SINGLE-CROSSOVER INTEGRATION IN THE LACTOBACILLUS-SAKE CHROMOSOME ANDINSERTIONAL INACTIVATION OF THE PTSI AND LACL GENES

Single-crossover homologous integration in Lactabacillus sake was stud ied. Integration was conducted,vith nonreplicative delivery vector pRV 300. This vector is composed of a pBluescript SK- replicon for propaga tion in Escherichia coli and an erythromycin resistance marker. Random chromosomal DNA fragments of L. sake 23K ranging between 0.3 and 3.4 kb were inserted into pRV300. The resulting plasmids were able to inte grate into the chromosome by homologous recombination as single copies and were maintained stably. The single cross-over integration frequen cy was logarithmically proportional to the extent of homology between 0.3 and 1.2 kb and reached a maximum value of 1.4 x 10(3) integrants/m u g of DNA. We used this integration strategy to inactivate the ptsI g ene, encoding enzyme I of the phosphoenolpyruvate:carbohydrate phospho transferase system, and the lacL gene, which is one of the two genes r equired for the synthesis of a functional P-galactosidase. The results indicated that our method facilitates genetic analysis of L. sake.