A SYSTEM TO GENERATE CHROMOSOMAL MUTATIONS IN LACTOCOCCUS-LACTIS WHICH ALLOWS FAST ANALYSIS OF TARGETED GENES

A system for generating chromosomal insertions in lactococci is descri bed. It is based on the conditional replication of lactococcal pWV01-d erived Ori(+) RepA(-) vector pORI19, containing lacZ alpha and the mul tiple cloning site of pUC19. Chromosomal AluI fragments of Lactococcus lactis were cloned in pORT19 in RepA(+) helper strain Escherichia col i EC101, The frequency of Campbell-type recombinants, following introd uction of this plasmid bank into L. lactis (RepA(-)), was increased by combining the system with temperature-sensitive pWV01 derivative pVE6 007. transformation of L. lactis MG1363(pVE6007) with the pORI19 bank of lactococcal chromosomal fragments at the permissive temperature all owed replication of several copies of a recombinant plasmid from the b ank within a cell because of the provision in trans of RepA-Ts from pV E6007. A temperature shift to 37 degrees C resulted in loss of pVE6007 and integration of the pORI19 derivatives at high frequencies. A bank of lactococcal mutants was made in this way and successfully screened for the presence of two mutations: one in the monocistronic 1.3-kb pe ptidoglycan hydrolase gene (acmA) and one in the hitherto uncharacteri zed maltose fermentation pathway. Reintroduction of pVE6007 into the M al(-) mutant at 30 degrees C resulted in excision of the integrated pl asmid and restoration of the ability to ferment maltose, The integrati on plasmid (pMAL) was rescued by using the isolated plasmid content of a restored Mal(+) colony to transform E. coli EC101. Nucleotide seque ncing of the 564-bp chromosomal fragment in pMAL revealed an internal part of an open reading frame of which the translated product showed s ignificant homology with ATP-binding proteins MalK of E. coli, Salmone lla typhimurium, and Enterobacter aerogenes and MsmK of Streptococcus mutans. This combined use of two types of conditional replicating pWV0 1-derived vectors represents a novel, powerful tool for chromosomal ge ne inactivation, targeting, cloning, and sequencing of the labelled ge ne.