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Characterization of the gene for chromosomal trimethoprim-sensitive dihydrofolate reductase of Staphylococcus aureus ATCC 25923.

ABSTRACT: The gene for the trimethoprim-sensitive (Tmps) chromosomal dihydrofolate reductase (DHFR) of Staphylococcus aureus ATCC 25923 was cloned and characterized. The structural gene encodes a polypeptide of 159 amino acid residues and has a calculated molecular weight of 18,251. The amino acid sequences of this Tmps DHFR and those of the trimethoprim-resistant type S1 DHFR encoded by transposon Tn4003 are 80% identical. In contrast to the trimethoprim-resistant enzyme, the Tmps DHFR can be highly overexpressed in Escherichia coli, with most of the recombinant protein occurring in a soluble and an active form.

SUBMITTER: Dale GE

PROVIDER: S-EPMC187982 | biostudies-other | 1993 Jul

REPOSITORIES: biostudies-other

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Characterization of the gene for chromosomal trimethoprim-sensitive dihydrofolate reductase of Staphylococcus aureus ATCC 25923.

Dale G E GE Then R L RL Stüber D D

Antimicrobial agents and chemotherapy 19930701 7

The gene for the trimethoprim-sensitive (Tmps) chromosomal dihydrofolate reductase (DHFR) of Staphylococcus aureus ATCC 25923 was cloned and characterized. The structural gene encodes a polypeptide of 159 amino acid residues and has a calculated molecular weight of 18,251. The amino acid sequences of this Tmps DHFR and those of the trimethoprim-resistant type S1 DHFR encoded by transposon Tn4003 are 80% identical. In contrast to the trimethoprim-resistant enzyme, the Tmps DHFR can be highly over ...[more]

PMID: 8363365

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Project description:Enterococci are usually susceptible in vitro to trimethoprim; however, high-level resistance (HLR) (MICs, >1,024 microg/ml) has been reported. We studied Enterococcus faecalis DEL, for which the trimethoprim MIC was >1,024 microg/ml. No transfer of resistance was achieved by broth or filter matings. Two different genes that conferred trimethoprim resistance when they were cloned in Escherichia coli (MICs, 128 and >1,024 microg/ml) were studied. One gene that coded for a polypeptide of 165 amino acids (MIC, 128 microg/ml for E. coli) was identical to dfr homologs that we cloned from a trimethoprim-susceptible E. faecalis strain, and it is presumed to be the intrinsic E. faecalis dfr gene (which causes resistance in E. coli when cloned in multiple copies); this gene was designated dfrE. The nucleotide sequence 5' to this dfr gene showed similarity to thymidylate synthetase genes, suggesting that the dfr and thy genes from E. faecalis are located in tandem. The E. faecalis gene that conferred HLR to trimethoprim in E. coli, designated dfrF, codes for a predicted polypeptide of 165 amino acids with 38 to 64% similarity with other dihydrofolate reductases from gram-positive and gram-negative organisms. The nucleotide sequence 5' to dfrF did not show similarity to the thy sequences. A DNA probe for dfrF hybridized under high-stringency conditions only to colony lysates of enterococci for which the trimethoprim MIC was >1,024 microg/ml; there was no hybridization to plasmid DNA from the strain of origin. To confirm that this gene causes trimethoprim resistance in enterococci, we cloned it into the integrative vector pAT113 and electroporated it into RH110 (E. faecalis OG1RF::Tn916DeltaEm) (trimethoprim MIC, 0.5 microg/ml), which resulted in RH110 derivatives for which the trimethoprim MIC was >1, 024 microg/ml. These results indicate that dfrF is an acquired but probably chromosomally located gene which is responsible for in vitro HLR to trimethoprim in E. faecalis.

Dataset Information

Characterization of the gene for chromosomal trimethoprim-sensitive dihydrofolate reductase of Staphylococcus aureus ATCC 25923.

Publications

Characterization of the gene for chromosomal trimethoprim-sensitive dihydrofolate reductase of Staphylococcus aureus ATCC 25923.

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