Open AccessInsertional mutagenesis in Lactococcus lactis subsp. lactis mediated by IS 946 *
Author(s) -
Dinsmore Polly K.,
Romero Dennis A.,
Klaenhammer Todd R.
Publication year - 1993
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1993.tb06001.x
Subject(s) - lactococcus lactis , insertional mutagenesis , mutagenesis , biology , microbiology and biotechnology , genetics , bacteria , mutation , gene , genome , lactic acid
Insertional mutagenesis by an IS 946 ‐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 (Em r ) 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 IS 946 , was demonstrated by digesting the chromosomal DNA from 20 integrants with Eco RI, 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 + Em s phenotype was restored. IS 946 ‐based vectors have the potential for development into effective insertional mutagens which may be valuable for localization and cloning of chrosomal genes in lactococci.