CsrA, an RNA-binding global regulator, is an essential protein in Vibrio cholerae. V. cholerae CsrA is regulated by three small RNAs (sRNAs), namely, CsrB, CsrC, and CsrD, which act to sequester and antagonize the activity of CsrA. Although the sRNAs were considered to be largely redundant, we found that they differ in expression, half-life, and the ability to regulate CsrA. Further, we identified a feedback loop in the Csr system in which CsrA increases the synthesis of these antagonistic sRNAs. Because the Csr sRNAs are positively regulated by VarA, we determined the effects of CsrA on VarA levels. The level of VarA was reduced in a csrA mutant, and we found that CsrA directly bound to varA mRNA in an electrophoretic mobility shift assay in vitro and in an CsrA-RNA immunoprecipitation assay in vivo. Thus, varA mRNA is an in vivo-verified direct target of CsrA in V. cholerae, and this is the first demonstration of CsrA directly binding to a varA/uvrY/gacA homolog. Additionally, we demonstrated that a varA translational fusion was less active in a csrA mutant than in wild-type V. cholerae, suggesting that CsrA enhances varA translation. We propose that this autoregulatory feedback loop, in which CsrA increases the production of the nonredundant Csr sRNAs by regulating the amount of VarA, provides a mechanism for fine-tuning the availability of CsrA and, thus, of its downstream targets. IMPORTANCE Vibrio cholerae is a major human pathogen, causing epidemics and pandemics of cholera. V. cholerae persists in the aquatic environment, providing a constant source for human infection. Success in transitioning from the environment to the human host and back requires the bacterium to rapidly respond and to adjust its gene expression and metabolism to these two very different habitats. Our findings show that CsrA, an RNA-binding regulatory protein, plays a central role in regulating these transitions. CsrA activity is controlled by the antagonistic sRNAs CsrB, CsrC, and CsrD, and these sRNAs respond to changes in the availability of nutrients. CsrA autoregulates its own activity by controlling these sRNAs via their primary regulator VarA. Thus, the change in CsrA availability in response to nutrient availability allows V. cholerae to alter gene expression in response to environmental cues.

CsrA is a posttranscriptional global regulator in Vibrio cholerae. Although CsrA is critical for V. cholerae survival within the mammalian host, the regulatory targets of CsrA remain mostly unknown. To identify pathways controlled by CsrA, RNA-seq transcriptome analysis was carried out by comparing the wild type and the csrA mutant grown to early exponential, mid-exponential, and stationary phases of growth. This enabled us to identify the global effects of CsrA-mediated regulation throughout the V. cholerae growth cycle. We found that CsrA regulates 22% of the V. cholerae transcriptome, with significant regulation within the gene ontology (GO) processes that involve amino acid transport and metabolism, central carbon metabolism, lipid metabolism, iron uptake, and flagellum-dependent motility. Through CsrA-RNA coimmunoprecipitation experiments, we found that CsrA binds to multiple mRNAs that encode regulatory proteins. These include transcripts encoding the major sigma factors RpoS and RpoE, which may explain how CsrA regulation affects such a large proportion of the V. cholerae transcriptome. Other direct targets include flrC, encoding a central regulator in flagellar gene expression, and aphA, encoding the virulence gene transcription factor AphA. We found that CsrA binds to the aphA mRNA both in vivo and in vitro, and CsrA significantly increases AphA protein synthesis. The increase in AphA was due to increased translation, not transcription, in the presence of CsrA, consistent with CsrA binding to the aphA transcript and enhancing its translation. CsrA is required for the virulence of V. cholerae and this study illustrates the central role of CsrA in virulence gene regulation. IMPORTANCE Vibrio cholerae, a Gram-negative bacterium, is a natural inhabitant of the aqueous environment. However, once ingested, this bacterium can colonize the human host and cause the disease cholera. In order to successfully transition between its aqueous habitat and the human host, the bacterium must sense changes in its environment and rapidly alter gene expression. Global regulators, including CsrA, play an integral role in altering the expression of a large number of genes to promote adaptation and survival, which is required for intestinal colonization. We used transcriptomics and a directed CsrA-RNA coimmunoprecipitation to characterize the CsrA regulon and found that CsrA alters the expression of more than 800 transcripts in V. cholerae. Processes regulated by CsrA include motility, the rugose phenotype, and virulence pathways. CsrA directly binds to the aphA transcript and positively regulates the production of the virulence regulator AphA. Thus, CsrA regulates multiple processes that have been linked to pathogenesis.

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