Transient transcriptional activation of the Vibrio cholerae El Tor virulence regulator ToxT in response to culture conditions

Vibrio cholerae EI. Tor require special in vitro culture conditions, consis ting of an initial static growth period followed by shift to shaking (AKI c onditions), for expression of cholera toxin (CT) and toxin coregulated pill (TCP), ToxT, a regulator whose initial transcription depends on the ToxR r egulator, positively modulates expression of CT and TCP, To help understand control of CT and TCP in El Tor vibrios, we monitored ctxAB and ToxR-depen dent toxT transcription by lime course primer extension assays. AKI conditi ons stimulated CT synthesis with an absence of ctxAB transcription during s tatic growth followed by induction upon shaking. ToxR-dependent toxT transc ription was induced at the end of the static growth period but was transien t, 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, transcriptio n of ctxAB was maintained during shaking. This finding suggested continued toxT expression, possibly through relay transcription from another promoter . The 12.6-kb distant upstream tcpA promoter responsible for expression of the TCP operon has been proposed to provide an alternate toxT message by re adthrough transcription. Activation of the tcpA promoter is supported by in creased expression of TcpA protein during the shaking phase of the culture. Readthrough transcription of toxT from tcpA would be compatible with rever se transcription-PCR evidence for a toxT mRNA at times when ToxR dependent transcription was no longer detectable by primer extension.