Open AccessEfficient insertional mutagenesis in lactococci and other gram-positive bacteria
Author(s) -
Emmanuelle Maguin,
Hervé Prévost,
S. Dusko Ehrlich,
Alexandra Gruss
Publication year - 1996
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.178.3.931-935.1996
Subject(s) - biology , lactococcus lactis , transposable element , plasmid , replicon , transposition (logic) , mutagenesis , transposon mutagenesis , insertional mutagenesis , genetics , mutant , streptococcus thermophilus , gene , bacteria , lactic acid , linguistics , philosophy , lactobacillus
In lactococci, the study of chromosomal genes and their regulation is limited by the lack of an efficient transposon mutagenesis system. We associated the insertion sequence ISS1 with the thermosensitive replicon pG+ host to generate a mutagenic tool that can be used even in poorly transformable strains. ISS1 transposition is random in different lactococcal strains as well as in Enterococcus faecalis and Streptococcus thermophilus. High-frequency random insertion (of about 1%) obtained with this system in Lactococcus lactis allows efficient mutagenesis, with typically one insertion per cell. After ISS1 replicative transposition, the chromosome contains duplicated ISS1 sequences flanking pG+ host. This structure allows cloning of the interrupted gene. In addition, efficient excision of the plasmid leaves a single ISS1 copy at the mutated site, thus generating a stable mutant strain with no foreign markers. Mutants obtained by this transposition system are food grade and can thus be used in fermentation processes.