title: Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum.
abstract: 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-P(32)-cat-lox71 cassette, where lox66 and lox71 are mutant variants of loxP and P(32)-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-P(32)-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.
authors.affiliation: Wegeningen Centre for Food Science, Microbial Functionality and Safety Programme, Health and Safety Department, P.O. Box 20, 6710 BA Ede, The Netherlands.
authors.forename: Jolanda M
authors.initials: JM
authors.lastname: Lambert
authors.forename: Roger S
authors.initials: RS
authors.lastname: Bongers
authors.forename: Michiel
authors.initials: M
authors.lastname: Kleerebezem
mesh_terms: D017353:Gene Deletion; D008040:Genetic Linkage / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics; D019426:Integrases / Q000378:metabolism*; D048191:Lactiplantibacillus plantarum / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis
chemical_list: D005819:Genetic Markers; C045073:Cre recombinase; D019426:Integrases