Candidate Essential Genes in Burkholderia cenocepacia J2315 Identified by Genome-Wide TraDIS.

Yee-Chin Wong,Moataz Abd El Ghany,Kok-Wei Lee,Arnab Pain,Raeece Naeem,Sheila Nathan,Yung-Chie Tan

doi:10.3389/fmicb.2016.01288

Abstract

Burkholderia cenocepacia infection often leads to fatal cepacia syndrome in cystic fibrosis patients. However, antibiotic therapy rarely results in complete eradication of the pathogen due to its intrinsic resistance to many clinically available antibiotics. Recent attention has turned to the identification of essential genes as the proteins encoded by these genes may serve as potential targets for development of novel antimicrobials. In this study, we utilized TraDIS (Transposon Directed Insertion-site Sequencing) as a genome-wide screening tool to facilitate the identification of B. cenocepacia genes essential for its growth and viability. A transposon mutant pool consisting of approximately 500,000 mutants was successfully constructed, with more than 400,000 unique transposon insertion sites identified by computational analysis of TraDIS datasets. The saturated library allowed for the identification of 383 genes that were predicted to be essential in B. cenocepacia. We extended the application of TraDIS to identify conditionally essential genes required for in vitro growth and revealed an additional repertoire of 439 genes to be crucial for B. cenocepacia growth under nutrient-depleted conditions. The library of B. cenocepacia mutants can subsequently be subjected to various biologically related conditions to facilitate the discovery of genes involved in niche adaptation as well as pathogenicity and virulence.

Highlights

Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc), is an opportunistic pathogen in cystic fibrosis (CF) patients where infection is often associated with deterioration of pulmonary function, resulting in a fatal necrotizing pneumonia known as cepacia syndrome (Mahenthiralingam et al, 2005)
To enhance the electrocompetency of B. cenocepacia J2315, we adopted the electrocompetent cell preparation protocol described by Dubarry et al (2010) where bacteria were cultured in medium supplemented with glycine and harvested at a lower density
We have demonstrated the successful application of TraDIS to identify B. cenocepacia J2315 essential genes

Summary

Introduction

Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc), is an opportunistic pathogen in cystic fibrosis (CF) patients where infection is often associated with deterioration of pulmonary function, resulting in a fatal necrotizing pneumonia known as cepacia syndrome (Mahenthiralingam et al, 2005). Members of the Bcc complex are intrinsically resistant to many antimicrobials with limited treatment choices that rarely result in complete eradication of the pathogen for chronic infections (Leitão et al, 2010; Sousa et al, 2011). Transmission of B. cenocepacia is often associated with contaminated hospital water as well as surfaces of medical devices, leading to nosocomial outbreaks (Nasser et al, 2004; Lee et al, 2013). It is unclear how this infectious bacterium gains its ability to adapt to the changing state of nutrient availability. Target-based drug discovery to combat Bcc infections still remains a major challenge in the absence of knowledge on the molecular mechanisms that contribute to the pathogen’s capacity to adapt and survive within a broad range of environments

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Discussion

Conclusion