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A more recent version of this article appeared on November 27, 2002
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M206893200v1
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Papers In Press, published online ahead of print September 27, 2002
J. Biol. Chem, 10.1074/jbc.M206893200
Submitted on July 10, 2002
Revised on September 6, 2002
Accepted on September 27, 2002

Promoter analysis and regulatory characteristics of vvhBA encoding cytolytic hemolysin of vibrio vulnificus

Hyun Kyung Choi, Na Young Park, Dae-il Kim, Hee Jong Chung, Sangryeol Ryu, and Sang Ho Choi

Department of Food Science and Technology, Chonnam National University, Kwang-Ju 500-757

Corresponding Author: shchoi{at}chonnam.ac.kr

Cytolytic hemolysin, a gene product of vvhA, is a potent virulence factor of the pathogenic bacterium V. vulnificus. We have previously shown that hemolysin production is repressed by adding glucose to culture media and that production can be restored by adding cyclic AMP. In this study, hemolysin activity and the level of vvh transcript were determined to reach a maximum in late exponential phase and were repressed when cells entered stationary phase. Northern blot and primer extension analyses revealed that vvhA is cotranscribed with a second gene, vvhB, located upstream of vvhA. Transcription of the vvhBA operon begins at a single site, and is under the direction of a single promoter, Pvvh. A crp null mutation decreased hemolysin production and the level of vvhBA transcript by reducing the activity of Pvvh, indicating that the Pvvh activity is under the positive control of CRP. A direct interaction between CRP and the regulatory region of the vvhBA operon was demonstrated by gel-mobility shift assays. The CRP binding site, centered at the 59.5 base pairs upstream of the transcription start site, was mapped by deletion analysis of the vvhBA promoter region and confirmed by DNase I protection assays. These results demonstrate that the vvhBA expression is activated by CRP in a growth-dependent manner, and that CRP exerts its effects by directly binding to DNA upstream of Pvvh.


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