In-frame and unmarked gene deletions in burkholderia cenocepacia via an allelic exchange system compatible with gateway technology
Harrison, Joe J.
Date of Issue2015
Singapore Centre on Environmental Life Sciences Engineering (SCELSE)
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.
Applied and environmental microbiology
© 2015 American Society for Microbiology (ASM). This paper was published in Applied and Environmental Microbiology and is made available as an electronic reprint (preprint) with permission of American Society for Microbiology (ASM). The paper can be found at the following official DOI: [http://dx.doi.org/10.1128/AEM.03909-14]. One print or electronic copy may be made for personal use only. Systematic or multiple reproduction, distribution to multiple locations via electronic or other means, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper is prohibited and is subject to penalties under law.