Induction of the dihydrofolate reductase of Streptococcus faecium var. durans by folic and by some of its derivatives

Abstract

For growth in a defined medium, Streptococcus faecium var. durans requires either folate or some of its derivatives, or a natural purine together with thymine. When folate at 2.3·10 −8–2.3·10 −10 M, was added to S. faecium cells growing in the presence of purines and thymine, a concentration dependent increase in dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP exodoreductase, EC 1.5.1.3) activity occurred. This increase was detectable in cell extracts within 30 min, and reached a maximal level of 6–10 times that of control in approx. 5 h. Dihydrofolate, tetrahydrofolate and folinic acid caused a similar increase in reductase activity. Chloramphenicol or actinomycin D, when added to growing cultures simultaneously with folate, prevented any increase in reductase activity. Furthermore, titration of the reductase with methotrexate showed that the increase in enzyme activity, which occurred in the presence of the cofactors, was accompanied by a parallel increase in the number of enzyme sites. Taken together, these findings indicate that the observed increase in reductase activity was due to new protein synthesis. In the absence of the cofactors no increase in enzyme activity occurred during the entire growth cycle of the culture. Methotrexate itself did not induce the dihydrofolate reductase. In the presence of this analog, folate was ineffective as an inducer, whereas its reduced derivatives brought about some increase in enzyme activity. When cells containing high levels of the induced enzyme were transferred from the folate-containing to the folate-free medium, the enzyme activity decreased slowly. Three passages in the folate-free medium were required before the constitutive enzyme level was restored. The progressive decrease in reductase activity which occurred during these passages, was paralleled by the increasing sensitivity of the subcultures to inhibition of their growth by methotrexate. By affecting the level of dihydrofolate reductase, folate and its derivatives can, thus, regulate the extent of their activation, and thereby, the extent of their cofactor function.