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Background  A number of allele replacement methods can be used to mutate bacterial genes. For instance, the Red recombinase system of phage Lambda has been used very efficiently to inactivate chromosomal genes in  E. coli  K-12, through recombination between regions of homology. However, this method does not work reproducibly in some clinical  E. coli  isolates.    Findings  The procedure was modified by using longer homologous regions (85 bp and 500-600 bp), to inactivate genes in the uropathogenic  E. coli  strain UTI89. An  lrhA  regulator mutant, and deletions of the  lac  operon as well as the complete  type 1  fimbrial gene cluster, were obtained reproducibly. The modified method is also functional in other recalcitrant  E. coli , like the avian pathogenic  E. coli  strain APEC1. The  lrhA  regulator and  lac  operon deletion mutants of APEC1 were successfully constructed in the same way as the UTI89 mutants. In other avian pathogenic  E. coli  strains (APEC3E, APEC11A and APEC16A) it was very difficult or impossible to construct these mutants, with the original Red recombinase-based method, with a Red recombinase-based method using longer (85 bp) homologous regions or with our modified protocol, using 500 - 600 bp homologous regions.    Conclusions  The method using 500-600 bp homologous regions can be used reliably in some clinical isolates, to delete single genes or entire operons by homologous recombination. However, it does not invariably show a greater efficiency in obtaining mutants, when compared to the original Red-mediated gene targeting method or to the gene targeting method with 85 bp homologous regions. Therefore the length of the homology regions is not the only limiting factor for the construction of mutants in these recalcitrant strains.

Reproducible gene targeting in recalcitrant Escherichia coli isolates