Uncovering the functions of genes in the pathogen acinetobacter baumannii

Currently, only approximately 2% of the genome of the multi-drug resistant organism Acinetobacter baumannii has been experimentally characterised. Unfortunately, this is not uncommon. The experimental verification of gene functions is now lagging behind the rapidly increasing amount of sequence data generated via high - throughput next generation sequencing technology. However, using cellular fluorescence as a proxy for phenotypic data, TraDISort (Transposon - Directed Insertion Site Sequencing and Fluorescence Activated Cell Sorting) is a novel high - throughput protocol able to provide phenotypic data for a multitude of mutants in one experiment. In this thesis, data from a TraDISort experiment performed on an A. baumannii transposon mutant library treated with a sub - inhibitory concentration of the DNA/RNA intercalating fluorescent biocide ethidium bromide was bioinformatically analysed. TraDI Sort uncovered significant ethidium bromide efflux systems in A. baumannii , exposed a novel gene potentially involved with ethidium bromide resistance , and consistently selected genes related to DNA, transcription, and translation. FACS gating procedures also predisposed TraDISort to select for mutants with altered morphologies, including genes involved with shape - regulation and cell - division, confirmed via phase - contrast microscopy. Combined with alternative compounds possessing different cellular targets and/or sites of accumulation, TraDISort possesses great potential in probing the functions of numerous genes with diverse functions.