Characterization of temperature-sensitive cytidine triphosphate synthase mutations in bacteria by high-performance liquid chromatography

Abstract

Cytidine triphosphate (CTP) synthase catalyzes the last step in pyrimidine ribonucleotide synthesis, namely the formation of CTP from UTP, ATP, and glutamine. Mutants devoid of CTP synthase activity require cytidine for growth and have been designated purG in an obligate cdd background. Using a ts mutation blocked in the conversion of UTP to CTP at 43°C, it was demonstrated that the conversion occurs by growing cells at 33°C or below where UTP and CTP pools are normal. Growth at 43°C shuts off the enzyme, while UTP accumulates and CTP is described significantly. By now feeding exogenous cytidine the CTP pool can be restored to the level found at the permissive temperature. Instacellular nucleoside triphosphates (CTP and UTP) were separated on a Partisil SAX10 cartridge, using a linear gradient of low buffer (7 m M ammonium dihydrogenphosphate, pH 3.8) to high buffer (250 m M ammonium dihydrogenphosphate, pH 4.5 with 500 m M potassium chloride). Nucleoside triphosphates were also separated after enzymativ conversion of UTP to CTP in solution by cell extracts using ion-pair reversed-phase chromatography on a C 18 cartridge eluted with a mixture of 95% buffer A (25 m M ammonium dihydrogenphosphate with 1 m M tetrabutylammonium phosphate, pH 7.0) and 5% buffer B (15% aqueous acetonitrile). Using the two different separation techniques, it was possible to monitor the level of UTP and CTP inside cells as well as the enzymatic conversion of UTP to CTP by the enzyme CTP synthase.