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J. Biol. Chem., Vol. 277, Issue 49, 47292-47299, December 6, 2002
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From the Cytolytic hemolysin, a gene product of
vvhA, is a putative virulence factor of the pathogenic
bacterium Vibrio vulnificus. We have previously shown that
hemolysin production is repressed by adding glucose to culture media
and that production can be restored by adding cAMP. 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 cAMP receptor protein (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 59.5 bp
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.
Promoter Analysis and Regulatory Characteristics of vvhBA
Encoding Cytolytic Hemolysin of Vibrio
vulnificus*
,,,,¶
Department of Food Science and Technology,
Department of Molecular Biotechnology, and Institute of Biotechnology,
Chonnam National University, Kwang-Ju, 500-757 and the
§ Department of Food Science and Technology, School of
Agricultural Biotechnology, Seoul National University, Suwon 441-744, South Korea
*
This work was supported by the 21C Frontier Microbial
Genomics and Applications Center Program, Ministry of Science & Technology (Grant MG02-0201-004-2-1-1) (to S. H. C.), Republic of
Korea.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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