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 described. It is based on the conditional replication of lactococcal pWV01-derived Ori RepA vector pORI19, containing lacZα and the multiple cloning site of pUC19. Chromosomal AluI fragments of Lactococcus lactis were cloned in pORI19 in RepA helper strain Escherichia coli EC101. The frequency of Campbell- type recombinants, following introduction of this plasmid bank into L. lactis (RepA), was increased by combining the system with temperature-sensitive pWV01 derivative pVE6007. Transformation of L. lactis MG1363(pVE6007) with the pORI19 bank of lactococcal chromosomal fragments at the permissive temperature allowed replication of several copies of a recombinant plasmid from the bank within a cell because of the provision in trans of RepA-Ts from pVE6007. A temperature shift to 37°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 peptidoglycan hydrolase gene (acmA) and one in the hitherto uncharacterized maltose fermentation pathway. Reintroduction of pVE6007 into the Mal mutant at 30°C resulted in excision of the integrated plasmid and restoration of the ability to ferment maltose. The integration plasmid (pMAL) was rescued by using the isolated plasmid content of a restored Mal colony to transform E. coli EC101. Nucleotide sequencing of the 564-bp chromosomal fragment in pMAL revealed an internal part of an open reading frame of which the translated product showed significant homology with ATP-binding proteins MalK of E. coli, Salmonella typhimurium, and Enterobacter aerogenes and MsmK of Streptococcus mutans. This combined use of two types of conditional replicating pWV01-derived vectors represents a novel, powerful tool for chromosomal gene inactivation, targeting, cloning, and sequencing of the labelled gene.