The Rpf/DSF system and the regulation of virulence in the plant pathogen Xanthomonas campestris

The full virulence of Xanthomonas campestris pv. campestris (Xcc) to plants depends upon cell-to-cell signalling mediated by the signal molecule DSF (for diffusible signal factor), that has been characterised as cis-11-methyl-2-dodecenoic acid. DSF-mediated signalling regulates motility, biofilm dynamics and the synthesis of particular virulence determinants. The synthesis and perception of the DSF signal molecule involves products of the rpf (regulation of pathogenicity factors) gene cluster. DSF synthesis is fully dependent on RpfF, which encodes a putative enoyl-CoA hydratase. A two-component system, comprising the complex sensor histidine kinase RpfC and the HD-GYP domain regulator RpfG, is implicated in DSF perception. The HD-GYP domain of RpfG is a phosphodiesterase working on cyclic di-GMP; DSF perception is thereby linked to the turnover of this intracellular second messenger. The full range of regulatory influences of the Rpf/DSF system and of cyclic di-GMP in Xcc has yet to be established. In order to further characterise the Rpf/DSF regulatory network in Xcc, a proteomic approach was used to compare protein expression in the wildtype and defined rpf mutants. This work shows that the Rpf/DSF system regulates a range of biological functions that are associated with virulence and biofilm formation but also reveals new functions mediated by DSF regulation. These functions include antibiotic resistance, detoxification and stress tolerance. Mutational analysis showed that several of these regulated protein functions contribute to virulence in Chinese radish. Interestingly, it was demonstrated that different patterns of protein expression are associated with mutations of rpfF, rpfC and rpfG. This suggests that RpfG and RpfC have broader roles in regulation other than perception and transduction of DSF. Taken together, this analysis indicates the broad and complex regulatory role of Rpf/DSF system and identifies a number of new functions under Rpf/DSF control, which were shown to play a role in virulence.

Identification and characterization of a glycosulfatase-encoding gene cluster in Bifidobacterium breve UCC2003

Bifidobacteria constitute a specific group of commensal bacteria, typically found in the gastrointestinal tract (GIT) of humans and other mammals. Bifidobacterium breve strains are numerically prevalent among the gut microbiota of many healthy breast-fed infants. In the current study, we investigated glycosulfatase activity in a bacterial nursling stool isolate, B. breve UCC2003. Two putative sulfatases were identified on the genome of B. breve UCC2003. The sulfated monosaccharide N-acetylglucosamine-6-sulfate (GlcNAc-6-S) was shown to support growth of B. breve UCC2003, while, N-acetylglucosamine-3-sulfate, N-acetylgalactosamine-3-sulfate and N-acetylgalactosamine-6-sulfate, did not support appreciable growth. Using a combination of transcriptomic and functional genomic approaches, a gene cluster, designated ats2, was shown to be specifically required for GlcNAc-6-S metabolism. Transcription of the ats2 cluster is regulated by a ROK-family transcriptional repressor. This study represents the first description of glycosulfatase activity within the Bifidobacterium genus. Bifidobacteria are saccharolytic organisms naturally found in the digestive tract of mammals and insects. Bifidobacterium breve strains utilize a variety of plant and host-derived carbohydrates which allow them to be present as prominent members of the infant gut microbiota as well as being present in the gastrointestinal tract of adults. In this study, we introduce a previously unexplored area of carbohydrate metabolism in bifidobacteria, namely the metabolism of sulfated carbohydrates. B. breve UCC2003 was shown to metabolize N-acetylglucosamine-6-sulfate (GlcNAc-6-S) through one of two sulfatase-encoding gene clusters identified on its genome. GlcNAc-6-S can be found in terminal or branched positions of mucin oligosaccharides, the glycoprotein component of the mucous layer that covers the digestive tract. The results of this study provide further evidence of this species' ability to utilize mucin-derived sugars, a trait which may provide a competitive advantage in both the infant and adult gut.