ABSTRACTTcpP and ToxR coordinately regulate transcription oftoxT, the master regulator of numerous virulence factors inVibrio cholerae. TcpP and ToxR are membrane-localized transcription factors, each with a periplasmic domain containing two cysteines. In ToxR, these cysteines form an intramolecular disulfide bond and a cysteine-to-serine substitution affects activity. We determined that the two periplasmic cysteines of TcpP also form an intramolecular disulfide bond. Disruption of this intramolecular disulfide bond by mutation of either cysteine resulted in formation of intermolecular disulfide bonds. Furthermore, disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP. While the decreased stability of TcpP-C207S resulted in a nearly complete loss oftoxTactivation and cholera toxin (CT) production, the second cysteine mutant, TcpP-C218S, was partially resistant to proteolytic degradation and maintained ∼50%toxTactivation capacity. TcpP-C218S was also TcpH independent, since deletion oftcpHdid not affect the stability of TcpP-C218S, whereas wild-type TcpP was degraded in the absence of TcpH. Finally, TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, suggesting that the single periplasmic cysteine in TcpH may assist with disulfide bond formation in TcpP by interacting with the periplasmic cysteines of TcpP. Consistent with this finding, a TcpH-C114S mutant was unable to stabilize TcpP and was itself unstable. Our findings demonstrate a periplasmic disulfide bond in TcpP is critical for TcpP stability and virulence gene expression.IMPORTANCETheVibrio choleraetranscription factor TcpP, in conjunction with ToxR, regulates transcription oftoxT, the master regulator of numerous virulence factors inVibrio cholerae. TcpP is a membrane-localized transcription factor with a periplasmic domain containing two cysteines. We determined that the two periplasmic cysteines of TcpP form an intramolecular disulfide bond and disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP and reduced virulence gene expression. Normally TcpH, another membrane-localized periplasmic protein, protects TcpP from degradation. However, we found that TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, indicating that the periplasmic cysteines of TcpP are required for functional interaction with TcpH and that this interaction is required for both TcpP and TcpH stability.