R plasmid dihydrofolate reductase with a dimeric subunit structure

Dihydrofolate reductase specified by plasmid R483 from a trimethoprim-resistant strain of Escherichia coli has been purified 2,000-fold to homogeneity using dye-ligand chromatography, gel filtration, and polyacrylamide gel electrophoresis. The protein migrated as a single band on nondenaturing polyacrylamide gel electrophoresis and had a specific activity of 250 mumol/mg min(-1). The molecular weight was estimated to be 32,000 by gel filtration and 39,000 by Ferguson analysis of polyacrylamide gel electrophoresis. When subjected to electrophoresis in the presence of sodium dodecyl sulfate, the protein migrated as a single 19,000-molecular weight species, a fact that suggests that the native enzyme is a dimer of similar or identical subunits. Antibody specific for R483-encoded dihydrofolate reductase did not cross-react with dihydrofolate reductase encoded by plasmid R67, T4 phage, E. coli RT500, or mouse L1210 leukemia cells. The amino acid sequence of the first 34 NH2-terminal residues suggests that the R483 plasmid dihydrofolate reductase is more closely related to the chromosomal dihydrofolate reductase than is the enzyme coded by plasmid R67.     

Trimethoprim resistance plasmids of different origin encode different drug-resistant dihydrofolate reductases.

Several plasmids mediating resistance to folic acid analogs were studied. The plasmids were in part newly isolated from clinical material and in part R factors studied earlier, such as R483, R721, R751, and R388. By gel chromatography, plasmid-carrying bacterial strains were all found to produce drug-resistant dihydrofolate reductases of a molecular weight distinctly larger than that of the chromosomal enzyme of the host. By gel electrophoresis and zymographic detection technique, analog inhibition characteristics, heat sensitivity, and pH optimum curves, the dihydrofolate reductases induced by R483, R751, and R388, respectively, could be clearly discerned as separate enzymes. Of the newly isolated plasmids all but one coded for a dihydrofolate reductase similar to that of R483. The aberrant one seemed to yield a new enzyme variant as judged from its drug inhibition characteristics and its pH optimum profile. Large differences in drug insensitivity were observed, thus the R751 and R388 enzymes were virtually insensitive to folic acid analogs, whereas the corresponding enzymes from the newly isolated plasmids, and from R483 showed a substantially higher sensitivity. On the other hand these latter enzymes were overproduced, in that the plasmid-carrying bacteria showed a 10- to 20-fold higher content of dihydrofolate reductase than the plasmid-free host strain. Among newly isolated trimethoprim-resistant strains, one was found which overproduced dihydrofolate reductase about 200-fold. In this case the enzyme was only slightly more resistant to folic acid analogs than the chromosomal Escherichia coli K-12 enzyme, and did not seem to be plasmid borne.     

R plasmid dihydrofolate reductase with subunit structure.

Dihydrofolate reductase, specified by the type II plasmid of a trimethoprim-resistant Escherichia coli, was purified 40-fold to homogeneity using a combination of gel filtration, DEAE-Sephacel chromatography, and hydrophobic chromatography. The final product shows a single protein band on polyacrylamide gel electrophoresis and has a specific activity of 1.0 unit/mg. The molecular weight of the purified enzyme is 36,000 as determined both by gel filtration and Ferguson analysis of polyacrylamide gel electrophoresis. In contrast, a single polypeptide with a molecular weight of 8,500 was observed on sodium dodecyl sulfate-gel electrophoresis. These experiments suggest that, unlike any bacteria or vertebrate dihydrofolate reductase previously examined, the type II R plasmid reductase is a tetramer composed of four identical subunits. A partial amino acid sequence determination shows no heterogeneity of the subunits and also no clear homology with any reductase sequence previously reported.     

Comparative topography of the catalytic sites of chromosomal dihydrofolate reductases and that of the enzyme encoded in R 67 plasmid from E. coli

The trimethoprim-resistant dihydrofolate reductase specified in E. coli by plasmid R 67, when compared, with other enzymes catalyzing the same reaction, to have a dissimilar primary, secondary, tertiary and quaternary structure. In regard to the tertiary structure, we show here that the pteridine binding site, in the plasmid-encoded enzyme, has a geometrical similarity with that of other chromosomal specified reductases.     

