Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum
Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum
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DOI：
10.1128/aem.01473-06
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发表时间：
2007-02-01
影响因子：
4.4
通讯作者：
Kjeerebezem, Michiel
中科院分区：
文献类型：
--
作者：
Lambert, Jolanda M.;Bongers, Roger S.;Kjeerebezem, Michiel
实现基因缺失变体的经典策略是基于将非复制载体双交叉整合到基因组中。此外，Cre-lox等重组系统已被广泛使用，主要用于真核生物。本研究构建了基于 Cre-lox 的植物乳杆菌多基因缺失系统，该系统可适用于革兰氏阳性菌。首先，构建了有效的诱变载体（pNZ5319），该载体允许直接克隆代表同源重组目标区域的平端PCR产物。使用这种诱变载体，可以根据抗生素抗性表型轻松选择双交叉基因替换突变体。在所得突变体中，靶基因被 lox66-P-32-cat-lox71 盒取代，其中 lox66 和 lox71 是 loxP 的突变变体，P-32-cat 是氯霉素抗性盒。 Lox 位点充当 Cre 酶的识别位点，Cre 酶是一种属于位点特异性重组酶整合酶家族的蛋白质。因此，双交叉突变体中短暂的 Cre 重组酶表达导致 lox66-P-32-cat-lox71 盒重组到双突变体 loxP 位点（称为 lox72）中，该位点显示出 Cre 的识别能力大大降低。通过在革兰氏阳性模式生物植物乳杆菌 WCFS1 染色体上的 melA 和 bsh1 位点构建单基因和双基因删除，证明了基于 Cre-lox 的多基因删除策略的有效性。此外，基于 Cre-lox 的系统在多基因替换中的效率是通过植物乳杆菌 WCFS1 中遗传紧密连锁的基因座 melA 和 lacS2 的连续诱变来确定的。事实上，99.4% 的分析克隆都经过了正确的 Cre-lox 分辨率，这一事实强调了此处描述的系统对于单一遗传背景中的多个基因替换和删除策略的适用性。
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 chloramphenicoll 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.