Bacteriophage Tail Components: II. Dihydrofolate Reductase in T4D Bacteriophage

The protein component of the T-even bacteriophage coat which binds the phage-specific dihydropteroyl polyglutamate has been identified as the phage-induced dihydrofolate reductase. Dihydrofolate reductase activity has been found in highly purified preparations of T-even phage ghosts and phage substructures after partial denaturation. The highest specific enzymatic activity was found in purified tail plate preparations, and it was concluded that this enzyme was a structural component of the phage tail plate. Phage viability was directly correlated with the enzymological properties of the phage tail plate dihydrofolate reductase. All reactions catalyzed by this enzyme which changed the oxidation state of the phage dihydrofolate also inactivated the phage. Properties of two T4D dihydrofolate reductase-negative mutants, wh1 and wh11, have been examined. Various lines of evidence support the view that the product of the wh locus of the phage genome is normally incorporated into the phage tail structure. The effects of various dihydrofolate reductase inhibitors on phage assembly in in vitro complementation experiments with various extracts of conditional lethal T4D mutants have been examined. These inhibitors were found to specifically block complementation when added to extracts which did not contain preformed tail plates. If tail plates were present, inhibitors such as aminopterin, did not affect further phage assembly. This specific inhibition of tail plate formation in vitro confirms the analytical and genetic evidence that this phage-induced "early" enzyme is a component of the phage coat.     

Bacteriophage T4 Virion Dihydrofolate Reductase: Approaches to Quantitation and Assessment of Function

This paper is concerned with the physiological role(s) of T4 phage-coded dihydrofolate reductase, which functions both in DNA precursor metabolism and as a virion protein. (i) We have detected enzyme activity in noninfectious particles produced under restrictive conditions by gene 11 mutants. This supports the conclusion of Kozloff et al. (J. Virol. 16:1401-1408, 1975) that the protein lies in the baseplate, covered by the gene 11 protein. (ii) We have obtained further evidence for virion dihydrofolate reductase as the target for neutralizing activity of T4 dihydrofolate reductase antiserum and as a determinant of the heat lability of the virion. This derives from our observation that the reductases specified by T4B and T4D differ in several properties. (iii) We have investigated several anomalous properties of T4 mutants bearing deletions that reportedly extend into or through the frd gene, which codes for dihydrofolate reductase. Evidence is presented that the deletions in fact do not extend through frd. These strains direct the synthesis of material that cross-reacts with antiserum to homogeneous dihydrofolate reductase. Moreover, they are all quite sensitive to the phage-neutralizing effects of this antiserum. In addition, they are restricted by several of the hospital strains, wild-type strains of Escherichia coli supplied by the California Institute of Technology group. (iv) We have attempted to detect dihydrofolate reductase among early-synthesized proteins present in T4 tails. Two such proteins are seen, one of which is evidently the gene 25 product and one that is a bacterial protein. Quantitation of our electrophoretic technique has allowed determination of the number of molecules of some T4 tail components present per virion. (v) Finally, we have compared the T4 dihydrofolate reductase with the corresponding enzyme specified by two plasmids conferring resistance to trimethoprim (Skold and Widh, J. Biol. Chem. 249:4324-4325, 1974). Although the enzymes are similar in some properties, they differ in several important respects, including immunological activity.     

Polyoma virus and cyclic AMP-mediated control of dihydrofolate reductase mRNA abundance in methotrexate-resistant mouse fibroblasts.

