Novel Mechanism of Resistance to Folate Analogues: Ribonucleoside Diphosphate Reductase Deficiency in Bacteriophage T4

Abstract

Some spontaneously occurring bacteriophage T4 mutants (far mutants) were able to form plaques in the presence of concentrations of folate analogues that completely inhibit plaque formation by wild-type phage T4. Some of these far mutants were shown to be ribonucleoside diphosphate (RDP) reductase (EC 1.17.4.1) deficient, and some independently isolated RDP reductase-deficient mutants (nrd mutants) were shown to be folate analogue resistant. The map positions of the RDP reductase-deficient far mutants were shown to be within the genes controlling the phage-induced RDP reductase activity.Escherichia coli infected by bacteriophage T4 offers several advantages as a model system in which to study mechanisms of resistance to antimetabolites. Since many of the T4-induced enzymes duplicate functions already present in the host cell, this system is similar to most other host-parasite relationships but is much easier to study in the laboratory because T4 and E. coli multiply rapidly in simple media. T4 is a genetically complex virus, and the results obtained with it may be comparable to higher biological systems. To date, about 100 T4 genes have been identified out of a maximum of approximately 200 potential genes. The products of over 30 of these genes have been identified as phage-induced enzymatic activities (review by Mathews [40]). Many types of T4 mutants are available for study, comparison, and confirmation of phenotype.Among the proteins encoded in the genome of phage T4 and synthesized early after infection is a six-enzyme system that controls the biosynthesis of thymidylic acid and the interconversion of pyrimidine nucleotides. The six enzymes are ribonucleoside diphosphate (RDP) reductase, thymidylate synthetase, dihydrofolate (FH2) reductase, deoxycytidine triphosphatase, deoxycytidylate (deoxycytidine monophosphate [dCMP]) deaminase, and thymidine kinase (5, 17, 27-29, 35, 42, 47, 56). The reactions catalyzed by these enzymes are diagramed in Fig. 1 dine at a low but detectable rate (34), is grown on agar plates containing synthetic medium and cytidine as the only pyrimidine (S + Cyd plates), T4 mutants deficient in the production of FH2 reductase activity, designated frd mutants (27), or thymidylate synthetase activity, designated td mutants (47), produce plaques that exhibit the white-halo phenotype (28). The white halo surrounding the plaques of these mutants is the result of an increase in bacterial cell growth at the periphery of the plaque. It is believed that a deficiency in either FH2 reductase or thymidylate synthetase activity allows deoxyuridine monophosphate (dUMP) or a related metabolite to accumulate within the mutant-infected cells (Fig. 1); lysis of these cells during the normal cycle of phage proliferation releases high levels of uracil or a related metabolite into the medium. On S + Cyd plates with E. coli OK305 as the host cell, this metabolite could either directly or indirectly stimulate cell growth by relieving the requirement for uracil. Mutations in the phage cd gene, which codes for...

Επιδημιολογική Διερεύνηση Της Αντοχής Στα Αντιβιοτικά Φυσιολογικής Εντερικής Χλωρίδας Της Παιδικής Ηλικίας

“…Gram αρνητικά βακτηρίδια ανθεκτικά στην τριμεθοπρίμη περιγράφηκαν πολύ σύντομα μετά την εισαγωγή του συνδυασμού τριμε-θοπρίμης-σουλφαμεθοξαζόλης στην κλινική πράξη στην Αγγλία το 1969 και στη Γαλλία το 1971 (55). Η αντοχή στην τριμεθοπρίμη είναι χρωμοσωμιακή ή πλασμιδιακή (56,57,58,59,60,61) και αναπτύσσε ται, είτε με ποιοτική ή ποσοτική μεταβολή του ενζύμου διυδροφυλλική αναγωγάση (DHFR), είτε με αλλαγές της διαπερατότητας του μικροβιακού κυττάρου ως προς το αντιβιοτικό (55).…”