Objective: Thymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from the airways of cystic fibrosis patients, often in combination with isogenic normal strains if patients were treated with SXT for extended periods. Sequence analysis showed alterations in thyA, wich encodes thymidylate synthase (TS), in TD-SCVs. TS (EC 2.1.1.45) catalyses the reductive methylation of dUMP to dTMP with concomitant conversion of 5,10-methylenetetrahydrofolate to dihydrofolate and is essential for DNA synthesis. We hypothesized that mutations in thyA alter the activity of TS. Therefore, we investigated the activity of TS of normal and TD-SCVs.
Methods: The TS of 2 laboratory S. aureus strains and 6 clinical strain pairs consisting of TD-SCVs and the normal isogenic phenotypes were cloned and heterologously expressed in E. coli. The TS was purified and the specific activity was determined by means of a photometric assay.
Results: The activity of the TS of the 2 laboratory strains were used to evaluate the photometric assay. In 4/6 clinical strain pairs, the TS of TD-SCVs displayed no activity whereas the TS of the normal strains were active. In one strain pair, the TS of both phenotypes were active while in another strain pair the TS of both phenotypes were inactive.
Conclusions: These results indicate that alterations in thyA are generally but not exclusively responsible for thymidine-dependency of TD-SCVs isolated from the airways of cystic fibrosis patients.