Using both sequence-and function-based metagenomic approaches, multiple antibiotic resistance determinants were identified within metagenomic libraries constructed from DNA extracted from bacterial chromosomes, plasmids, or viruses within an activated sludge microbial assemblage. Metagenomic clones and a plasmid that in Escherichia coli expressed resistance to chloramphenicol, ampicillin, or kanamycin were isolated, with many cloned DNA sequences lacking any significant homology to known antibiotic resistance determinants.

Metagenomic analyses can provide extensive information on the structure, composition, and predicted gene functions of diverse environmental microbial assemblages. Each environment presents its own unique challenges to metagenomic investigation and requires a specifically designed approach to accommodate physicochemical and biotic factors unique to each environment that can pose technical hurdles and/or bias the metagenomic analyses. In particular, soils harbor an exceptional diversity of prokaryotes that are largely undescribed beyond the level of ribotype and are a potentially vast resource for natural product discovery. The successful application of a soil metagenomic approach depends on selecting the appropriate DNA extraction, purification, and if necessary, cloning methods for the intended downstream analyses. The most important technical considerations in a metagenomic study include obtaining a sufficient yield of high-purity DNA representing the targeted microorganisms within an environmental sample or enrichment and (if required) constructing a metagenomic library in a suitable vector and host. Size does matter in the context of the average insert size within a clone library or the sequence read length for a highthroughput sequencing approach. It is also imperative to select the appropriate metagenomic screening strategy to address the specific question(s) of interest, which should drive the selection of methods used in the earlier stages of a metagenomic project (e.g., DNA size, to clone or not to clone). Here, we present both the promising and problematic nature of soil metagenomics and discuss the factors that should be considered when selecting soil sampling, DNA extraction, purification, and cloning methods to implement based on the ultimate study objectives.

The viral metagenome within an activated sludge microbial assemblage was sampled using culture-dependent and culture-independent methods and compared to the diversity of activated sludge bacterial taxa. A total of 70 unique cultured bacterial isolates, 24 cultured bacteriophages, 829 bacterial metagenomic clones of 16S rRNA genes, and 1,161 viral metagenomic clones were subjected to a phylogenetic analysis.Bacteriophages play an active role in the ecology of natural environments, influencing prokaryotic population dynamics (5, 15) and mediating lateral gene transfer between diverse bacterial species, for example. Activated sludge (AS) microbial assemblages in wastewater treatment plants have been shown to harbor great numbers of viruses with a wide range of genome sizes (7,9,10, 16). Historically, the focus of wastewater viral studies has been on specific host-virus interactions, the application of phages as tools in microbial source tracking, or the use of phages to improve the efficiency of the wastewater treatment process (e.g., foam and pathogen control) (2,4,8,12,17). Despite the interest in the wastewater viral community, a census of the activated sludge total viral community has not, to our knowledge, been investigated using both culture-based and metagenomic approaches.Taxonomic assignment of viral metagenomic clone sequences. Samples were collected from the AS aeration basin at the H. C. Morgan Water Pollution Control Facility in Auburn, AL, treated with 10% beef extract buffer (to separate viruses from aggregates), precipitated with polyethylene glycol, and subjected to cesium chloride gradient centrifugation. Viral DNA was then extracted by benzonase, proteinase K, and sodium dodecyl sulfate treatments and purified by phenolchloroform extraction and isopropanol precipitation. An AS viral metagenomic library was constructed at the Lucigen Corporation (Middleton, WI) using a linker-amplified shotgun library approach (3, 18), and 1,161 cloned insert DNA sequences were determined using a single-vector primer. Trimmed viral insert sequences were classified as known (E value Ͻ 0.001; n ϭ 694), unknown (n ϭ 97), or novel (n ϭ 370) as described previously (18). The known sequences were assigned to their taxonomic affiliations by BLASTx comparisons to the GenBank nonredundant nucleotide (nr/nt) database and selection of the top BLASTx hits for phylogenetic affiliation (Fig. 1).The dominance of viral metagenomic sequences with significant homology to bacterial DNA (nearly 60% of the known sequences) is likely the consequence of the high frequency of prophage sequences within bacterial genomes deposited in GenBank databases (1). Considerable care was taken to prevent inclusion of prokaryotic (or eukaryotic) chromosomal DNA within the viral metagenomic library (i.e., CsCl purification and nuclease treatment were performed), and the high percentage of viral metagenomic sequences with homology to bacterial genomes has been observed in other environments (3). Of the 191 known sequences with homology to viral genomes...

Polyketides are structurally diverse secondary metabolites, many of which have antibiotic or anticancer activity. Type I modular polyketide synthase (PKS) genes are typically large and encode repeating enzymatic domains that elongate and modify the nascent polyketide chain. A fosmid metagenomic library constructed from an agricultural soil was arrayed and the macroarray was screened for the presence of conserved ketosynthase [b-ketoacyl synthase (KS)] domains, enzymatic domains present in PKSs. Thirty-four clones containing KS domains were identified by Southern hybridization. Many of the KS domains contained within metagenomic clones shared significant similarity to PKS or nonribosomal peptide synthesis genes from members of the Cyanobacteria or the Proteobacteria phyla. However, analysis of complete clone insert sequences indicated that the BLAST analysis for KS domains did not reflect the true phylogenetic origin of many of these metagenomic clones that had a %G1C content and significant sequence similarity to genes from members of the phylum Acidobacteria. This conclusion of an Acidobacteria origin for several clones was further supported by evidence that cultured soil Acidobacteria from different subdivisions have genetic loci closely related to PKS domains contained within metagenomic clones, suggesting that Acidobacteria may be a source of novel polyketides. This study also demonstrates the utility of combining data from culture-dependent and -independent investigations in expanding our collective knowledge of microbial genomic diversity.

INTRODUCTIONThe successful construction of large-insert community DNA (i.e., metagenomic) libraries from natural environments is dependent on several parameters, including effective cell lysis, DNA purity, and a high transformation efficiency. One problem associated with constructing metagenomic libraries from soil microbes is the co-isolation of contaminants, leading to the degradation of DNA as a result of nuclease activity. Because the isolation of intact genetic pathways from soil microbes is necessary to characterize their genetic and functional diversity, obtaining high-purity, high-molecular-weight (HMW) DNA for library construction is absolutely critical. This protocol describes the steps for the indirect extraction of bacterial DNA from soil, embedding the DNA in an agarose matrix, using a formamide and high-salt treatment to eliminate nucleases, size-selecting DNA by restriction digestion and pulsed-field gel electrophoresis (PFGE), and cloning the HMW DNA into a large-insert vector. The resulting metagenomic libraries contain high-purity, stable, HMW DNA that can be screened for various genetic loci (sequence-based) or phenotypic traits (function-based).