Date of Award
The study of bacterial natural products offers important insights into the physiologies and behaviors of individual species of bacteria as well as the contributions of these organisms to complex microbial communities. Such information frequently has important downstream applications in medicine, industry, agriculture, biotechnology, basic sciences and applied microbiology. The majority of bacteria found in nature cannot be cultivated in a laboratory setting, however, leaving a wealth of genetic and chemical diversity unexplored by traditional microbiological methods. Culture-independent approaches for studying diverse microbial populations affords indirect access to these resources. Within the wide array of culture-independent approaches currently in use (collectively termed â€œmetagenomicsâ€), the strategies of functional metagenomics provide access to important functions, phenotypes and activities encoded on fragments of environmental DNA (eDNA) as evidenced by the expression of these traits in model cultured bacterial hosts. Functional metagenomic techniques can be tailored towards finding metagenome-derived small molecule biosynthetic capabilities, thereby allowing access to novel metabolites encoded within the genomes of uncultured bacteria. From a practical standpoint, however, eDNA-derived biosynthetic gene clusters may frequently go undetected in many functional metagenomic screens due to the inherent expressions limitations associated with individual heterologous hosts. One way to circumvent these limitations is to develop novel technologies that allow for cloned eDNA to be screened in a wide variety of bacterial hosts, each possessing its own unique expression capabilites. I begin this thesis by describing the development of a novel cloning system that permits the functional metagenomic screening of eDNA libraries hosted by multiple Gram-negative bacterial species, along with the application of this technology to natural product discovery through the targeting of clones that express small molecule associated phenotypes (Craig, Chang et al. 2009). The results of these screens support the hypothesis that phylogenetically diverse host species differ in their abilities to heterologously express foreign genes and gene clusters (Craig, Chang et al. 2010). Important genes and secondary metabolites associated with individual eDNA clones identified in these screens are also described in detail (Craig and Brady 2011). I conclude this thesis with a bioinformatic and functional analysis of eDNA clones that encode for the biosynthesis of N-acyl amino acids (Craig, Cherry et al. 2011). This analysis indicates that N-acyl amino acid synthase enzymes are found predominantly within bacterial species that harbor the putative PEP-CTERM/exosortase protein-sorting system.
Craig, Jeffrey William, "The Application of Functional Metagenomics to Natural Products Research" (2012). Student Theses and Dissertations. 159.