In-Frame and Unmarked Gene Deletions in Burkholderia cenocepacia via an Allelic Exchange System Compatible with Gateway Technology

Applied and Environmental Microbiology
Mustafa FazliTim Tolker-Nielsen

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.

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Citations

Oct 23, 2015·Nature Protocols·Laura R HmeloJoe J Harrison
Mar 10, 2019·Applied and Environmental Microbiology·Tingting WangXin Deng
Jun 29, 2018·Applied Microbiology and Biotechnology·MaFeng LiuAnChun Cheng
Jan 29, 2019·Frontiers in Microbiology·Anja M RichterTim Tolker-Nielsen
Aug 21, 2020·Proceedings of the National Academy of Sciences of the United States of America·Eisha MhatreVaughn S Cooper
Jun 22, 2021·International Journal of Environmental Health Research·Jasna HrenovicBranka Bedenic

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