Powerful methods to establish chromosomal markers in Lactococcus lactis: an analysis of pyrimidine salvage pathway mutants obtained by positive selections.

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.