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Infection and Immunity, May 1999, p. 2178-2183, Vol. 67, No. 5
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Ana I. Medrano,1,2 Victor J. DiRita,3 Gabriela Castillo,2 and Joaquin Sanchez1,2,*

Facultad de Medicina, UAEM, Cuernavaca, Morelos, Mexico 62210,1 Centro de Investigación sobre Enfermedades Infecciosas, INSP, Cuernavaca, Morelos, Mexico 62508,2 and Unit for Laboratory Animal Medicine and Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 481093

Received 18 November 1998/Returned for modification 12 January 1999/Accepted 9 February 1999

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 monitored ctxAB and ToxR-dependent toxT transcription by time course primer extension assays. AKI conditions stimulated CT synthesis with an absence of ctxAB transcription during static growth followed by induction upon shaking. ToxR-dependent toxT 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 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 readthrough transcription. Activation of the tcpA promoter is supported by increased expression of TcpA protein during the shaking phase of the culture. Readthrough transcription of toxT from tcpA would be compatible with reverse transcription-PCR evidence for a toxT mRNA at times when ToxR-dependent transcription was no longer detectable by primer extension.

* Corresponding author. Mailing address: Facultad de Medicina, UAEM, Av. Universidad 1001, Col. Chamilpa, Cuernavaca, Mor. Mexico 62210. Phone: (52) 73 297009. Fax: (52) 73 297031 E-mail: joaquin.sanchez{at}microbio.gu.se.

Infection and Immunity, May 1999, p. 2178-2183, Vol. 67, No. 5
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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