The amino acid sequence of the trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67.

The amino acid sequence of a trimethoprim-resistant dihydrofolate reductase (EC 1.5.1.3) specified by the R-plasmid R67 is described. The sequence was deduced from automatic and manual sequence analysis of the intact protein, the fragments produced by cyanogen bromide cleavage, and peptides derived from the largest cyanogen bromide fragment by digestion with trypsin, Staphylococcus aureus V8 proteus, chymotrypsin, and Lysobacter enzymogenes alpha-lytic protease. The complete sequence comprises 78 residues in a single polypeptide chain of molecular weight 8444. No evidence of heterogeneity was obtained, indicating that all subunits of the native enzyme are identical. Comparison of the sequence with that of all known dihydrofolate reductases shows no significant sequence homology.     

Characterization of a R plasmid-associated, trimethoprim-resistant dihydrofolate reductase and determination of the nucleotide sequence of the reductase gene

The trimethoprim-resistant dihydrofolate reductase associated with the R plasmid R388 was isolated from strains that over-produce the enzyme. It was purified to apparent homogeneity by affinity chromatography and two consecutive gel filtration steps under native and denaturing conditions. The purified enzyme is composed of four identical subunits with molecular weights of 8300. A 1100 bp long DNA segment which confers resistance to trimethoprim was sequenced. The structural gene was identified on the plasmid DNA by comparing the amino acid composition of the deduced proteins with that of the purified enzyme. The gene is 234 bp long and codes for 78 amino acids. No homology can be found between the deduced amino acid sequence of the R388 dihydrofolate reductase and those of other prokaryotic or eukaryotic dihydrofolate reductases. However, it differs in only 17 positions from the enzyme associated with the trimethoprim-resistance plasmid R67.     

Two distinct types of trimethoprim-resistant dihydrofolate reductase specified by R-plasmids of different compatibility groups.

R-Plasmids from a number of trimethoprim-resistant Escherichia coli and Citrobacter sp. were studied after transfer to E. coli K12 hosts. Each was found to specify a dihydrofolate reductase which was resistant to trimethoprim and Methotrexate, and which could be completely separated from the host chromosomal enzyme by gel filtration. Two distinct types of R-plasmid dihydrofolate reductases were identified. Type I enzymes, typified by the R483 enzyme previously described (Sk?ld, O., and Widh, A. (1974) J. Biol. Chem. 249, 4324-4325), are synthesized in amounts severalfold higher than the chromosomal enzyme. The 50% inhibitory concentrations (I50) of trimethoprim, Methotrexate, and aminopterin are increased several thousandfold over the corresponding values for the chromosomal enzyme. Type II R-plasmid dihydrofolate reductases are synthesized in about the same amount, or less, as the chromosomal enzyme, but are practically several hundredfold higher than those for the type I enzymes. Both types of R-plasmid dihydrofolate reductase showed little difference from the chromosomal enzyme in the binding of dihydrofolate, NADPH, folic acid, and 2,4-diaminopyrimidine.     

Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS257

Trimethoprim resistance mediated by the Staphylococcus aureus multi-resistance plasmid pSK1 is encoded by a structure with characteristics of a composite transposon which we have designated Tn4003. Nucleotide sequence analysis of Tn4003 revealed it to be 4717 bp in length and to contain three copies of the insertion element IS257 (789-790 bp), the outside two of which are flanked by directly repeated 8-bp target sequences. IS257 has imperfect terminal inverted repeats of 27-28 bp and encodes for a putative transposase with two potential alpha-helix-turn-alpha-helix DNA recognition motifs. IS257 shares sequence similarities with members of the IS15 family of insertion sequences from Gram-negative bacteria and with ISS1 from Streptococcus lactis. The central region of the transposon contains the dfrA gene that specifies the S1 dihydrofolate reductase (DHFR) responsible for trimethoprim resistance. The S1 enzyme shows sequence homology with type I and V trimethoprim-resistant DHFRs from Gram-negative bacteria and with chromosomally encoded DHFRs from Gram-positive and Gram-negative bacteria. 5' to dfrA is a thymidylate synthetase gene, designated thyE.     

