1 Department of Systems Biology, Technical University of Denmark
Using different 5-fluoropyrimidine analogues, positive selection procedures for obtaining mutants blocked in pyrimidine and purine salvage genes of Lactococcus lactis were established. Strains lacking the following enzyme activities due to mutations in the corresponding genes were isolated: uracil phosphoribosyltransferase (upp), uridindcytidine kinase (udk), pyrimidine nucleoside phosphorylase (pdp), cytidine/deoxycytidine deaminase (dd), thymidine kinase (tdk) and purine nucleoride phosphorylase (pup). Based on an analysis of the mutants obtained, the pathways by which L. lactis metabolizes uracil and the different pyrimidine nucleosides were verified. The substrate specificities of the different enzymes were determined. It was demonstrated that a single pyrimidine nucleoside phosphorylase accounts for the phosphorolytical cleavage of uridine, deoxyuridine and thymidine, and a single purine nucleoside phosphorylase has activity towards both the ribonucleoside and deoxyribonucleoside derivatives of adenine, guanine and hypoxanthine. No phosphorylase activity towards xanthosine appeared to be present. The selection procedures developed during this work may be employed in establishing markers on the chromosome of many related lactic acid bacteria.