Insertional mutagenesis in Lactococcus lactis subsp. lactis mediated by IS946*

Insertional mutagenesis by an IS946-based integration vector (pTRK145) was demonstrated in Lactococcus lactis subsp. lactis. The suicide vector pTRK145 does not replicate in Gram-positive bacteria and, therefore, expression of its erythromycin resistance (Emr) determinant in lactococci requires integration into the bacterial genome. Random integration of pTRK145 in L. lactis MG1363, a Rec+ strain with regions of homology to IS946, was demonstrated by digesting the chromosomal DNA from 20 integrants with EcoRI, which cuts pTRK145 once. Hybridization with a pTRK145-specific probe identified junction fragments which varied in size, indicating random insertion. The utility of pTRK145 was demonstrated by screening integrants for a mutant deficient in maltose utilization (Mal−). A Mal− mutation coincident with pTRK145 integration was isolated. Upon excision of the insert in vivo, the Mal+ Ems phenotype was restored. IS946-based vectors have the potential for development into effective insertional mutagens which may be valuable for localization and cloning of chrosomal genes in lactococci.