Examination of the Global Regulon of CsrA in Xanthomonas citri subsp. citri Using Quantitative Proteomics and Other Approaches

The RNA-binding protein CsrA is a global posttranscriptional regulator and controls many physiological processes and virulence traits. Deletion of csrA caused loss of virulence, reduced motility and production of xanthan gum and sub-stantial increase in glycogen accumulation, as well as enhanced bacterial aggregation and cell adhesion in Xantho-monas spp. How CsrA controls these traits is poorly understood. In this study, an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis was conducted to compare the protein profile of wild-type strain Xanthomonas citri subsp. citri and the isogenic Delta csrA strain. A total of 2,374 proteins were identified, and 284 were considered to be differentially expressed proteins (DEPS), among which 151 proteins were up-regulated and 133 were downregulated in the Delta csrA strain with respect to the wild-type strain. Enrichment analysis and a protein-protein interaction network analysis showed that CsrA regulates bacterial secretion systems, flagella, and xanthan gum biosynthesis. Several proteins encoded by the gumB operon were down-regulated, whereas proteins associated with flagellum assembly and the type IV secretion system were up-regulated in the Delta csrA strain relative to the Xcc306 strain. These results were confirmed by beta-glucuronidase assay or Western blot. RNA secondary structure prediction and a gel-shift assay indicated that CsrA binds to the Shine-Dalgarno sequence of virB5. In addition, the iTRAQ analysis identified 248 DEPs that were not previously identified in transcriptome analyses. Among them, CsrA regulates levels of eight regulatory proteins (ColR, GacA, GlpR, KdgR, MoxR, PilH, RecX, and YgiX), seven TonB-dependent receptors, four outer membrane proteins, and two ferric enterobactin receptors. Taken together, this study greatly expands understanding of the regulatory network of CsrA in X. citri subsp. citri.