Characterization of soil bacterial assemblies in Brazilian savanna-like vegetation reveals acidobacteria dominance.

The Brazilian Cerrado is the second largest biome in Brazil and is considered a biodiversity hotspot. In this work, we compared the bacterial communities in Cerrado soil associated with four types of native vegetation (Cerrado Denso, Cerrado sensu stricto, Campo Sujo, and Mata de Galeria) by ribosomal RNA intergenic spacer analysis, terminal fragment restriction length polymorphism and pyrosequencing. The fingerprinting results were very similar. The bacterial communities of Cerrado Denso and Cerrado sensu stricto grouped together and were distinct from those in Campo Sujo and Mata de Galeria. Pyrosequencing generated approximately 40,000 16S rRNA gene sequences per sample and allowed the identification of 17 phyla in soil samples under Cerrado vegetation. Acidobacteria were dominant in all areas studied with a relative frequency of 40-47 %, followed closely by Proteobacteria accounting for 34-40 % of the sequences. Results from all molecular techniques used suggested that the bacterial communities of Cerrado sensu stricto and Cerrado Denso are very similar to each other, while Campo Sujo forms a separate group, and Mata de Galeria is the most distinct with higher species richness. This is the first extensive study of native Cerrado soil microbiota, an important but endangered biome.

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.

Novel florfenicol and chloramphenicol resistance gene discovered in Alaskan soil by using functional metagenomics.

Functional metagenomics was used to search for florfenicol resistance genes in libraries of cloned DNA isolated from Alaskan soil. A gene that mediated reduced susceptibility to florfenicol was identified and designated pexA. The predicted PexA protein showed a structure similar to that of efflux pumps of the major facilitator superfamily.

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.

Ralstonia solanacearum Race 3, Biovar 2 Strains Isolated from Geranium Are Pathogenic on Potato.

Ralstonia solanacearum race 3, biovar 2 is a soilborne bacterium that causes potato brown rot disease in temperate and subtropical climates. Recent outbreaks of this disease in Europe have caused serious losses, but the pathogen had not been identified in the United States. However, in 1999, strains of R. solanacearum were isolated from wilting geraniums growing in Wisconsin greenhouses. Physiological and biochemical tests of the Wisconsin strains and a similar strain from South Dakota demonstrated that the strains belong to R. solanacearum subgroup biovar 2, which is largely synonymous with the race 3 subgroup, a classification based on host range. These results were confirmed by polymerase chain reaction analyses in which race 3, biovar 2-specific primers amplified a fragment of the expected size. This is the first report of race 3, biovar 2 in the United States, and it is the first known occurrence of race 3, biovar 2 in Wiscon-sin. The geranium strains were highly pathogenic on both geranium and potato. The presence of R. solanacearum race 3, biovar 2 in the United States raises concern that the bacterium could move from ornamental plants into potato fields, where it could cause both direct economic damage and quarantine problems. A commercial indirect enzyme-linked immunosorbent assay for R. solanacearum produced some false negatives for these strains, indicating that current indexing may not be sufficient to identify this destructive pathogen.