Reliable method for directed gene inactivation in wild type Escherichia coli strains

The entire genome sequence of the uropathogenic Escherichia coli model strain UTI89 (serotype O18:K1:H7), was recently determined (Chen et al., 2006). A function can be assigned to the relevant open reading frames, e.g. by characterizing the appropriate mutant strains. Methods for precise gene inactivation are important tools for studies in bacterial genetics. A number of allele replacement methods can be used to mutate bacterial genes. Datsenko and Wanner (2000) described an elegant method for the construction of deletion mutants in E. coli K-12 strains, based on homologous recombination mediated by the Red system of phage lambda. Derbise et al. (2003) described a PCR-based procedure, allowing the rapid deletion of chromosomal genes in Yersinia. We found that, although some mutants could be obtained, these methods did not work reliably in E. coli UTI89.
Here we report on the development and validation of an efficient method to inactivate specific genes in E. coli UTI89. This method allows the rapid and precise deletion of any given region - one gene or an entire operon - in E. coli. This is mediated by electroporation of a linear DNA fragment with about 500 bp sequences identical to the regions flanking the desired deletion. Red-mediated homologous recombination subsequently substitutes this region by the same P1-FRT-cat-FRT-P2 insert as in the mutants constructed by the protocol of Datsenko and Wanner (2000). This can be selected using the chloramphenicol resistance marker. Finally, it is possible to remove the cat gene by the expression of the FLP recombinase.
A UTI89Delta-lrhA::CmR mutant was constructed, to prove that the described method is functional to construct mutants of one gene in E. coli UTI89. The LysR-type regulator LrhA (LysR homologue A), encoded by lrhA, is a regulator of genes involved in flagellation, motility, chemotaxis, type 1 fimbriae production and biofilm formation. The mutant showed the expected hypermotile and hyperagglutinating phenotype.
UTI89Delta-lacZYA::CmR mutants, in which the whole lac operon was deleted, were also constructed, to prove that the described method is also functional to make larger deletions and to confirm its specificity.
In addition, we investigated whether the described method is also functional in other E. coli strains like the avian pathogenic E. coli O45 strain APEC1 (Vandemaele et al., 2003). APEC1Delta-lrhA::CmR and APEC1delta-lacZYA::CmR mutants were successfully constructed in the same way as the UTI89 mutants.