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

Abstract

e 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. Burkholderia cenocepacia is a member of a group of closely related Gram-negative bacteria referred to as the Burkholderia cepacia complex (Bcc). The Bcc contains at least 18 different species that thrive in diverse ecological niches, including clinical, industrial, and natural environments. These bacteria possess very large genomes separated into multiple replicons and hence are considered one of the most versatile groups of Gram-negative bacteria (1, 2). Some Bcc species have biotechnological potential for use in processes such as the enhancement of plant growth or breakdown of pollutants, while others are opportunistic pathogens causing life-threatening infections in immunocompromised individuals and in patients with cystic fibrosis (CF) (3). Although all members of the Bcc have been isolated from CF patients, B. cenocepacia accounts for the majority of these isolates, comprising the most virulent and transmissible strains associated with a poor clinical course and a high mortality rate (4). Therefore, research on the virulence mechanisms of Bcc bacteria has focused largely on B. cenocepacia.The genomes of several B. cenocepacia strains have recently been sequenced (5-7), enabling bioinformatics-based predictions of virulence determinants in this pathogen. Although a number of genes associated with virulence in B. cenocepacia have been identified (4, 8, 9) and tested in various infection models (10, 11), it seems likely that the list of genes implicated in virulence is far from complete and will expand with genetic tools becoming available to manipulate B. cenocepacia strains. The deletion of genes potentially associated with virulence is a powerful way to investigate their function in bacterial physiology and pathogenesis. Most of the virulence traits of B. cenocepacia, such as antibiotic resistance, motility, biofilm formation, cell invasion, and intracellular survival, are multifactorial, ...

Elevated intracellular cyclic‐di‐GMP level in Shewanella oneidensis increases expression of c‐type cytochromes

“…These results indicate that we have successfully constructed a high c‐di‐GMP S. oneidensis MR‐1 strain, containing the pYedQ 2 plasmid, which exhibits key characteristics such as lowered swimming motility, greater biofilm growth and increased intracellular c‐di‐GMP concentration. Although previous studies have demonstrated that the yedQ gene (from E. coli ) is active in other types of bacteria such as Burkholderia cenocepacia and Pseudomonas aeruginosa (Chen et al ., 2015; Fazli et al ., 2015), our work is the first study to provide strong evidence that yedQ is active in MR‐1, which enabled us to investigate how increased intracellular c‐di‐GMP can influence other cellular functions in MR‐1.…”

Genes Required for the Anti-fungal Activity of a Bacterial Endophyte Isolated from a Corn Landrace Grown Continuously by Subsistence Farmers Since 1000 BC

“…Chitinase encoding genes were previously found to be upregulated by c-di-GMP ( Zogaj et al, 2012 ). Furthermore, binding of c-di-GMP resulted in transcriptional regulation of anti-pathogen genes required for biofilm formation, adhesion, motility and synthesis of bacterial virulence factors ( Landini et al, 2010 ; Srivastava and Waters, 2012 ; Römling et al, 2013 ; An et al, 2014 ; Chen et al, 2015 ; Fazli et al, 2015 ; Steinberg and Kolodkin-Gal, 2015 ). Here, for the first time, to the best of our knowledge, the YajQ/c-di-GMP signaling system has been shown to underlie a mutualistic endophyte-host interaction of critical importance to its host plant.…”

One gene, multiple ecological strategies: A biofilm regulator is a capacitor for sustainable diversity

“… 18 and 21 with modifications described in SI Appendix . Isogenic mutants were created using methods described by Fazli et al ( 55 ). Fitness was measured as described in ref.…”