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Applied and Environmental Microbiology, February 2007, p. 1126-1135, Vol. 73, No. 4
0099-2240/07/$08.00+0     doi:10.1128/AEM.01473-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Cre-lox-Based System for Multiple Gene Deletions and Selectable-Marker Removal in Lactobacillus plantarum{triangledown}

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

Wageningen Centre for Food Science, Microbial Functionality and Safety Programme, and NIZO food research, Health and Safety Department, P.O. Box 20, 6710 BA Ede, The Netherlands

Received 27 June 2006/ Accepted 18 November 2006

The classic strategy to achieve gene deletion variants is based on double-crossover integration of nonreplicating vectors into the genome. In addition, recombination systems such as Cre-lox have been used extensively, mainly for 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 on 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-crossover 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, a protein that belongs to the integrase family of site-specific recombinases. Thus, transient Cre recombinase expression in double-crossover 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 analyzed 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.

* Corresponding author. Mailing address: NIZO food research, Health and Safety Department, P.O. Box 20, 6710 BA Ede, The Netherlands. Phone: 31 (0) 318 659 511. Fax: 31 (0) 318 650 400. E-mail: Michiel.Kleerebezem{at}nizo.nl.

{triangledown} Published ahead of print on 1 December 2006.

Applied and Environmental Microbiology, February 2007, p. 1126-1135, Vol. 73, No. 4
0099-2240/07/$08.00+0     doi:10.1128/AEM.01473-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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