title: Lambda Red-mediated Recombineering in the Attaching and Effacing Pathogen Escherichia albertii.
abstract: 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.
authors: Department of Biology, Saint Joseph's University, 5600 City Avenue, Philadelphia, PA 19131 USA ; Department of Mathematics, Saint Joseph's University, 5600 City Avenue, Philadelphia, PA 19131 USA.
authors: Marisa
authors: M
authors: Egan
authors: Department of Biology, Saint Joseph's University, 5600 City Avenue, Philadelphia, PA 19131 USA ; Present address: Microbiology Department, Perelman School of Medicine, University of Pennsylvania, 3610 Hamilton Walk, 221 Johnson Pavilion, Philadelphia, PA 19104 USA.
authors: Jasmine
authors: J
authors: Ramirez
authors: Department of Biology, Saint Joseph's University, 5600 City Avenue, Philadelphia, PA 19131 USA ; Present address: Bluemle Life Sciences Building, Thomas Jefferson University, 233 South Tenth Street, Philadelphia, PA 19107 USA.
authors: Christian
authors: C
authors: Xander
authors: Howard Hughes Medical Institute, Columbia University Medical Center, Columbia, USA ; Columbia University Medical Center, 1051 Riverside Drive, New York, NY 10032 USA.
authors: Chirag
authors: C
authors: Upreti
authors: Department of Biology, Saint Joseph's University, 5600 City Avenue, Philadelphia, PA 19131 USA.
authors: Shantanu
authors: S
authors: Bhatt
keywords: Bet; Escherichia albertii; Exo; Gam; LEE; Recombineering