The metF gene in Escherichia coli and Salmonella typhimurium is under negative transcriptional control by the MetJ repressor. Expression of an S. typhimurium metF-lacZ gene fusion is repressed up to 10-fold by methionine addition to the growth medium in E. coli hosts encoding wild-type MetJ repressor; this repression is not seen in metJ mutants. metR mutations which eliminate the MetR activator protein result in two-to threefold-more-severe repression by the MetJ repressor. In a metJ metR double mutant, however, the level of metF-lacZ expression is the same as in a metj mutant, suggesting that MetR antagonizes MetJ-mediated methionine repression of the metF promoter. A DNA footprint analysis showed that MetR binds to a DNA fragment carrying the metF promoter and protects two separate regions from DNase I digestion: a 46-bp region from position -50 to -95 upstream of the transcription initiation site and a 24-bp region from about position +62 to +85 downstream of the transcription initiation site and within the metF structural gene. Nucleotide changes in each of the MetR-binding sites away from the consensus sequence disrupt MetR-mediated regulation of the metF-lacZ fusion.The folate branch of the methionine pathway in Salmonella typhimunum includes the metE, metH, and metF genes. The metF gene product (5,10-methylenetetrahydrofolate reductase) is involved in the biosynthesis of 5-methyltetrahydrofolate from 5,10-methylenetetrahydrofolate (20 (24), and pFlac carries a fusion of the S. typhimunum metF control region and the first 12 amino acid codons of the structural gene to the eighth codon of lacZ (25).Media and growth conditions. Luria agar, Luria broth, and glucose minimal medium (GM) have been described previously (23). Supplements were added at the following concentrations: amino acids, 50 ,ug/ml; vitamin B1, 1 ,ug/ml; ampicillin, 150 pug/ml; and 5-bromo-4-chloro-3-indolyl-P-Dgalactopyranoside (X-Gal), 40 ,ug/ml. 13-Galactosidase enzyme assay. ,B-Galactosidase activity was assayed as described by Miller (17), using the chloroform-sodium dodecyl sulfate lysis procedure. Results are the averages of two or more assays in which each sample was measured in triplicate.DNA manipulation. Restriction enzyme digestions, ligations, plasmid and phage DNA isolations, and DNA fragment isolations were done as described by Maniatis et al. (14). Transformations were done by using polyethylene glycol-and dimethyl sulfoxide-prepared competent cells (5).Site-directed mutagenesis. A 746-bp EcoRI-BamHI fragment carrying the metF control region was isolated from plasmid pFlac (25)