FIG 1.

The E. faecalis thymidylate synthase R177E C197W double mutant functions as a dominant negative. Wild-type E. faecalis strains carrying either an empty expression vector or the expression vector with thyA R177E C197W were subjected to 10-fold serial dilutions and inoculated on MH agar (A) or the semidefined MM9YE agar (B) that had been supplemented with Em and thymine where indicated, followed by incubation at 37°C for ∼16 h. Wild-type E. faecalis does not require thymine for growth (top) due to the activity of TS. Expression of thyA R177E C197W impairs the function of wild-type TS, thereby preventing growth unless exogenous thymine is provided. The E. faecalis host strains were OG1 and T1. The plasmids were as follows: vector, pBK200; thyA-DN, pCJK237 carrying thyA R177E C197W.