Mao, Weimin (1997-02). Genetic analysis of the stationary-phase induced and [sigma]S-independent promoter of the mcb operon in Escherichia coli. Master's Thesis.
Thesis
Pmcb is the promoter of the mcb operon, which contains the genes needed for production of the peptide antibiotic microcin B 17. In Escherichia coli, transcription from Pmcb is induced when cells are starved for carbon, nitrogen, or phosphorus, or enter stationary phase in rich medium. This induction is independent of all regulatory pathways currently known to be involved in regulating expression of other stationary-phase induced genes. To understand the molecular mechanisms controlling the expression from Pmcb during the starvation response in E. coli, genetic and molecular biology methods were used. A series of promoter deletion mutants was made to define the minimal promoter. Analysis of the mutants showed that while sequences upstream of-54 region were required for maximal promoter activity, sequences downstream of-54 region were sufficient for stationary-phase induction. The 5' ends of transcripts from several of the Pmcb deletion mutants were mapped, and they all have similar 5'ends, suggesting that they use the same transcriptional start site. Random mutagenesis was performed to identify the bases important for promoter activity. Mutations at-8,-11,-30, -31 and-32 greatly decreased promoter activity. These sites might have contact with RNAP or a transcriptional activator. Mutations between-105 and-138 define the potential binding site(s) for Pmcb regulators. The regulatory proteins OmpR and EmrR affect transcription from Pmcb. My results showed that the site(s) for OmpR-dependent activation is between- 105 and-1 38, while the site(s) for repression by EmrR lies downstream of-54. In the absence of both OmpR and EmrR, there is still about 6-to 8-fold stationary-phase induction, indicating that there are other factors involved in the regulation of Pmcb.
