Characterization of the sigmaE-dependent rpoEp3 promoter of Salmonella enterica serovar Typhimurium.
Using a two-plasmid system, we recently identified sigma(E)-dependent promoters directing expression of the sigma(E) regulon genes in Salmonella enterica serovar Typhimurium (S. Typhimurium). Comparison of the promoters revealed a consensus sequence almost identical to the sigma(E)-dependent rpoEp3 promoter directing expression of rpoE. This two-plasmid system was previously optimized to identify nucleotides critical for the rpoEp3 promoter activity. However, two highly conserved nucleotides in the sigma(E) consensus sequence were not identified by this screening. In the present study, we have improved the two-plasmid screening system using a new optimized error-prone PCR mutagenesis. Together with site-directed mutagenesis, we further identified nucleotides critical for activity of the rpoEp3 promoter and quantified the effect of the particular mutation upon promoter activity. All the identified critical nucleotides of the rpoEp3 promoter (in capital) were located in the -35 (ggAACtt) and -10 (gTCtaA) regions and corresponded to the most conserved nucleotides in the sigma(E) consensus sequence. The expression of the wild-type and mutated rpoEp3 promoters was confirmed in S. Typhimurium and was found to exhibit a different pattern of sigma(E) activation compared with Escherichia coli, with a peak rpoEp3 promoter activity in early stationary phase followed by a decrease in late stationary phase.
Skovierova
Henrieta
H
Institute of Molecular Biology, Center of Excellence for Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovak Republik.
Rezuchova
Bronislava
B
Homerova
Dagmar
D
Roberts
Mark
M
Kormanec
Jan
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D016384:Consensus Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D008156:Luciferases / Q000032:analysis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic / Q000502:physiology; D018085:Regulon / Q000235:genetics; D012486:Salmonella typhimurium / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D012808:Sigma Factor / Q000235:genetics* / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D001616:beta-Galactosidase / Q000032:analysis
D012808:Sigma Factor; D014157:Transcription Factors; C063202:sporulation-specific sigma factors; D008156:Luciferases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D001616:beta-Galactosidase