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Exopolysaccharides and several extracellular enzymes of Xanthomonas campestris pv. campestris (Xcc), the causative agent of black rot in crucifers, are virulence determinants. It is known that Clp (cAMP receptor protein-like protein) and RpfF (an enoyl-CoA hydratase homologue required for the synthesis of the diffusible signal factor, DSF) regulate production of these factors. In this study, plate assay revealed that Xcc possesses pectin methylesterase activity and that its expression is controlled by Clp and RpfF. Mutational analysis has demonstrated that pmeA encodes a pectin methylesterase. Using the 5' RACE method, the pmeA transcription initiation site was mapped. Transcriptional fusion assays showed that pmeA transcription is positively regulated by Clp and RpfF, subject to catabolite repression which is independent of Clp or RpfF, and repressed under conditions of high osmolarity or oxygen limitation. This study not only extends previous work on Clp and RpfF regulation by showing that they both influence the expression of pmeA in Xcc, but also, for the first time, characterizes pectin methylesterase gene expression in Xanthomonas.

Biosynthesis of xanthan polysaccharide, a virulence factor of phytopathogenic Xanthomonas campestris pv. campestris (Xcc), involves the gum operon and the cyclic AMP receptor protein (CRP) homologue Clp. Clp was shown to have the same DNA binding specificity as the CRP at positions 5, 6, and 7 (GTG motif) of the left arm. Therefore, Clp binding sites (CBSs) have typically been identified by pattern searching of the Xcc genome using the consensus CRP binding sequence. Here, results of a reporter assay and electrophoretic mobility shift assay suggest that Clp upregulates the gum operon by binding to two non-consensus sites, in which a more conserved right arm may compensate for the lack of conservation in the left arm, a high GC content in the central region (6 bp) may be important for binding, and binding may be enhanced if the GC-rich central region is palindromic. These suggest that atypical CBSs exist in Xcc promoters and that Clp, while retaining the capacity to bind typical CBSs, has evolved to bind atypical CBS because: 1) Clp shares only moderate homology with the CRP and is modulated by cyclic di-GMP; and 2) Xcc has a higher GC content (65%) than Escherichia coli (50%).

Gram-negative phytopathogenic Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in crucifers. The ability of Xcc to incite this disease in plants depends on a number of factors, including exopolysaccharides, extracellular enzymes and biofilm production. In this study, transposon mutagenesis led to identification of the prc gene, encoding a tail-specific protease, which plays a role in Xcc pathogenesis. Mutation of prc resulted in decreased virulence, extracellular protease production and bacterial attachment, with restoration to the levels of wild type by the intact prc gene. From subsequent quantitative RT-PCR analysis and reporter assay, the major extracellular protease gene prt1, biofilm-related gene galE encoding a UDP-galactose 4-epimerase and two putative adhesin genes (yapH and XC_4290 encoding autotransporter-like protein H and hemagglutinin, respectively) were found to be reduced in the prc mutant. Results of transcriptome profiling of Xcc wild type and prc mutant by RNA sequencing (RNA-Seq) showed that mutation of prc in Xcc leads to alteration in the transcriptional levels (more than twofold) of 91 genes. These differentially expressed genes were associated with a wide range of biological functions such as carbohydrate transport and metabolism, cell wall/membrane biogenesis, posttranslational modification, protein turnover and chaperones, inorganic ion transport and metabolism and signal transduction mechanisms. The results of this study facilitate the functional understanding of and provide new information about the regulatory role of prc.