title: Genome-wide saturation mutagenesis of Burkholderia pseudomallei K96243 predicts essential genes and novel targets for antimicrobial development.
abstract: Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 10(6) transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied.
  
  
  OBJECTIVE
  Burkholderia pseudomallei is a lethal human pathogen that is considered a potential bioterrorism threat and has limited treatment options due to an unusually high natural resistance to most antibiotics. We have identified a set of genes that are required for bacterial growth and thus are excellent candidates against which to develop potential novel antibiotics. To validate our approach, we constructed four mutants in which gene expression can be turned on and off conditionally to confirm that these genes are required for the bacteria to survive.
authors: Madeleine G
authors: MG
authors: Moule
authors: Claudia M
authors: CM
authors: Hemsley
authors: Qihui
authors: Q
authors: Seet
authors: José Afonso
authors: JA
authors: Guerra-Assunção
authors: Jiali
authors: J
authors: Lim
authors: Mitali
authors: M
authors: Sarkar-Tyson
authors: Taane G
authors: TG
authors: Clark
authors: Patrick B O
authors: PB
authors: Tan
authors: Richard W
authors: RW
authors: Titball
authors: Jon
authors: J
authors: Cuccui
authors: Brendan W
authors: BW
authors: Wren
mesh_terms: D016957:Burkholderia pseudomallei / Q000235:genetics*; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005798:Genes, Bacterial; D020043:Genes, Essential; D016680:Genome, Bacterial; D016254:Mutagenesis, Insertional; D017422:Sequence Analysis, DNA
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial