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Appl. Environ. Microbiol. doi:10.1128/AEM.01473-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

A Cre-lox based system for multiple gene deletions and selectable marker removal in Lactobacillus plantarum

Jolanda M. Lambert, Roger S. Bongers, and Michiel Kleerebezem*

Wageningen Centre for Food Science, Microbial Functionality and Safety Programme; NIZO food research, Health & Safety department, PO Box 20, 6710 BA Ede, The Netherlands

* To whom correspondence should be addressed. Email: Michiel.Kleerebezem{at}nizo.nl.


  Abstract

The classic strategy to achieve gene deletion variants is based on double cross-over integration of non-replicating vectors into the genome. In addition, recombination systems such as Cre-lox have been used extensively mainly in eukaryotic organisms. This study presents the construction of a Cre-lox based system for multiple gene deletions in Lactobacillus plantarum that could be adapted for use in Gram-positive bacteria. First, an effective mutagenesis vector (pNZ5319) was constructed that allows direct cloning of blunt-end PCR products representing homologous recombination target regions. Using this mutagenesis vector, double cross-over gene replacement mutants could be readily selected based on their antibiotic resistance phenotype. In the resulting mutants, the target gene is replaced by a lox66-P32-cat-lox71 cassette, where lox66 and lox71 are mutant variants of loxP and P32-cat is a chloramphenicol resistance cassette. The lox sites serve as recognition sites for the Cre enzyme, which is a protein that belongs to the integrase family of site-specific recombinases. Thus, transient Cre recombinase expression in double-cross over mutants leads to recombination of the lox66-P32-cat-lox71 cassette into a double mutant loxP site, called lox72, which displays strongly reduced recognition by Cre. The effectiveness of the Cre-lox based strategy for multiple gene deletions was demonstrated by construction of both single and double gene deletions at the melA and bsh1 loci on the chromosome of the Gram-positive model organism Lactobacillus plantarum WCFS1. Furthermore, the efficiency of the Cre-lox based system in multiple gene replacements was determined by successive mutagenesis of the genetically closely linked loci melA and lacS2 in L. plantarum WCFS1. The fact that 99.4 % of the clones that were analysed had undergone correct Cre-lox resolution emphasizes the suitability of the system described here for multiple gene replacement and deletion strategies in a single genetic background.




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