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Journal of Bacteriology, May 2005, p. 3013-3019, Vol. 187, No. 9
0021-9193/05/$08.00+0     doi:10.1128/JB.187.9.3013-3019.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Requirements for Vibrio cholerae HapR Binding and Transcriptional Repression at the hapR Promoter Are Distinct from Those at the aphA Promoter

Wei Lin, Gabriela Kovacikova, and Karen Skorupski*

Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, New Hampshire 03755

Received 1 December 2004/ Accepted 31 January 2005

Virulence gene expression in certain strains of Vibrio cholerae is regulated in response to cell density by a quorum-sensing cascade that influences the levels of the LuxR homolog HapR through small regulatory RNAs that control the stability of its message. At high cell density, HapR represses the expression of the gene encoding the virulence gene activator AphA by binding to a site between –85 and –58 in the aphA promoter. We show here that a second binding site for HapR lies within the hapR promoter from which it functions to repress its own transcription. This site, as determined by gel mobility shift assay and DNaseI footprinting, is located between +8 and +36 from the transcriptional start and is not strongly conserved with the site at the aphA promoter. At low cell density, when the expression of a transcriptional hapR-lacZ fusion was low, no autorepression was observed. However, at high cell density, when the expression of the hapR-lacZ fusion was approximately 15-fold higher, the presence of HapR reduced its expression. Introduction of a single base pair change within the binding site at +18 prevented HapR binding in gel mobility shift assays. In the absence of HapR, this mutation did not significantly influence the expression of the hapR promoter, but in its presence, the expression of the promoter was increased at high cell density. These results indicate that HapR autorepresses from a single binding site in the hapR promoter and suggest a model for the temporal regulation of its expression as its intracellular levels increase.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, NH 03755. Phone: (603) 650-1623. Fax: (603) 650-1318. E-mail: karen.skorupski{at}dartmouth.edu.

Journal of Bacteriology, May 2005, p. 3013-3019, Vol. 187, No. 9
0021-9193/05/$08.00+0     doi:10.1128/JB.187.9.3013-3019.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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