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Efficient insertional mutagenesis in lactococci and other gram-positive bacteria.

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  • 1. Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Domaine de Vilvert, Jouy en Josas, France.
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    • Maguin E 1
    (1 author)

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Journal of Bacteriology, 01 Feb 1996, 178(3):931-935
https://doi.org/10.1128/jb.178.3.931-935.1996 PMID: 8550537 PMCID: PMC177749

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Abstract 

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.

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J Bacteriol. 1996 Feb; 178(3): 931–935.
PMCID: PMC177749
PMID: 8550537

Efficient insertional mutagenesis in lactococci and other gram-positive bacteria.

E Maguin, H Prévost, S D Ehrlich, and A Gruss
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Abstract

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.

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Selected References

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