As a model cell culture system for studying polyoma-mediated control of host gene expression, we isolated methotrexate-resistant 3T6 cells in which one of the virus-induced enzymes, dihydrofolate reductase, is a major cellular protein. In highly methotrexate-resistant cell lines dihydrofolate reductase synthesis accounts for over 10% that of soluble portein, corresponding to an increase of approximately 100-fold over the level in parental cells. This increase in dihydrofolate reductase synthesis is due to a corresponding increase in the abundance of dihydrofolate reductase mRNA and gene sequences. We have used these cells to show that infection with polyoma virus results in a 4- to 5-fold increase in the relative rate of dihydrofolate reductase synthesis and a corresponding increase in dihydrofolate reductase mRNA abundance. The increase in dihydrofolate reductase synthesis begins 15 to 20 h after infection and continues to increase until cell lysis. These observations represent the first direct evidence that viral infection of eukaryotic cells results in the increased synthesis of a specific cellular enzyme and an increase in the abundance of a specific cellular mRNA. In order to gain additional insight into the control of dihydrofolate reductase synthesis we examined other parameters affecting dihydrofolate reductase synthesis. We found that the addition of fresh serum to stationary phase cells results in a 2-fold stimulation of dihydrofolate reductase synthesis, beginning 10 to 12 h after serum addition. Serum stimulation of dihydrofolate reductase synthesis is completely inhibited by the presence of dibutyryl cyclic AMP as well as by theophylline or prostaglandin E1, compounds which cause an increase in intracellular cyclic AMP levels. In fact, the presence of dibutyryl cyclic AMP and theophylline results in a 2- to 3-fold decrease in the rate of dihydrofolate reductase synthesis and the abundance of dihydrofolate reductase mRNA. However, in contrast to the effect on serum stimulation, dibutyryl cyclic AMP and theophylline do not inhibit polyoma virus induction of dihydrofolate reductase synthesis or dihydrofolate reductase mRNA levels. These observations suggest that dihydrofolate reductase gene expression is controlled by at least two regulatory pathways: one involving serum that is blocked by high levels of cyclic AMP and another involving polyoma induction that is not inhibited by cyclic AMP.     

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.     

Dihydrofolate Reductase Activity in Strains of Streptococcus faecium var. durans Resistant to Methasquin and Amethopterin

Resistance to the antifolates methasquin and amethopterin has been studied in new strains of Streptococcus faecium var. durans. Two methasquin-resistant strains (SF/MQ, SF/MQ(T)) and an amethopterin-resistant strain (SF/AM) were selected independently from the wild-type S. faecium var. durans (SF/O). SF/MQ(T) is a thymine auxotroph. Total dihydrofolate reductase activity was elevated in each of the resistant strains. The greatest increase (36-fold) was observed in extracts of SF/AM. The methasquin-resistant strains, SF/MQ and SF/MQ(T), had 29-fold and 8-fold, respectively, more dihydrofolate reductase activity than the parental strain. Total dihydrofolate reductase activity of SF/O was separable by gel filtration into two components: a folate reductase (11%) and a specific dihydrofolate reductase (89%). Folate reductase activity was associated with 88% of the total dihydrofolate reductase activity of SF/MQ(T), with specific dihydrofolate reductase activity accounting for the remaining 12%. In SF/MQ and SF/AM, folate reductase activity was associated with 97% of the total dihydrofolate reductase activity. Studies of the inhibition by methasquin and amethopterin of partially purified folate reductase and specific dihydrofolate reductase of the mutant strains suggested that resistance was not accompanied by changes in the affinities of these enzymes for either antifolate.     

Increased levels of dihydrofolate reductase mRNA can be measured in normal, growth-stimulated mouse fibroblasts

Levels of mRNA for the enzyme dihydrofolate reductase (EC 1.5.1.3) were determined in growth-stimulated 3T6 cells which contained wild-type dosage of the gene coding for this enzyme. As in the case of methotrexate-resistent cells having highly amplified levels of genes for dihydrofolate reductase, an increase in dihydrofolate reductase mRNA by a factor of 2–4 can be determined when cells enter the S phase. This increase is inhibited by sodium butyrate (which inhibits growth-stimulated 3T6 cells in mid G1 phase) but not by hydroxyurea (which inhibits in early S phase). We conclude that with the available methods it is possible to study the regulation of S phase-specific enzymes after growth stimulation at the level of the mRNA, even if gene amplification is not possible or desirable.     

Bacteriophage T4-coded dihydrofolate reductase: synthesis, turnover, and location of the virion protein.

