Discovery and Characterization of Novel Antibiotic Synthesis and Resistance Determinants from Diverse Microbial Metagenomes
Type of Degreedissertation
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The application of metagenomics to investigate the genetic and functional diversity of as-yet-uncultured microorganisms from natural environments has been one of the most significant breakthroughs in molecular microbiology. Using a variety of metagenomic approaches, we conducted several studies of wastewater and soil communities, which are known to harbor a dynamic and complex assemblage of microorganisms expressing an array of metabolic activities. In the first study, we explored the bacterial and viral diversity in activated sludge (AS), its influent (IN, and a laboratory-scale sequencing batch reactor (SBR) originally seeded from the AS aeration basin using both culture-based and metagenomic approaches. A total of 91 unique cultured bacterial isolates, 28 cultured bacteriophages, 1,103 bacterial 16S rDNA clones, and 1,779 viral metagenomic clones were subjected to various phylogenetic and functional analyses, including BLAST comparisons, microscopic observations, metabolic profiling, and biochemical characterization. These surveys represent the most comprehensive census of wastewater microbial diversity than ever performed. The second study focused on discovery of antibiotic resistance determinants encoded on bacterial, plasmid, and viral metagenomes. All three genetic sources were screened for resistance to any of 12 antibiotics, and clones or plasmids of interest were subjected to bioinformatic and biochemical analyses. We identified several resistance genes of interest, including six clones that exhibited high levels of chloramphenicol (Cm) resistance but that share no homology with any known CmR genes. The potential for the lateral transfer of antibiotic resistance genes was also addressed, and several putative mobile genetic elements were identified from all three metagenomes, and possible in vivo transposition of kanamycin and ampicillin resistance from the multi-drug resistant plasmid pAS1 to E. coli was observed. The third study describes the discovery of novel pathways from a soil metagenomic library involved in the biosynthesis of polyketides, a structurally diverse group of secondary metabolites that often exhibits antimicrobial activity. Although many of the cloned pathways shared homology with known genes, others appear to be distantly related to genes associated with polyketide synthesis. More specifically, many of the pathways may have originated from the Cyanobacterial lineage, and one clone exhibited significant similarity with Solibacter usitatus, an as-yet-uncultured species from the poorly-characterized bacterial division Acidobacteria. Because metagenomics is an evolving field that depends on the emergence of new approaches and technologies for success, we also developed two protocols for the characterization of diverse bacterial metagenomes. The first protocol describes a novel process for the recovery, purification, and cloning of pure, high-molecular-weight metagenomic DNA from soil and is a valuable contribution toward overcoming the challenges of constructing large-insert libraries from complex environments. The second protocol enables the fluorescence in situ hybridization (FISH) of multiple probes to bacteria in pure or mixed cultures and environmental samples using an in-solution approach. This method provides a more cost- and time-effective alternative to traditional FISH analyses, and the probed sample can be used directly in downstream applications such as flow cytometry or fluorometry.
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