Analysis of the regulatory role of Rpf/DSF cell-cell signaling system in the virulence of Xanthomonas campestris using proteomics

The black rot pathogen Xanthomonas campestris (Xcc) utilizes molecules of the diffusible signal factor (DSF) family as signals to regulate diverse processes contributing to virulence. DSF signal synthesis and transduction requires proteins encoded by the rpf gene cluster. RpfF catalyzes DSF synthesis whereas the RpfCG two-component system links the perception of DSF to alteration in the level of the second messenger cyclic di-GMP. As this nucleotide can exert a regulatory influence at the post-transcriptional and post-translational levels, we have used comparative proteomics to identify Rpf-regulated processes in Xcc that may not be revealed by transcriptomics. The abundance of a number of proteins was altered in rpfF, rpfC or rpfG mutants compared to the wild type. These proteins belonged to several functional categories to include biosynthesis and intermediary metabolism, regulation, oxidative stress or antibiotic resistance and DNA replication. For many of these proteins, the alteration in abundance was not associated with alteration in transcript level. A directed mutational analysis allowed us to describe a number of new virulence factors amongst these proteins to include elongation factor P and a putative outer membrane protein, which are both widely conserved in bacteria.