An efficient method of selectable marker gene excision by Xer recombination for gene replacement in bacterial chromosomes

Abstract

A simple, effective method of unlabeled, stable gene insertion into bacterial chromosomes has been developed. This utilizes an insertion cassette consisting of an antibiotic resistance gene flanked by dif sites and regions homologous to the chromosomal target locus. dif is the recognition sequence for the native Xer site-specific recombinases responsible for chromosome and plasmid dimer resolution: XerC/XerD in Escherichia coli and RipX/CodV in Bacillus subtilis. Following integration of the insertion cassette into the chromosomal target locus by homologous recombination, these recombinases act to resolve the two directly repeated dif sites to a single site, thus excising the antibiotic resistance gene. Previous approaches have required the inclusion of exogenous site-specific recombinases or transposases in trans; our strategy demonstrates that this is unnecessary, since an effective recombination system is already present in bacteria. The high recombination frequency makes the inclusion of a counter-selectable marker gene unnecessary.

FIG. 1.

Deletion of a target chromosomal gene and subsequent removal of the selectable marker (e.g., antibiotic resistance) gene by Xer recombination at flanking dif sites (Xer-cise). Shaded regions represent homology between the integration cassette and genes flanking the target gene.

FIG. 2.

Integration of PCR products and antibiotic resistance gene excision in E. coli. (A) Deletion of msbB using a PCR product with homology to the regions flanking chromosomal msbB to generate DH1M. (B) Agarose gel of PCR products generated using primers SML and SMR. Lane 1, negative control (no template DNA); lane 2, wild-type msbB locus; lane 3, integrant; lane 4, deletion mutant. (C) Chromosomal insertion of rbpA using a PCR product with homology to the ubiB-fadR intergenic region to generate DH1R. (D) Agarose gel of PCR products generated using primers UbiB F and UbiB R. Lane 1, negative control (no template DNA); lane 2, wild-type ubiB-fadR locus; lane 3, integrant; lane 4, recombinant with inserted rbpA.

FIG. 3.

Wild-type and deleted loci of extracellular protease genes mpr (A) and nprE (B) in B. subtilis.