The Burkholderia cepacia complex (Bcc) are a group of closely related species known for their intrinsic multidrug resistance, large multipart genomes and ability to infect people with cystic fibrosis. The clinical relevance of the Bcc and their large multipart genomes make the study of their essential genes of broad interest. Essential genes are those required for survival in standard laboratory conditions this makes them potential targets for novel antibiotics against a group of species where few existing antibiotics are effective. Furthermore, while essential gene studies have been carried out in a number of bacterial species, only one of these species had multiple chromosomes and none had a genome as large as the Bcc. In my research I identified essential genes in B. cenocepacia K56-2, a member of the Bcc, by using transposon mutagenesis to deliver a rhamnose inducible promoter randomly into the genome and screening for a conditional growth (CG) phenotype. The utility of the CG mutant library was confirmed by showing that, when grown in suboptimal concentrations of rhamnose, only mutants that under-expressed the target of the antibiotic were hypersensitive. The CG mutant library included transposon insertions upstream from widely conserved, well-characterized essential genes suggesting that the system is capable of recovering essential gene mutants. A number of genes with either no or mixed records of essentiality in other microorganisms were also recovered. Among these was one of the three electron transfer flavoproteins (ETFs) in B. cenocepacia. The ETFs are a family of proteins found in a large number of eukaryotic, archaeal and bacterial species, which are required for the metabolism of specific substrates or for symbiotic nitrogen fixation in some bacteria. Despite these non-essential functions, high throughput screens have identified ETFs as putatively essential in several species. I showed that ETF expression is required for both viability and growth both on complex media and on media containing a variety of single carbon sources. Furthermore, cells depleted of ETF were determined to be nonviable and the morphologic shape of the cells changed from short rods to small spheres. In depth studies of essential genes are only possible for organisms with sequenced genomes. Of the 18 named species that currently comprise the Bcc, only 7 have been sequenced limiting the possibility of cross species comparative genomics. Therefore, I have assembled the first draft genomes of B. contaminans isolates, a species that has emerged as the dominant Bcc species recovered from the CF populations of Argentina and Spain. Identifying and characterizing essential genes in the Bcc, and sequencing additional Bcc species for comparative genomics are important first steps in understanding these clinically important bacteria. / February 2016
| Identifer | oai:union.ndltd.org:MANITOBA/oai:mspace.lib.umanitoba.ca:1993/30912 |
| Date | 08 1900 |
| Creators | Bloodworth, Ruhullah |
| Contributors | Cardona, Silvia (Microbiology), Brassinga, Ann Karen (Microbiology) Oresnik, Ivan (Microbiology) Domaratzki, Michael (Computer Science) McClean, Siobhán (Institute of Technology, Dublin, Ireland) |
| Publisher | Wiley, Microbiolog Society, American Society of Microbiology |
| Source Sets | University of Manitoba Canada |
| Detected Language | English |
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