Dihydrofolate reductase plays a dual role in bacteriophage T4, first, as an enzyme of thymidylate metabolism, and second, as a protein component of the tail baseplate. Antibody to the purified enzyme has been used to study its synthesis and intracellular turnover. The antibody specifically precipitates one protein from T4D-infected cell extracts. This has been identified as dihydrofolate reductase, although the polypeptide molecular weight (22,000) is lower than that earlier determined for this enzyme. The protein comigrates on gels with pY, a genetically undefined protein component of the baseplate. However, it is not pY, for pY is synthesized late in infection, whereas virtually no dihydrofolate reductase synthesis occurs later than 10 min after infection at 37 degrees C. Dihydrofolate reductase, once formed, is neither degraded nor converted to proteins of higher or lower molecular weight. Thus, it is probably incorporated into virions at the same molecular weight as that of the soluble enzyme. 125I-radiolabeled antibody binds to the wedge substructure of the baseplate, and this binding is blocked by preincubation with purified T4 dihydrofolate reductase. Thus, the enzyme protein seems to be a component of the wedge.     

Growth of a Dihydrofolate Reductaseless Mutant of Bacteriophage T4

A mutant of bacteriophage T4 was isolated which was unable to induce virus-specific dihydrofolate reductase in infected cells. The mutant was able to form several other early enzymes of pyrimidine metabolism. Growth of the mutant in a wild-type host, Escherichia coli B, was compared with that of the parent strain, T4BO(1), and T4td8, a mutant which lacks the ability to induce thymidylate synthetase. Growth studies were carried out in minimal medium, which gave higher growth rates and phage yields than the supplemented media used in previous studies. The reductase mutant formed deoxyribonucleic acid and plaque-forming particles at a rate slightly higher than the synthetase mutant but 1.5-to 2-fold lower than that of the wild-type phage under all conditions studied. The addition of thymine to a culture infected by the mutant increased the growth rate significantly, suggesting that the genetic lesion leads to a partial thymidylate deficiency. Like other viral genes controlling steps in thymidylate metabolism, the dihydrofolate reductase gene appears to be useful but not completely essential for growth.     

Analysis of T4 bacteriophage deletion mutants that lack td and frd genes.

The roles of bacteriophage T4-encoded thymidylate synthase and dihydrofolate reductase as virion structural components have been further investigated. Two mutants, del(63-32)7 and del(63-32)9, bearing deletions in the gene 63 to 32 region of the T4 genome, were characterized by Southern blotting analysis, as well as by enzyme and immunological assays. Our results have confirmed the original report of Homyk and Weil (Virology 61:505-523, 1974) that del7 and del9 each carries a deletion of about 4.0 kilobases, which totally eliminates the frd gene, encoding dihydrofolate reductase, and the td gene, encoding thymidylate synthase. With the well-characterized deletion mutants, along with newly prepared antisera against T4-encoded thymidylate synthase and dihydrofolate reductase, we have reevaluated the experimental results supporting the idea that T4-induced dihydrofolate reductase and thymidylate synthase are essential T4 baseplate components and antigenic determinants of phage particles. These deletion mutant phages are not targets for neutralization by antisera against either dihydrofolate reductase or thymidylate synthase purified from cloned genes. Furthermore, these newly prepared antisera also cannot neutralize the infectivity of T4D. Those results suggest that the phage-neutralizing components in the old antisera used in the earlier studies were not antibodies against either dihydrofolate reductase or thymidylate synthase but were antibodies against minor components of the purified enzyme preparations. Study of the biological properties of the deletion mutants indicates that T4-induced thymidylate synthase and dihydrofolate reductase play significant roles in growth of the phage beyond their known roles in nucleotide biosynthesis, even though they are apparently not essential for phage viability. The deletion mutants should be useful in defining these roles.     

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.     

