title: Development of a thermally regulated broad-spectrum promoter system for use in pathogenic gram-positive species.
abstract: 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.
authors.affiliation: Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina 29403, USA. schofida@musc.edu
authors.forename: David A
authors.initials: DA
authors.lastname: Schofield
authors.forename: Caroline
authors.initials: C
authors.lastname: Westwater
authors.forename: Brian D
authors.initials: BD
authors.lastname: Hoel
authors.forename: Phillip A
authors.initials: PA
authors.lastname: Werner
authors.forename: James S
authors.initials: JS
authors.lastname: Norris
authors.forename: Michael G
authors.initials: MG
authors.lastname: Schmidt
mesh_terms: D017101:Bacteriophage P1 / Q000235:genetics; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006095:Gram-Positive Cocci / Q000235:genetics / Q000472:pathogenicity*; D006358:Hot Temperature; D006801:Humans; D007763:Lac Operon; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012097:Repressor Proteins; D014158:Transcription, Genetic; D014764:Viral Proteins
chemical_list: D012097:Repressor Proteins; D014764:Viral Proteins; C076896:repressor protein C1, bacteriophage