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Applied and Environmental Microbiology, June 2003, p. 3385-3392, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3385-3392.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Development of a Thermally Regulated Broad-Spectrum Promoter System for Use in Pathogenic Gram-Positive Species

David A. Schofield,* Caroline Westwater, Brian D. Hoel, Phillip A. Werner, James S. Norris, and Michael G. Schmidt

Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina 29403

Received 10 February 2003/ Accepted 25 March 2003

Selectively regulating gene expression is an essential molecular tool that is lacking for many pathogenic gram-positive bacteria. In this report, we describe the evaluation of a series of promoters regulated by the bacteriophage P1 temperature-sensitive C1 repressor in Enterococcus faecium, Enterococcus faecalis, and Staphylococcus aureus. Using the lacZ gene to monitor gene expression, we examined the strength, basal expression, and induced expression of synthetic promoters carrying C1 operator sites. The promoters exhibited extremely low basal expression and, under inducing conditions, gave high levels of expression (100- to 1,000-fold induction). We demonstrate that the promoter system could be modulated by temperature and showed rapid induction and that the mechanism of regulation occurred at the level of transcription. Controlled expression with the same constructs was also demonstrated in the gram-negative bacterium Escherichia coli. However, low basal expression and the ability to achieve derepression were dependent on both the number of mismatches in the C1 operator sites and the promoter driving c1 expression. Since the promoters were designed to contain conserved promoter elements from gram-positive species and were constructed in a broad-host-range plasmid, this system will provide a new opportunity for controlled gene expression in a variety of gram-positive bacteria.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, BSB 201, Medical University of South Carolina, 173 Ashley Ave., Charleston, SC 29403. Phone: (843) 792-7703. Fax: (843) 792-2464. E-mail: schofida{at}musc.edu.

Applied and Environmental Microbiology, June 2003, p. 3385-3392, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3385-3392.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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