Bacteriophage-coded thymidylate synthetase. Evidence that the T4 enzyme is a capsid protein

It has previously been shown that T4 bacteriophage-coded dihydrofolate reductase is a capsid protein, specifically an element of the tail plate. This paper presents evidence that thymidylate synthetase is also a structural protein. Antiserum prepared against purified T4 thymidylate synthetase neutralizes T4 infectivity. Evidence is presented that structural thymidylate synthetase is the target of the antiphage component of the serum.The td gene in T4 codes for thymidylate synthetase. We have crossed the td gene from phage T6 into T4 and eliminated other T6 genetic material from the hybrid phage by extensive backcrossing. The hybrid phage, T4td^T6, is inactivated at 60 °C significantly more rapidly than the parent phage, T4D. Thus, the td gene is a determinant of a physical property of the virion, providing direct confirmation that thymidylate synthetase is a capsid protein. At present the role of the virion-bound enzyme is unknown.     

Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability. I. Dihydrofolate reductase, thymidylate synthetase and formyltetrahydrofolate synthetase in amethopterin-resistant and -sensitive strains of Pediococcus cerevisiae.

Pediococcus cerevisiae/AMr, resistant to amethopterin, possesses a higher dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) activity than the parent, a folate-permeable and thus amethopterin-susceptible strain and than the wild-type. The properties of dihydrofolate reductase from the three strains have been compared. Temperature, pH optima, heat stability, as well amethopterin binding did not reveal significant differences between the enzymes from the susceptible and resistant strains. The enzyme from the wild-type was 10 times more sensitive to inhibition by amethopterin and more susceptible to heat denaturation. The apparent Km values for dihydrofolate in enzymes from the three strains were in the range of 4.8--7.2 muM and for NADPH 6.5--8.0 muM. The amethopterin-resistant strain exhibited cross-resistance to trimethoprim and was about 40-fold more resistant to the latter than the sensitive parent and the wild-type. The resistance to trimethoprim appears to be a direct result of the increased dihydrofolate reductase activity. Inhibition of dihydrofolate reductase activity by this drug was similar in the three strains. 10--20 nmol caused 50% inhibition of 0.02 enzyme unit. Trimethoprim was about 10 000 times less effective inhibitor of dihydrofolate reductase than amethopterin. The cell extract of the AMr strain possessed a folate reductase activity three times higher than that of the sensitive strain. The activities of other folate-related enzymes like thymidylate synthetase and 10-formyltetrahydrofolate synthetase (formate: tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) were similar in the three strains studied.     

Dihydrofolate reductase. The stereochemistry of inhibitor selectivity

X-ray structural results are reported for 10 triazine and pyrimidine inhibitors of dihydrofolate reductase, each one studied as a ternary complex with NADPH and chicken dihydrofolate reductase. Analysis of these data and comparison with structural results from the preceding paper (Matthews, D.A., Bolin, J.T., Burridge, J.M., Filman, D.J., Volz, K.W., Kaufman, B. T., Beddell, C.R., Champness, J.N., Stammers, D.K., and Kraut, J. (1985) J. Biol. Chem. 260, 381-391) in which we contrasted binding of the antibiotic trimethoprim (TMP) to chicken dihydrofolate reductase on the one hand with its binding to Escherichia coli dihydrofolate reductase on the other, permit identification of differences that are important in accounting for TMP's selectivity. The crystallographic evidence strongly suggests that loss of a potential hydrogen bond between the 4-amino group of TMP and the backbone carbonyl of Val-115 when TMP binds to chicken dihydrofolate reductase but not when it binds to the E. coli reductase is the major factor responsible for this drug's more potent inhibition of bacterial dihydrofolate reductase. A key finding of the current study which is important in understanding why TMP binds differently to chicken and E. coli dihydrofolate reductases is that residues on opposite sides of the active-site cleft in chicken dihydrofolate reductase are about 1.5-2.0 A further apart than are structurally equivalent residues in the E. coli enzyme.     

Isolation of a dihydrofolate reductase-deficient mutant of Escherichia coli.

