Functional Genomics of Soil Bacteria using a Metagenomics Approach
Type of Degreedissertation
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Soil microbial communities are an abundant resource for natural product discovery. Traditional methods such as cultivation of soil microorganisms from soil under laboratory conditions have lead to discovery of new compounds but the vast majority of microorganisms are as yet unculturable and hence many prokaryotic phyla have yet to be explored for bioactive secondary metabolites. One of the significant breakthroughs to overcome this limitation is the application of metagenomics to investigate the genetic and functional diversity of as-yet-uncultured microorganisms from natural environments. Metagenomic analyses can provide extensive information on the structure, composition, and predicted gene functions of diverse environmental microbial assemblages. Our studies used a metagenomic approach to identify large-insert clones that express an antimicrobial activity. Bacterial artificial chromosome (BAC) vectors have been used to clone and express DNA fragments from single genomes and from entire microbial communities. Cloning and expression of large insert DNA in different host organisms can be of significance in the functional analysis and is facilitated by shuttle BAC vectors which permit the transfer and replication of BAC genomic libraries in the host organism of choice. In the first study, we designed and constructed a novel Gram negative shuttle BAC vector that enables enables stable replication of cloned DNA in diverse Gram-negative species. This vector possesses an inducible copy system to increase the number of plasmids per cell. Thus, the vector that is maintained as a single copy can be induced by addition of arabinose thereby getting a ~100-fold amplification of the DNA and potentially better expression of the cloned DNA due to a gene dosage affect. The pGNS-BAC vector can be used for high efficiency cloning of large fragments of genomic DNA transferred from Escherichia coli to other Gram-negative bacteria. The second study describes screening a soil metagenomic library to identify recombinant clones producing an antimicrobial activity. Here we used a culture-independent and function based method to characterize the soil “metagenome” to access novel antibiotics of potential medical importance. Three different libraries were screened using various tester strains. After multiple rounds of screening and validation tests we identified several clones with antimicrobial activity. Clones of interest were further characterized using preliminary biochemical studies and genetic analysis. The third study focused on detailed characterization of one of the clones (clone P6L4) identified from the screening of the large-insert library. The anti-MRSA activity derived from this clone was consistent and reproducible in all the bioassays that were performed. Basic biochemical and genetic analysis revealed that the anti-MRSA activity is likely due to the esterase produced by this clone which counteracts the action of the chloramphenicol acetyl transferase which in turn leads to growth inhibition of the MRSA by chloramphenicol.
- Dissertation Kavita 07-07-12.pdf