EFFICIENT INSERTIONAL MUTAGENESIS IN LACTOCOCCI AND OTHER GRAM-POSITIVE BACTERIA

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 replic on pG(+) host to generate a mutagenic tool that can be used even in po orly transformable strains. ISS1 transposition is random in different lactococcal strains as well as in Enterococcus faecalis and Streptococ cus thermophilus. High-frequency random insertion (of about 1%) obtain ed with this system in Lactococcus lactis allows efficient mutagenesis , with typically one insertion per cell. After ISS1 replicative transp osition, the chromosome contains duplicated ISS1 sequences flanking pG (+) host. This structure allows cloning of the interrupted gene. In ad dition, efficient excision of the plasmid leaves a single ISS1 copy at the mutated site, thus generating a stable mutant strain with no fore ign markers. Mutants obtained by this transposition system are food gr ade and can thus be used in fermentation processes.