A novel, non-invasive promoter probe vector: cloning of the osmoregulated proU promoter of Escherichia coli K12

Abstract

We have constructed a novel promoter probe plasmid pSB40, containing a unique lac-alpha-tetracycline marker gene tandem, which allows for both positive and negative selection of active promoters. Promoters cloned in pSB40 can be readily mobilized as EcoRI cassettes. Using this vector we have performed a non-invasive analysis of the E. coli chromosome for promoters regulated by osmotic upshift. Only one such promoter, subsequently identified as part of the proU operon, was isolated. A sequence of 253 bp, sufficient to mediate osmotic regulation of the proU promoter, was defined. This E. coli promoter was normally regulated in Salmonella typhimurium, Klebsiella and Citrobacter but not in Shigella. A proU-luxAB fusion plasmid was constructed and used to monitor in vivo real-time kinetics of proU induction following osmotic upshock.