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Identification of genomic alterations and altered expression profiles using novel baterial genome display techniques Malloff, Chad Alexander

Abstract

The efficacy of antimicrobial agents against common bacterial pathogens has been diminishing significantly in recent years. Numerous clinical isolates and lab strains with altered virulence and drug resistances have been identified. The identification of novel and acquired genes involved in pathogenesis will be imperative in the development of new drugs. Large scale genomic and expression profiling of pathogenic phenotypes will prove invaluable for gene identification. We have developed three whole genome-scanning techniques to aid in the identification of acquired and novel genes. First, two-dimensional bacterial genomic display (2DBGD) was developed, from current two-dimensional DNA electrophoresis (2DDE) techniques, for the high-throughput comparison of bacterial genomes. This technique was used to generate high-resolution displays that enable the direct comparison of > 800 genomic fragments simultaneously. 2DBGDs allowed the identification of large insertions, encoding for antibiotic resistance, in strains of the respiratory pathogens B. pertussis and P. aeruginosa, and a 7 nucleotide substitution in a 20 kbp plasmid. 2DBGDs are capable of detecting acquired DNA, however, only in very closely related strains. If used to compare more distantly related bacteria (different species) numerous small changes (ie. small deletions and point mutations), unrelated to the interesting phenotype, would encumber the comparison of 2DBGDs. For this reason a second method, bacterial comparative genomic hybridization (BCGH), was developed to directly compare the genomes of more distantly related bacteria to identify gain or loss of genomic DNA. The utility of BCGH was also evaluated by comparing two strains of the opportunistic pathogen P. aeruginosa. Detection of a single copy gene insertion responsible for gentamicin resistance validates this method. Hybridization of the BCGH template with cDNA probes generated from the comparison strains was also used to confirm the identity of the gentamicin resistance gene. Lastly we used a bacterial artificial chromosome (BAC) library of B. pertussis to generate a template of BAC fingerprints spanning 87.2% of the genome. This template was used for the comparison of cDNA populations that represent different virulence phases in B. pertussis. Known virulence genes were identified and numerous candidate genes demonstrated alternate expression. These novel genome wide scanning techniques will enable the identification of novel and acquired genes that may be used as targets for combating the reoccurrence of bacterial pathogens.

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