Reproducible gene targeting in recalcitrant <it>Escherichia coli </it>isolates

• Main Authors: De Greve Henri, Hernalsteens Jean-Pierre, Deboeck Francine, Derous Veerle
• Format: Article
• Language: English
• Published: BMC 2011-06-01
• Series: BMC Research Notes
• Online Access: http://www.biomedcentral.com/1756-0500/4/213

<p>Abstract</p> <p>Background</p> <p>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 <it>E. coli </it>K-12, through recombination between regions of homology. However, this method does not work reproducibly in some clinical <it>E. coli </it>isolates.</p> <p>Findings</p> <p>The procedure was modified by using longer homologous regions (85 bp and 500-600 bp), to inactivate genes in the uropathogenic <it>E. coli </it>strain UTI89. An <it>lrhA </it>regulator mutant, and deletions of the <it>lac </it>operon as well as the complete <it>type 1 </it>fimbrial gene cluster, were obtained reproducibly. The modified method is also functional in other recalcitrant <it>E. coli</it>, like the avian pathogenic <it>E. coli </it>strain APEC1. The <it>lrhA </it>regulator and <it>lac </it>operon deletion mutants of APEC1 were successfully constructed in the same way as the UTI89 mutants. In other avian pathogenic <it>E. coli </it>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.</p> <p>Conclusions</p> <p>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.</p>