A strain of Escherichia coli was isolated in which dihydrofolate reductase was not detected by an enzyme assay or by competition for antibody. This strain requires methionine, glycine, a purine, and thymidine for growth in addition to the auxotrophic requirements of the parent strain. It was found to be useful as a recipient of plasmids harboring dihydrofolate reductase genes.     

Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability I. Dihydrofolate reductase,thymidylate synthethase and formyltetrahydrofolate synthetase in amethopterin-resistant and -sensitive strains of Pediococcus cerevisiae

Pediococcus cerevisiae/AMr, resistant to amethopterin, possesses a higher dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP⁺ oxidoreductase, EC 1.5.1.3) activity than the parent, a folate-permeable and thus amethopterin-susceptible strain and than the wild-type. The properties of dihydrofolate reductase from the three strains have been compared. Temperature, pH optima, heat stability, as well amethopterin binding did not reveal significant differences between the enzymes from the susceptible and resistant strains. The enzyme from the wild-type was 10 times more sensitive to inhibition by amethopterin and more susceptible to heat denaturation. The apparent K_m values for dihydrofolate in enzymes from the three strains were in the range of 4.8–7.2 μM and for NADPH 6.5–8.0 μM. The amethopterin-resistant strain exhibited cross-resistance to trimethoprim and was about 40-fold more resistant to the latter than the sensitive parent and the wild-type. The resistance to trimethoprim appears to be a direct result of the increased dihydrofolate reductase activity. Inhibition of dihydrofolate reductase activity by this drug was similar in the three strains. 10–20 nmol caused 50% inhibition of 0.02 enzyme unit. Trimethoprim was about 10 000 times less effective inhibitor of dihydrofolate reductase than amethopterin. The cell extract of the AMr strain possessed a folate reductase activity three times higher than that of the sensitive strain. The activities of other folate-related enzymes like thymidylate synthethase and 10-formyltetra-hydrofolate synthetase (formate: tetrahydrofolate ligase (ADP)-forming), EC 6.3.4.3) were similar in the three strains studied.     

Construction of a fol mutant strain of Escherichia coli for use in dihydrofolate reductase mutagenesis experiments.

A strain of Escherichia coli with the fol gene deleted and a kan gene inserted in its place was created for use in cloning and isolation of mutant dihydrofolate reductase. Southern blot analysis and dihydrofolate reductase enzyme assays confirmed the delta fol::kan genotype. A thyA mutation accompanied the fol deletion and is required for survival of a dihydrofolate reductase-deficient strain.     

Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability : II. Permeability changes to amethopterin and other folates in the drug-resistant mutant

Pediococcus cerevisiae/AMr, resistant to amethopterin, possesses a higher dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP⁺ oxidoreductase, EC 1.5.1.3) activity than the parent, a folate-permeable and thus amethopterin-susceptible strain and than the wild-type. The properties of dihydrofolate reductase from the three strains have been compared. Temperature, pH optima, heat stability, as well amethopterin binding did not reveal significant differences between the enzymes from the susceptible and resistant strains. The enzyme from the wild-type was 10 times more sensitive to inhibition by amethopterin and more susceptible to heat denaturation. The apparent K_m values for dihydrofolate in enzymes from the three strains were in the range of 4.8–7.2 μM and for NADPH 6.5–8.0 μM. The amethopterin-resistant strain exhibited cross-resistance to trimethoprim and was about 40-fold more resistant to the latter than the sensitive parent and the wild-type. The resistance to trimethoprim appears to be a direct result of the increased dihydrofolate reductase activity. Inhibition of dihydrofolate reductase activity by this drug was similar in the three strains. 10–20 nmol caused 50% inhibition of 0.02 enzyme unit. Trimethoprim was about 10 000 times less effective inhibitor of dihydrofolate reductase than amethopterin. The cell extract of the AMr strain possessed a folate reductase activity three times higher than that of the sensitive strain. The activities of other folate-related enzymes like thymidylate synthethase and 10-formyltetra-hydrofolate synthetase (formate: tetrahydrofolate ligase (ADP)-forming), EC 6.3.4.3) were similar in the three strains studied.