Construction and validation of two metagenomic DNA libraries from Cerrado soil with high clay content.

A challenge of metagenomic studies is in the extraction and purification of DNA from environmental samples. The soils of the Cerrado region of Brazil present several technical difficulties to DNA extraction: high clay content (>55% w/w), low pH (4.7) and high iron levels (146 ppm). Here we describe for the first time the efficient recovery and purification of microbial DNA associated with these unusual soil characteristics and the construction and validation of two metagenomic libraries: a 150,000 clones library with insert size of approximately 8 kb and a 65,000 clones library with insert size of approximately 35 kb. The construction of these metagenomic libraries will allow the biotechnological exploitation of the microbial community present in the soil from this endangered biome.

Summer workshop in metagenomics: one week plus eight students equals gigabases of cloned DNA.

We designed a week-long laboratory workshop in metagenomics for a cohort of undergraduate student researchers. During this course, students learned and utilized molecular biology and microbiology techniques to construct a metagenomic library from Puerto Rican soil. Pre-and postworkshop assessments indicated student learning gains in technical knowledge, skills, and confidence in a research environment. Postworkshop construction of additional libraries demonstrated retention of research techniques by the students.

Recovery, purification, and cloning of high-molecular-weight DNA from soil microorganisms.

We describe here an improved method for isolating, purifying, and cloning DNA from diverse soil microbiota. Soil microorganisms were extracted from soils and embedded and lysed within an agarose plug. Nucleases that copurified with the metagenomic DNA were removed by incubating plugs with a high-salt and -formamide solution. This method was used to construct large-insert soil metagenomic libraries.

Signal mimics derived from a metagenomic analysis of the gypsy moth gut microbiota.

Bacterial signaling is an important part of community life, but little is known about the signal transduction pathways of the as-yet-uncultured members of microbial communities. To address this gap, we aimed to identify genes directing the synthesis of signals in uncultured bacteria associated with the midguts of gypsy moth larvae. We constructed a metagenomic library consisting of DNA extracted directly from the midgut microbiota and analyzed it using an intracellular screen designated METREX, which detects inducers of quorum sensing. In this screen, the metagenomic DNA and a biosensor reside in the same cell. The biosensor consists of a quorum-sensing promoter, which requires an acylhomoserine lactone or other small molecule ligand for activation, driving the expression of the reporter gene gfp. We identified an active metagenomic clone encoding a monooxygenase homologue that mediates a pathway of indole oxidation that leads to the production of a quorum-sensing inducing compound. The signal from this clone induces the activities of LuxR from Vibrio fischeri and CviR from Chromobacterium violaceum. This study is the first to identify a new structural class of quorum-sensing inducer from uncultured bacteria.

Intracellular screen to identify metagenomic clones that induce or inhibit a quorum-sensing biosensor.

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.