Identification of the essential genome of Burkholderia cenocepacia K56-2 to uncover novel antibacterial targets

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dc.contributor.authorGislason, April Shelley
dc.contributor.examiningcommitteeMark, Brian (Microbiology) Oresnik, Ivan (Microbiology) Whyard, Steve (Biological Sciences) Yost, Christopher (Biology, University of Regina)en_US
dc.contributor.supervisorCardona, Silvia (Microbiology)en_US
dc.date.accessioned2017-10-25T15:59:04Z
dc.date.available2017-10-25T15:59:04Z
dc.date.issued2013-04en_US
dc.date.issued2014-03en_US
dc.date.issued2016-12en_US
dc.degree.disciplineMicrobiologyen_US
dc.degree.levelDoctor of Philosophy (Ph.D.)en_US
dc.description.abstractBurkholderia 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.en_US
dc.description.noteFebruary 2018en_US
dc.identifier.citationCardona, S.T., Selin, C., and Gislason, A.S. (2014). Genomic tools to profile antibiotic mode of action. Crit. Rev. Microbiol. 4, 465–472.en_US
dc.identifier.citationGislason, A.S., Choy, M., Bloodworth, R.A., Qu, W., Stietz, M.S., Li, X., Zhang, C., and Cardona, S.T. (2016). Competitive Growth Enhances Conditional Growth Mutant Sensitivity to Antibiotics and Exposes a Two-Component System as an Emerging Antibacterial Target in Burkholderia cenocepacia. Antimicrob. Agents Chemother. 61, 10.1128/AAC.00790-16.en_US
dc.identifier.urihttp://hdl.handle.net/1993/32675
dc.language.isoengen_US
dc.publisherMicrobiologyOpenen_US
dc.publisherCritical Reviews in Microbiologyen_US
dc.publisherAntimicrobial Agents and Chemotherapyen_US
dc.rightsopen accessen_US
dc.subjectBurkholderiaen_US
dc.subjectTn-seqen_US
dc.subjectEssential genesen_US
dc.subjectTransposon mutagenesisen_US
dc.subjectAntibiotic targetsen_US
dc.subjectCell envelopeen_US
dc.subjectMicrobiologyen_US
dc.subjectIlluminaen_US
dc.subjectAntibiotic resistanceen_US
dc.subjectDrug effluxen_US
dc.subjectTwo component regulatory systemsen_US
dc.subjectGram-negativeen_US
dc.subjectAntibiotic profilingen_US
dc.titleIdentification of the essential genome of Burkholderia cenocepacia K56-2 to uncover novel antibacterial targetsen_US
dc.typedoctoral thesisen_US
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