Sunday, July 24, 2011
Grand Ballroom, 5th fl (Sheraton New Orleans)
Emergence of multidrug resistant pathogens has led to an increased need for new antibiotic classes. Significant numbers of clinically-used antibiotics are natural products derived from cultured microorganisms. Studies have shown that the diverse microbial communities in soils are potentially a great resource for novel natural products but due to limitations of culturing methods a majority of them are unexplored. To tap into this vast resource, we combined randomly sheared, large-insert cloning with the ability to express clones in multiple heterologous hosts. High molecular weight DNA was isolated from diverse soil microorganisms, randomly sheared to >100 kb, and cloned into a bacterial artificial chromosome (BAC) shuttle vector. The resulting library had 110 kb average insert size. Functional screening of 19,200 clones in Escherichia coli identified 28 clones with inhibitory activity against methicillin-resistant Staphyloccocus aureus (MRSA). The library was screened in 96-well microtitre plate format with in situ lysis method for detecting both intra- and extracellular compounds. A negative control (empty vector) was used in all bioassays. Pyrosequencing and annotation of insert sequences of 12 anti-MRSA clones revealed many predicted genes involved in polyketide synthesis in GenBank database and numerous novel genes. Transformation of naïve E. coli with BAC DNA isolated from these clones confirmed presence of anti-MRSA activity. Extraction, purification, and elucidation of the biochemical structure of metagenomic clone-derived compounds have yielded novel chemistry for at least one clone. This study demonstrates a proof-of-concept for using very large-insert cloning methods alongwith functional metagenomics approach to identify antimicrobial compounds active against MRSA.