Characterization of a staphylococcal trimethoprim resistance gene and its product.
Resistance to trimethoprim (Tp) is mediated by a plasmid-encoded gene in staphylococci. The gene is responsible for high-level resistance (MIC, greater than 1,000 micrograms/ml) in both its native host and when cloned on high-copy-number vectors in Escherichia coli. Analysis of subclones of the staphylococcal Tp gene on E. coli expression vectors and estimation of the size of full and truncated proteins produced in E. coli minicells generated an approximate size limit of 505 base pairs for the gene and 18,500 daltons for the gene product. Crude extracts of E. coli containing the cloned gene had dihydrofolate reductase (DHFR) specific activity that was more than 100 times greater than that of control cells and more than 1,000 times more resistant to trimethoprim inhibition. The amount of trimethoprim required for a 50% reduction in the specific activity of staphylococcal DHFR differed from those of cells containing DHFR types I, II, or III, enzymes mediating Tp in members of the family Enterobacteriaceae. In addition, the size of the monomeric staphylococcal DHFR protein was larger than that of any of the gram-negative DHFRs both compared with published sequence data and as observed by direct comparison on polyacrylamide gels. Finally, there was no homology between a DNA fragment containing the cloned staphylococcal gene and DNA encoding any of the gram-negative DHFRs. Thus, the staphylococcal Tp gene codes for a single protein with DHFR activity that appears to be unrelated to DHFR genes that mediate Tp in members of the Enterobacteriaceae.
Coughter
J P
JP
Department of Microbiology/Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0001.
Johnston
J L
JL
Archer
G L
GL
D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000032:analysis; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D009693:Nucleic Acid Hybridization; D011815:R Factors; D012689:Sequence Homology, Nucleic Acid; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D014295:Trimethoprim / Q000494:pharmacology*
D004269:DNA, Bacterial; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D004262:DNA Restriction Enzymes