CLONING AND CHARACTERIZATION OF UPP, A GENE ENCODING URACIL PHOSPHORIBOSYLTRANSFERASE FROM LACTOCOCCUS-LACTIS

Uracil phosphoribosyltransferase catalyzes the key reaction in the sal vage of uracil in many microorganisms. The gene encoding uracil phosph oribosyltransferase (upp) was cloned from Lactococcus lactis subsp. cr emoris MG1363 by complementation of an Escherichia coli mutant. The ge ne was sequenced, and the putative amino acid sequence was deduced. Th e promoter was mapped by both primer extension and analysis of beta-ga lactosidase expressed from strains carrying fusion between upp promote r fragments and the lacLM gene. The results showed that the upp gene w as expressed from its own promoter. After in vitro construction of an internal deletion, a upp mutant was constructed by a double-crossover event. This implicated the utilization of a plasmid with a thermosensi tive origin of replication and a new and easy way to screen for double crossover events in both gram-positive and gram-negative bacterial st rains. The phenotype of the uracil phosphoribosyltransferase-deficient strain was established. Surprisingly, the upp strain is resistant onl y to very low concentrations of 5-fluorouracil. Secondary mutants in t hymidine phosphorylase and thymidine kinase mere isolated by selection for resistance to high concentrations of 5-fluorouracil.