Characterization of the gene for the chromosomal dihydrofolate reductase (DHFR) of Staphylococcus epidermidis ATCC 14990: the origin of the trimethoprim-resistant S1 DHFR from Staphylococcus aureus?

The gene for the chromosomally encoded dihydrofolate reductase (DHFR) of Staphylococcus epidermidis ATCC 14990 has been cloned and characterized. The structural gene encodes a polypeptide of 161 amino acid residues with a calculated molecular weight of 18,417. This trimethoprim-sensitive (Tmps) DHFR, SeDHFR, differs in only three amino acids (Val-31-->Ile, Gly-43-->Ala, and Phe-98-->Tyr) from the trimethoprim-resistant (Tmpr) S1 DHFR encoded by transposon Tn4003. Since in addition the S. epidermidis gene also forms part of an operon with thyE and open reading frame 140 as in Tn4003, the chromosomally located gene encoding the Tmps SeDHFR is likely to be the molecular origin of the plasmid-located gene encoding the Tmpr S1 DHFR. Site-directed mutagenesis and kinetic analysis of the purified enzymes suggest that a single Phe-->Tyr change at position 98 is the major determinant of trimethoprim resistance.     

A new mechanism of plasmid trimethoprim resistance. Characterization of an inducible dihydrofolate reductase

The dihydrofolate reductase encoded by plasmid pUK1123, which confers only a moderate level of trimethoprim resistance on its host, has been isolated and characterized. This enzyme, designated type IV, differs markedly from all previously described plasmid dihydrofolate reductases. It has a relatively high molecular weight of 46,700 as measured by gel filtration and, unlike previous plasmid dihydrofolate reductases, its synthesis is induced in the presence of increasing concentrations of trimethoprim. It is only slightly resistant to trimethoprim but is competitively inhibited by this drug with an inhibitor binding constant of 63 nM. In addition, the enzyme has a relatively low affinity for the substrate, dihydrofolate (Km = 37 microM). This is the first report of a plasmid trimethoprim resistance mechanism resulting from the induced synthesis of a large molecular weight dihydrofolate reductase which is only slightly resistant to trimethoprim. The possible origins of the type IV enzyme are discussed.     

Crystal structure of a novel trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67

Crystalline R67 dihydrofolate reductase (DHFR) is a dimeric molecule with two identical 78 amino acid subunits, each folded into a beta-barrel conformation. The outer surfaces of the three longest beta strands in each protomer together form a third beta barrel having six strands at the subunit interface. A unique feature of the enzyme structure is that while the intersubunit beta barrel is quite regular over most of its surface, an 8-A "gap" runs the full length of the barrel, disrupting potential hydrogen bonds between beta-strand D in subunit I and the adjacent corresponding strand of subunit II. It is proposed that this deep groove is the NADPH binding site and that the association between protein and cofactor is modulated by hydrogen-bonding interactions along one face of this antiparallel beta-barrel structure. A hypothetical model is proposed for the R67 DHFR-NADPH-folate ternary complex that is consistent with both the known reaction stereoselectivity and the weak binding of 2,4-diamino inhibitors to the plasmid-specified reductase. Geometrical comparison of this model with an experimentally determined structure for chicken DHFR suggests that chromosomal and type II R-plasmid specified enzymes may have independently evolved similar catalytic machinery for substrate reduction.     

