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Polly K. Dinsmore, Dennis A. Romero, Todd R. Klaenhammer, Insertional mutagenesis in Lactococcus lactis subsp. lactis mediated by IS946, FEMS Microbiology Letters, Volume 107, Issue 1, February 1993, Pages 43–48, https://doi.org/10.1111/j.1574-6968.1993.tb06001.x
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Abstract
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.
Paper FSR92-15 of the Journal Series of the Department of Food Science, North Carolina State University, Raleigh, NC 27695, USA.
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Author notes
Present address: Promega, 2800 Woods Hollow Road, Madison, WI 53711-5399, USA.