Integration host factor (IHF) modulates the expression of the pyrimidine-specific promoter of the carAB operons of Escherichia coli K12 and Salmonella typhimurium LT2.
We report the identification of Integration Host Factor (IHF) as a new element involved in modulation of P1, the upstream pyrimidine-specific promoter of the Escherichia coli K12 and Salmonella typhimurium carAB operons. Band-shift assays, performed with S-30 extracts of the wild type and a himA, hip double mutant or with purified IHF demonstrate that, in vitro, this factor binds to a region 300 bp upstream of the transcription initiation site of P1 in both organisms. This was confirmed by deletion analysis of the target site. DNase I, hydroxyl radical and dimethylsulphate footprinting experiments allowed us to allocate the IHF binding site to a 38 bp, highly A+T-rich stretch, centred around nucleotide -305 upstream of the transcription initiation site. Protein-DNA contacts are apparently spread over a large number of bases and are mainly located in the minor groove of the helix. Measurements of carbamoyl-phosphate synthetase (CPSase) and beta-galactosidase specific activities from car-lacZ fusion constructs of wild type or IHF target site mutants introduced into several genetic backgrounds affected in the himA gene or in the pyrimidine-mediated control of P1 (carP6 or pyrH+/-), or in both, indicate that, in vivo, IHF influences P1 activity as well as its control by pyrimidines. IHF stimulates P1 promoter activity in minimal medium, but increases the repressibility of this promoter by pyrimidines. These antagonistic effects result in a two- to threefold reduction in the repressibility of promoter P1 by pyrimidines in the absence of IHF binding. IHF thus appears to be required for maximal expression as well as for establishment of full repression. IHF could exert this function by modulating the binding of a pyrimidine-specific regulatory molecule.
Charlier
D
D
Research Institute, CERIA-COOVI, Brussels, Belgium.
Roovers
M
M
Gigot
D
D
Huysveld
N
N
Piérard
A
A
Glansdorff
N
N
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics*; D002474:Cell-Free System; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004755:Enterobacteriaceae / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D034982:Integration Host Factors; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009876:Operon; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011743:Pyrimidines / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000235:genetics; D017384:Sequence Deletion
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D011743:Pyrimidines; C440508:integration host factor, Salmonella; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)