Incorporation of the ampicillin resistance transposon into the Escherichia coli K-12 chromosome and into plasmids]

The mutant pEG1 of R-factor RP4 with temperature-sensitive defect in replication, carrying a transposable ampicillin resistance element Tn1 was used to define the frequency of insertion of this element into Escherichia coli K-12 chromosome and some other plasmids. Our results indicate that the frequency of colony forming by bacteria with pEG1-factor on ampicillin medium in non-permissive conditions corresponds to the frequency of Tn1 insertion into bacterial chromosome or some other plasmid (in case when the strains are carrying a second plasmid). The frequency of Tn1 insertion into the chromosome is about 4.10(-4). The defect in recA gene produce no serious change in the frequency of Tn1 insertion into the bacterial chromosome. The translocation of Tn1 element from pEG1-factor to R483, R6 and ColE1 plasmids occurs at 10 to 100-fold-higher frequency than from the plasmid to the chromosome. The insertion of Tn1 into the F'-factor KLF10 and R-factor R64-11 occurs at far lower frequency than that to plasmids R6, R483, or ColE1.     

Nucleotide sequence of dihydrofolate reductase genes from trimethoprim-resistant mutants of Escherichia coli. Evidence that dihydrofolate reductase interacts with another essential gene product

Summary 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 K_i 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-sensitve 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.     

Expression of the plasmid-encoded type I dihydrofolate reductase gene in cultured mammalian cells: a novel selectable marker

A recombinant plasmid has been designed to express the gene encoding a type I methotrexate-resistant dihydrofolate reductase, derived from the bacterial plasmid R483, in DHFR- Chinese hamster ovary cells. Vectors containing the wild type gene, whose coding sequence initiates with a GTG codon, fail to direct the synthesis of detectable levels of protein. Substitution of the GTG codon by an AG codon using in vitro mutagenesis overcomes this block; cells transfected with the modified vector synthesize a functional prokaryotic protein that sustains the growth of these cells in the presence of dihydrofolic acid in the culture media. This property is consistent with the inability of the type I enzyme to reduce folate to dihydrofolate, and enabled the development of a selection strategy whereby prokaryotic and mammalian DHFRs genes could be used sequentially as independently selectable markers.     

The purification and properties of the trimethoprim-resistant dihydrofolate reductase mediated by the R-factor, R388.

The R-factor R388 mediates the production of a trimethoprim-resistant dihydrofolate reductase. This enzyme has a different molecular weight and pH profile to the trimethoprim-sensitive enzyme of the Escherichia coli host. The R-factor mediated enzyme was separated completely from the host E. coli enzyme by DEAE-cellulose ion-exchange chromatography. The purified R-factor enzyme was about 20 000 times less susceptible to trimethoprim than the E. coli enzyme and although it was inhibited competitively by trimethoprim, its inhibitor constant (Ki) was 20 000 times greater than that of the host enzyme. The R388 and E. coli enzymes also differed in their substrate specificity requirements. In addition, the R388 enzyme suprisingly conferred high level resistance to the broad spectrum dihydrofolate reductase inhibitor, amethopterin. The possible origins of the R388 enzyme are discussed.     

Genetic and Biophysical Study of R Plasmids Conferring Sulfonamide Resistance in Shigella Strains Isolated in 1952 and 1956

The conjugative plasmids determining sulfonamide resistance in five Shigella strains, each isolated from a different patient, have been characterized. One S. flexneri 2a strain, isolated in 1952, harbored an fi(+) plasmid of molecular weight 53 x 10(6), which specified synthesis of F-like pili and bore determinants for sulfonamide resistance (Su) and bacteriocinogeny (Col). This plasmid was compatible with plasmids of groups F(I), F(II), I(alpha), and P. A second S. flexneri 2a strain isolated in 1952 harbored an fi(-) plasmid of molecular weight 59 x 10(6), bearing the Su determinant and compatible with all plasmids tested. This strain also harbored an fi(+) group-F(II) plasmid of molecular weight 42 x 10(6), which bore the Col determinant and specified synthesis of F-like pili. Three S. dysenteriae 2 strains isolated in 1956 carried apparently identical fi(-) plasmids of molecular weight 58 x 10(6), which bore the Su determinant, could form transconjugants in Pseudomonas but not in Proteus, and were incompatible with the P-group plasmid RP4.