Tight regulation and modulation via a C1-regulated promoter in Escherichia coli and Pseudomonas aeruginosa.
We describe the development and analysis of a novel promoter system regulated by the bacteriophage P1 temperature-sensitive C1 repressor. Using transcriptional fusions to the lacZ reporter gene to monitor gene expression, we show that the ratio of induction/repression can be up to 1500-fold in Escherichia coli. The promoters exhibited extremely tight repression and could be modulated over a range of temperatures. The utility of the promoter system was tested in Pseudomonas aeruginosa. C1 effectively repressed transcription; however, only modest induction was achieved. To increase the levels of induction, the amount of c1 was modulated at the mRNA level by using a LacI-regulated promoter. This resulted in a 59-fold induction in gene expression under inducing conditions. As the promoter system was constructed in a broad-host range vector and utilized the C1 repressor from a broad-host range phage, the system will provide the potential for controlled gene expression in Gram-negative bacteria.
Schofield
David A
DA
Department of Microbiology and Immunology, BSB-201, 173 Ashley Avenue, Medical University of South Carolina, Charleston SC 29403, USA. schofida@musc.edu
Westwater
Caroline
C
Dolan
Joseph W
JW
Norris
James S
JS
Schmidt
Michael G
MG
D017101:Bacteriophage P1 / Q000235:genetics; D001483:Base Sequence; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D007763:Lac Operon; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000235:genetics*
D004269:DNA, Bacterial