814 is required for full virulence and extracellular polysaccharide production. In this work, the reporter plasmid pL3814sac was constructed by fusing the promoter region of XC3814 to the coding region of the gene sacB, and introduced into Xcc wild-type strain 8004. The resulted strain 8004/pL3814sac was mutagenized randomly by the transposon EZ::Tn5, and 3 mutant strains insensitive to sucrose were isolated. One of the mutants was due to the disruption of the open reading frame XC3882, which was assigned to code a hypothetical protein. To verify whether XC3882 has an impact on the expression level of XC3814, the reporter plasmid pGUS3814 was constructed by fusing the promoter region of XC3814 to the coding region of the gusA gene. This construct was introduced into the wild-type strain 8004 and the XC3882 mutant strain 190A10, which was derived from the transposon Tn5gusA5 insertion. The GUS activity, produced by pGUS3814 in the XC3882 mutant background, was reduced by 81.3% compared to that in the wild type background. These results indicate that the expression of XC3814 is influenced by XC3882.

The role of glucose kinase in carbohydrate utilization and extracellular polysaccharide production in Xanthomonas campestris pathovar campestris.

The genome of the Xanthomonas campestris pathovar campestris (Xcc) strain 8004 encodes three uncharacterized proteins, XC1166, XC1223 and XC1976, annotated as glucose kinase (Glk) by bioinformatic studies. Here we have investigated the biochemical characteristics and physiological roles of these proteins with particular reference to the synthesis of extracellular polysaccharide (EPS). XC1166, XC1223 and XC1976 were overexpressed as fusion proteins with a His(6) affinity tag and purified by nickel affinity chromatography. The standard Glk activity assay revealed that all three proteins possessed apparent Glk activity, with XC1976-His(6) being the most active; the specific activity values were 1.16x10(6) U mg(-1) for XC1166-His(6), 4.36x10(7) U mg(-1) for XC1223-His(6) and 2.63x10(8) U mg(-1) for XC1976-His(6). TLC analysis showed, however, that only XC1976-His(6) could phosphorylate glucose. Insertional mutants of XC1166, XC1223 and XC1976 were generated using the suicide plasmid pK18mob. Although mutant strains with insertions in XC1166 or XC1223 had Glk activity similar to that of the wild-type strain, the XC1976 mutant had only about 6% of the wild-type activity. Mutation in XC1976 had complex effects on EPS production. In media containing arabinose, glucose, galactose, sucrose or maltose, the XC1976 mutant produced about 40-75% of the wild-type level of EPS, whereas in medium containing fructose, the mutant showed a 30% increase in EPS production compared to the wild-type strain. The XC1976 mutant also showed attenuated virulence on the host plant Chinese radish (Raphanus sativus). The results indicate that XC1976 has the most significant role for the parameters tested.