A SINGLE-POINT MUTATION IN CTP SYNTHETASE OF CHLAMYDIA-TRACHOMATIS CONFERS RESISTANCE TO CYCLOPENTENYL CYTOSINE

A Chlamydia trachomatis strain (L2/CPEC) resistant to the cytotoxic ef fects of cyclopentenyl cytosine (CPEC) was isolated by a stepwise sele ction procedure. This strain showed an approximate 350-fold increase i n resistance to CPEC. Sequencing of the gene encoding CTP synthetase f rom this resistant strain revealed a single point mutation, resulting in a change of amino acid 149 from Asp to Glu, This appeared to be the only mutation in L2/CPEC, because no changes in CTP transport, CTP sy nthetase expression, or incorporation of CPEC into DNA or RNA could be detected, The mutation in the chlamydial CTP synthetase resulted in a loss of CTP feedback inhibition. This was demonstrated both in vivo u sing Escherichia coil cells carrying the cloned gene, and an in vitro assay using partially purified preparations of CTP synthetase, As a re sult of the loss of feedback inhibition, E. coil cells carrying the CP EC(R) CTP synthetase showed a 22-fold increase in their CTP pools, How ever, examination of the CTP pools of L2/CPEC revealed no change in CT P levels when compared with wild type C. trachomatis.