Nucleotide sequence of dihydrofolate reductase genes from trimethoprim-resistant mutants of Escherichia coli. Evidence that dihydrofolate reductase interacts with another essential gene product.
We report the construction of recombinant plasmids containing the dihydrofolate reductase structural gene (fol) from several trimethoprim-resistant mutants of Escherichia coli. Strains carrying some of these plasmids produced approximately 6% of their soluble cell protein as dihydrofolate reductase and are therefore excellent sources of the purified enzyme for inhibitor binding or mechanistic studies. The nucleotide sequence of the fol region from each of the plasmids was determined. A plasmid derived from a Ki mutant which produced a dihydrofolate reductase with lowered affinity for trimethoprim contained a mutation in the structural gene that altered the sequence of the polypeptide in a conserved region which is adjacent to the dihydrofolate binding site. Two other independently-isolated mutants which overproduced dihydrofolate reductase had a mutation in the -35 region of the fol promoter. One of them, strain RS35, was also temperature-sensitive for growth in minimal medium. This phenotype was shown to be the result of an additional mutation in a locus unlinked to fol by P1 transduction. The fol regions from two temperature-independent revertants of strain RS35 were sequenced. One of these had a mutation within the dihydrofolate reductase structural gene which altered some properties of the enzyme. This confirmed some previous enzymological data which suggested that some revertants of strain RS35 had mutations in fol (Sheldon 1977). These results suggest that dihydrofolate reductase interacts physically with some other essential gene product in E. coli.
D001483:Base Sequence; D004269:DNA, Bacterial; D004352:Drug Resistance, Microbial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D005798:Genes, Bacterial / Q000187:drug effects; D006358:Hot Temperature; D009154:Mutation; D010957:Plasmids; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic; D014295:Trimethoprim / Q000494:pharmacology*
D004269:DNA, Bacterial; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase