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Background  The ability to introduce site-specific mutations in bacterial pathogens is essential towards understanding their molecular mechanisms of pathogenicity. This has been greatly facilitated by the genetic engineering technique of recombineering. In recombineering, linear double- or single-stranded DNA molecules with two terminal homology arms are electroporated into hyperrecombinogenic bacteria that express a phage-encoded recombinase. The recombinase catalyzes the replacement of the endogenous allele with the exogenous allele to generate selectable or screenable recombinants. In particular, lambda red recombinase has been instrumental in engineering mutations to characterize the virulence arsenal of the attaching and effacing (A/E) pathogens enteropathogenic  Escherichia coli  (EPEC), enterohemorrhagic  E. coli  (EHEC), and  Citrobacter rodentium. Escherichia albertii  is another member of this taxon; however, the virulence of  E. albertii  remains cryptic despite accumulating evidence that  E. albertii  is an emerging pathogen. Multiple retrospective studies have reported that a substantial number of EPEC and EHEC isolates (~15 %) that were previously incriminated in human outbreaks actually belong to the  E. albertii  lineage. Thus, there is increased urgency to reliably identify and rapidly engineer mutations in  E. albertii  to systematically characterize its virulence determinants. To the best of our knowledge not a single chromosomal gene has been altered by targeted mutagenesis in  E. albertii  since it was first isolated almost 25 years ago. This is disconcerting because an  E. albertii  outbreak could cause significant morbidity and mortality owing to our inadequate understanding of its virulence program.    Results  In this report we describe a modified lambda red recombineering protocol to mutagenize  E. albertii . As proof of principle, we successfully deleted three distinct virulence-associated genetic loci –  ler ,  grlRA , and  hfq  – and replaced each wild type allele by a mutant allele with an encodable drug resistance cassette bracketed by FRT sites. Subsequently, the FRT-site flanked drug resistance marker was evicted by FLP-dependent site-specific recombination to generate excisants containing a solitary FRT site.    Conclusions  Our protocol will enable researchers to construct marked and unmarked genome-wide mutations in  E. albertii,  which, in turn, will illuminate its molecular mechanisms of pathogenicity and aid in developing appropriate preventative and therapeutic approaches to combat  E. albertii  outbreaks.

Lambda Red-mediated Recombineering in the Attaching and Effacing Pathogen Escherichia albertii