title: In-Frame and Unmarked Gene Deletions in Burkholderia cenocepacia via an Allelic Exchange System Compatible with Gateway Technology.
abstract: Burkholderia cenocepacia is an emerging opportunistic pathogen causing life-threatening infections in immunocompromised individuals and in patients with cystic fibrosis, which are often difficult, if not impossible, to treat. Understanding the genetic basis of virulence in this emerging pathogen is important for the development of novel treatment regimes. Generation of deletion mutations in genes predicted to encode virulence determinants is fundamental to investigating the mechanisms of pathogenesis. However, there is a lack of appropriate selectable and counterselectable markers for use in B. cenocepacia, making its genetic manipulation problematic. Here we describe a Gateway-compatible allelic exchange system based on the counterselectable pheS gene and the I-SceI homing endonuclease. This system provides efficiency in cloning homology regions of target genes and allows the generation of precise and unmarked gene deletions in B. cenocepacia. As a proof of concept, we demonstrate its utility by deleting the Bcam1349 gene, encoding a cyclic di-GMP (c-di-GMP)-responsive regulator protein important for biofilm formation.
authors: Department of Biology, Faculty of Science, University of Copenhagen, Copenhagen, Denmark.
authors: Mustafa
authors: M
authors: Fazli
authors: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
authors: Joe J
authors: JJ
authors: Harrison
authors: Department of Biosciences, University of Milan, Milan, Italy.
authors: Michela
authors: M
authors: Gambino
authors: Costerton Biofilm Center, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Republic of Singapore.
authors: Michael
authors: M
authors: Givskov
authors: Costerton Biofilm Center, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark ttn@sund.ku.dk.
authors: Tim
authors: T
authors: Tolker-Nielsen
mesh_terms: D057508:Burkholderia cenocepacia / Q000235:genetics*; D017353:Gene Deletion; D055786:Gene Knockout Techniques / Q000379:methods*; D005827:Genetics, Microbial / Q000379:methods*; D059765:Homologous Recombination