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PMID: 8550537 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

Journal of bacteriology ·Vol. 178 ·No. 3 ·1996-02-00 ·Pages 931-5

Maguin E, Prévost H, Ehrlich SD, Gruss A

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.

MeSH Terms
Base Sequence Cloning, Molecular DNA Transposable Elements Enterococcus faecalis/genetics Lactococcus/genetics Molecular Sequence Data Mutagenesis, Insertional Streptococcus/genetics
Chemicals
DNA Transposable Elements
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Maguin E
Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Domaine de Vilvert, Jouy en Josas, France.
Prévost H
Ehrlich S D
Gruss A
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.178.3.931-935.1996
Published
1996-02-00
Pages
931-5
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC177749
Subset
IM
External Links
PubMed source DOI: 10.1128/jb.178.3.931-935.1996 PMC Full Text View journal details
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