Vibrio cholerae CsrA Directly Regulates varA To Increase Expression of the Three Nonredundant Csr Small RNAs

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.