The Molecular Mechanisms Of Post-transcriptional Regulator Protein RsmA_Xcc Regulating Biofilm Formation In Xanthomonas Campestris Pathovar Campestris

The CsrA/RsmA protein belongs to a new class of RNA-binding proteins that affects translation of its gene targets by binding mRNAs. CsrA is a global regulator, It regulates multiple unrelated pathways such as central carbon metabolism, motility and biofilm formation, virulence and pathogenesis, quorum sensing and oxidative stress response. Our work has shown that deletion of the rsmA gene of Xanthomonas campestris pathovar campestris (Xcc) resulted in complete loss of virulence on host plant Chinese radish. The rsmAXcc mutant displayed significant reduction in the production of exopolysaccharide, but enhanced bacterial aggregation and cell adhesion,indicating that RsmA xcc plays important role in exopolysaccharide production, biofilm formation and pathogenesis. But the mechanisms of RsmAXcc is still unknown. In this study, we investigated the molecular mechanisms of RsmAxcc regulating biofilm formation in Xcc.Cyclic diguanylate(c-di-GMP) is an common, bacterial second messenger that controls the transition from a free-ling, motile to biofilm one. c-di-GMP is produced by DGC proteins containing the GGDEF domain and degraded by PDE proteins bearing the EAL or HD-GYP domains. The genome of Xcc encodes 37 proteins with potential roles in c-di-GMP turnover. We result show that the cellular levels of c-di-GMP in the rsmAXcc mutant was elevated over that seen in the wild-type. The EMSA assays showed that RsmAXcc interacted directly with the transcripts of five genes (XC1803, XC1824,XC2228,XC2715 and XC2866).The possibility that RsmAXcc regulates the expression of five genes was investigated using fusions gusA. The result show that RsmA regulates the express of XC1803, XC2715 and XC286 6protion, which encode GGDEF domain proteins. A triple disruption of XC1803, XC2715 and XC2866 in the rsmAXcc mutant background gave rise to planktonic dispersed growth in liquid medium. Double mutations of XC1824 and XC2228 showed no discernable difference in biofilm phenotype. These finding indicate that RsmAXcc regulates biofilm formation through its influence on the expression of XC1803, XC2715 and XC2866 (synthesis of c-di-GMP).manAXcc gene encoding the biofilm dispersing enzyme endo-mannanase, Xag-type EPS are essential for Xcc biofilm formation, clpXcc encodes a c-di-GMP responsive transcriptional regulator of the CRR-FNR family. The level of manAXcc transcript in both rsmAXcc and clp Xcc mutants was reduced compared to the wild-type. In contrast, mutation of rsmAXcc or clpxcc led to increased transcription of xagAXcc.These result indicate that RsmAXcc regulates biofilm formation through its influence on the expression of manAXcc. xagAXcc and xagBXcc-nevertheless Clp hinging to manAXcc and xagAxcc promoters is inhibited by c-di-GMP.Xcc produces at least two types of EPS, one is xanthan encoded by the gum genes, and the other is encoded by the xag genes. ManA, which is capable of dispersion of the aggregates formed by rpf mutants, is not able to degrade soluble xanthan. When manAXcc was constitutively expressed in the rsmAXcc Mutant background a different pattern of cell growth was observed. Bacterial cells gave rise to planktonic dispersed growth in liquid medium, and displayed significant reduction in the production of exopolysaccharide. These result indicate that Xag-type EPS may be dispersed by Man A;The gumBXcc mutant in the rsmAxcc mutant background showed no discernable difference in biofilm phenotype. The date indicating synthesis of xanthan is not necessary for the formation of aggregates in rsmAxcc mutants of Xcc.