Upregulation of thymidine kinase activity compensates for loss of thymidylate synthase activity in Mycoplasma pneumoniae

Abstract

Thymidylate, an essential building block of DNA, is synthesized either from deoxyuridylate by thymidylate synthase (TS) or thymidine (dT) by thymidine kinase (TK). Thymidylate kinase (TMPK) phosphorylates dTMP to dTTP. Thymidine phosphorylase (TP) catalyses reversible phosphorolysis of dT. Using transposon mutagenesis M. pneumoniae TS gene (thyA/MPN320) was interrupted and requirement of these enzymes was studied. We found that TK activity and transcript levels and TP activity, but not TMPK or TS activity, are growth-phase-regulated, with induction at the exponential growth phase and a decline after the stationary phase. Inactivation of thyA results in upregulation of TK transcript and a 10-fold increase in TK activity, reduced TMPK level and it had no effect on TP activity. The level of [3H]-dT uptake and incorporation into DNA in the thyA mutant correlates with increases in TK activity, suggesting that dT uptake and metabolism is TK-dependent and that upregulation of TK activity in the thyA mutant compensates for the lack of ThyA activity. [3H]-dU uptake was low compared with dT, and incorporation of radioactivity into DNA in the thyA mutant indicates the presence of an alternative TS. Our results suggest that TK and TMPK are potential targets for the development of Mycoplasma-specific antibiotics.