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The epidemic diarrheal disease cholera is caused by the Gram-negative bacterium Vibrio cholerae. V. cholerae virulence factors include the toxin-coregulated pilus (TCP) and cholera toxin, which are major factors responsible for host colonization and production of diarrhea. Expression of cholera toxin and TCP genes is controlled by the ToxR regulon. The ToxR regulon includes the transcriptional activators ToxR, TcpP, and ToxT. ToxT directly initiates transcription of the cholera toxin and TCP genes. TcpP and ToxR are necessary for expression of toxT. TcpP and ToxR activity requires TcpH and ToxS, respectively. Additionally, ToxR is able to directly initiate transcription of the cholera toxin genes independent of TcpP and ToxT. TCP is required early in infection to colonize the small intestine, then cholera toxin is expressed later in infection to produce diarrhea. We tested whether stringent response, the low nutrient stress response, was involved in regulation of virulence genes. Using an infant mouse model, we found that V. cholerae strains with deletions of the stringent response genes were unable to colonize the small intestine. We further tested these stringent response-null mutants and found that stringent response was necessary for TCP expression, although effects on cholera toxin expression were not significant. We then tested whether stringent response regulation of TCP occurred through the ToxR regulon. We found that stringent response induced toxT and tcpPH expression, while repressing toxRS. This differential regulation of ToxR and TcpP may explain the differential expression of TCP and cholera toxin in vivo.

Biofilm formation is a key factor in Vibrio cholerae environmental survival and host colonization. Production of biofilm enables V. cholerae to survive and persist in aquatic environments and aids in the passage through the gastric acid barrier to allow access to the small intestine. The genes involved in biofilm formation are regulated by the transcriptional activators vpsR and vpsT, which are in turn transcriptionally regulated by a number of environmental signals. In this study, the role of the stringent response in biofilm formation was examined. V. cholerae mutants deficient in stringent response had a reduced ability to form biofilms, although they were not completely deficient in biofilm formation. There are three (p)ppGpp synthases in V. cholerae: RelA, SpoT, and RelV. All three synthases were necessary for vpsR transcription, with RelV showing the strongest effect. RelA was the only synthase that was necessary for vpsT expression. Stringent response regulation of vpsR and vpsT was shown to partially occur through rpoS. Biofilm formation in V. cholerae is controlled by a complex regulatory apparatus, with negative regulators of biofilm gene expression, such as quorum sensing, and positive regulators of biofilm genes, including stringent response, interacting to ensure that biofilm formation is coordinated with the environment.