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Vibrio cholerae El Tor require special in vitro culture conditions, consisting of an initial static growth period followed by shift to shaking (AKI conditions), for expression of cholera toxin (CT) and toxin coregulated pili (TCP). ToxT, a regulator whose initial transcription depends on the ToxR regulator, positively modulates expression of CT and TCP. To help understand control of CT and TCP in El Tor vibrios, we monitoredctxAB and ToxR-dependenttoxT transcription by time course primer extension assays. AKI conditions stimulated CT synthesis with an absence ofctxAB transcription during static growth followed by induction upon shaking. ToxR-dependenttoxT transcription was induced at the end of the static growth period but was transient, stopping shortly after shaking was initiated but, interestingly, also if the static phase was prolonged. Immunoblot assays showed that ToxR protein levels were not coincidentally transient, implying a protein on/off switch mechanism for ToxR. Despite the transient activation by ToxR, transcription ofctxAB was maintained during shaking. This finding suggested continuedtoxT expression, possibly through relay transcription from another promoter. The 12.6-kb distant upstreamtcpA promoter responsible for expression of the TCP operon has been proposed to provide an alternatetoxT message by readthrough transcription. Activation of thetcpA promoter is supported by increased expression of TcpA protein during the shaking phase of the culture. Readthrough transcription oftoxT fromtcpA would be compatible with reverse transcription-PCR evidence for atoxT mRNA at times when ToxR-dependent transcription was no longer detectable by primer extension.

Transient Transcriptional Activation of the Vibrio cholerae El Tor Virulence Regulator ToxT in Response to Culture Conditions