Identification of the essential genome of Burkholderia cenocepacia K56-2 to uncover novel antibacterial targets
Gislason, April Shelley
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Burkholderia cenocepacia K56-2 is a clinical isolate of the Burkholderia cepacia complex, a group of intrinsically antibiotic resistant opportunistic pathogens, for which few treatment options are available. The identification of essential genes is an important step for exploring new antibiotic targets. Previously, a conditional growth mutant library was created using transposon mutagenesis to insert a rhamnose-inducible promoter into the genome of B. cenocepacia. It was then demonstrated that conditional growth mutants under-expressing an antibiotic target were sensitized to sub-lethal concentrations of that antibiotic. This thesis describes the use of next generation sequencing to measure the enhanced sensitivity of conditional growth mutants after being grown together competitively. Evidence is provided to show that the sensitivity of a GyrB conditional growth mutant to its cognate antibiotic, novobiocin, was enhanced due to competitive growth. Screening eight antibiotics with known mechanisms of action against a pilot conditional growth mutant library revealed a previously uncharacterized two-component system involved antibiotic resistance in B. cenocepacia. With the goal of exploring new Burkholderia antibacterial targets, the essential genome of B. cenocepacia K56-2 was identified by sequencing a high density transposon mutant library. Comparison of the B. cenocepacia K56-2 essential gene set with the essential genomes predicted for B. thailandensis, B. pseudomallei, and B. cenocepacia J2315 revealed strain-specific essential genes and 158 essential genes that are common to species in the Burkholderia genus. Three genes were identified that are essential in Burkholderia but not in other bacterial essential genomes identified so far, suggesting that specific cell envelope functions are critical to survival of Burkholderia species. To increase the diversity of the conditional growth mutant library, a method to enrich conditional growth mutants from the high density mutant library was developed and used to isolate 830 conditional growth mutants. With the addition of these mutants, the conditional growth mutant library currently represents 23.2% of the essential operons in B. cenocepacia K56-2 and 36.7% of the essential genes that are common to species in the Burkholderia genus. In summary, this thesis has contributed to create genomic tools available for antibiotic research and has identified novel targets for fighting Burkholderia related infections.