ISSN:
1574-6968
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Biology
Notes:
We previously described a two-plasmid system for the identification of promoters recognized by Salmonella enterica serovar Typhimurium (S. Typhimurium) σE. The S. Typhimurium σE-dependent rpoEp3 promoter was active in the E. coli two-plasmid system only after arabinose-induced expression of S. Typhimurium rpoE. In the present study, we have exploited this two-plasmid system for the identification of nucleotides critical for activity of the rpoEp3 promoter. A library of randomly mutated DNA fragments containing the rpoEp3 promoter was cloned upstream of a lacZα reporter gene and screened for activity in the presence of S. Typhimurium σE. The clones exhibiting reduced LacZ activity were sequenced to identify the mutations. The activity of the mutated rpoEp3 promoters were studied further using a luciferase-based promoter-probe plasmid. All of the important nucleotides of the rpoEp3 promoter (in capital) were located in the −35 (ggAActt) and −10 (TctaA) regions. The critical nucleotides were also the most conserved in known σE-dependent promoters. The study also revealed the importance of the 16-bp spacing between −10 and −35 region, as reducing the spacing to 15-bp greatly reduced activity of the promoter. This method should be generally applicable for the identification of important nucleotides in the cognate promoters of other σ factors.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1111/j.1574-6968.2004.tb09760.x
