title: Intracellular screen to identify metagenomic clones that induce or inhibit a quorum-sensing biosensor.
abstract: The goal of this study was to design and evaluate a rapid screen to identify metagenomic clones that produce biologically active small molecules. We built metagenomic libraries with DNA from soil on the floodplain of the Tanana River in Alaska. We extracted DNA directly from the soil and cloned it into fosmid and bacterial artificial chromosome vectors, constructing eight metagenomic libraries that contain 53,000 clones with inserts ranging from 1 to 190 kb. To identify clones of interest, we designed a high throughput "intracellular" screen, designated METREX, in which metagenomic DNA is in a host cell containing a biosensor for compounds that induce bacterial quorum sensing. If the metagenomic clone produces a quorum-sensing inducer, the cell produces green fluorescent protein (GFP) and can be identified by fluorescence microscopy or captured by fluorescence-activated cell sorting. Our initial screen identified 11 clones that induce and two that inhibit expression of GFP. The intracellular screen detected quorum-sensing inducers among metagenomic clones that a traditional overlay screen would not. One inducing clone carries a LuxI homologue that directs the synthesis of an N-acyl homoserine lactone quorum-sensing signal molecule. The LuxI homologue has 62% amino acid sequence identity to its closest match in GenBank, AmfI from Pseudomonas fluorescens, and is on a 78-kb insert that contains 67 open reading frames. Another inducing clone carries a gene with homology to homocitrate synthase. Our results demonstrate the power of an intracellular screen to identify functionally active clones and biologically active small molecules in metagenomic libraries.
authors.affiliation: Department of Plant Pathology, University of Wisconsin-Madison, 1630 Linden Dr., Madison, WI 53706, USA.
authors.forename: Lynn L
authors.initials: LL
authors.lastname: Williamson
authors.forename: Bradley R
authors.initials: BR
authors.lastname: Borlee
authors.forename: Patrick D
authors.initials: PD
authors.lastname: Schloss
authors.forename: Changhui
authors.initials: C
authors.lastname: Guan
authors.forename: Heather K
authors.initials: HK
authors.lastname: Allen
authors.forename: Jo
authors.initials: J
authors.lastname: Handelsman
mesh_terms: D000413:Alaska; D001419:Bacteria / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015374:Biosensing Techniques; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015698:Genomic Library; D023281:Genomics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015398:Signal Transduction; D012988:Soil Microbiology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; C085427:LuxI protein, Bacteria; D014157:Transcription Factors; D049452:Green Fluorescent Proteins