title: Genome modifications and cloning using a conjugally transferable recombineering system.
abstract: The genetic modification of primary bacterial disease isolates is challenging due to the lack of highly efficient genetic tools. Herein we describe the development of a modified PCR-based,λRed-mediated recombineering system for efficient deletion of genes in Gram-negative bacteria. A series of conjugally transferrable plasmids were constructed by cloning anoriTsequence and different antibiotic resistance genes into recombinogenic plasmid pKD46. Using this system we deleted ten different genes from the genomes ofEdwardsiella ictaluriandAeromonas hydrophila. A temperature sensitive and conjugally transferableflprecombinase plasmid was developed to generate markerless gene deletion mutants. We also developed an efficient cloning system to capture larger bacterial genetic elements and clone them into a conjugally transferrable plasmid for facile transferring to Gram-negative bacteria. This system should be applicable in diverse Gram-negative bacteria to modify and complement genomic elements in bacteria that cannot be manipulated using available genetic tools.
authors.affiliation: Department of Biological Sciences, Auburn University, Auburn, AL 36849, United States.
authors.forename: Mohammad J
authors.initials: MJ
authors.lastname: Hossain
authors.affiliation: Department of Biological Sciences, Auburn University, Auburn, AL 36849, United States.
authors.forename: Charles M
authors.initials: CM
authors.lastname: Thurlow
authors.affiliation: School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn AL 36849, United States.
authors.forename: Dawei
authors.initials: D
authors.lastname: Sun
authors.affiliation: Department of Biological Sciences, Auburn University, Auburn, AL 36849, United States.
authors.forename: Shamima
authors.initials: S
authors.lastname: Nasrin
authors.affiliation: Department of Biological Sciences, Auburn University, Auburn, AL 36849, United States.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Liles
keywords: Bacterial pathogens; Genetic modification; Recombineering