Environmental whole-genome amplification to access microbial populations in contaminated sediments

Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using phi29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and "clusters of orthologous groups" (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible. The reported SSU rRNA sequences and library clone end sequences are listed with their respective GenBank accession numbers, DQ 404590 to DQ 404652, DQ 404654 to DQ 404938, and DX 385314 to DX 389173.     

The molecular diversity of freshwater picoeukaryotes from an oligotrophic lake reveals diverse, distinctive and globally dispersed lineages

The recent discovery of a diverse phylogenetic assemblage of picoeukaryotes from environments such as oceans, salt marshes and acidic habitats, has expanded the debates about the extent and origin of microbial eukaryotes. However, the diversity of these eukaryote microorganisms, that overlap bacteria in size, and their environmental and biogeographical ubiquity remains poorly understood. Here we survey picoeukaryotes (microbial eukaryotes of 0.2-5 microm in size) from an oligotrophic (nutrient deficient) freshwater habitat using ribosomal RNA gene sequences. Three taxonomic groups the Heterokonta, Cryptomonads and the Alveolata dominated the detected diversity. Most sequences represented previously unsampled species, with several being unassignable to known taxonomic groups and plausibly represent new or unsampled phyla. Many freshwater phylogenetic groups identified in this study appeared unrelated to picoeukaryotic sequences identified in marine ecosystems, suggesting that aspects of eukaryote microbial diversity are specific to certain aquatic environments. Conversely, at least five phylogenetic clusters comprised sequences from freshwater and globally dispersed and often contrasting environments, supporting the concept that a number of picoeukaryotic lineages are widely distributed.     

Environmental Whole-Genome Amplification To Access Microbial Populations in Contaminated Sediments

Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using 29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and “clusters of orthologous groups” (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible.     

Novel high-rank phylogenetic lineages within a sulfur spring (Zodletone Spring, Oklahoma), revealed using a combined pyrosequencing-sanger approach

The utilization of high-throughput sequencing technologies in 16S rRNA gene-based diversity surveys has indicated that within most ecosystems, a significant fraction of the community could not be assigned to known microbial phyla. Accurate determination of the phylogenetic affiliation of such sequences is difficult due to the short-read-length output of currently available high-throughput technologies. This fraction could harbor multiple novel phylogenetic lineages that have so far escaped detection. Here we describe our efforts in accurate assessment of the novelty and phylogenetic affiliation of selected unclassified lineages within a pyrosequencing data set generated from source sediments of Zodletone Spring, a sulfide- and sulfur-rich spring in southwestern Oklahoma. Lineage-specific forward primers were designed for 78 putatively novel lineages identified within the pyrosequencing data set, and representative nearly full-length small-subunit (SSU) rRNA gene sequences were obtained by pairing those primers with reverse universal bacterial primers. Of the 78 lineages tested, amplifiable products were obtained for 52, 32 of which had at least one nearly full-length sequence that was representative of the lineage targeted. Analysis of phylogenetic affiliation of the obtained Sanger sequences identified 5 novel candidate phyla and 10 novel candidate classes (within Fibrobacteres, Planctomycetes, and candidate phyla BRC1, GN12, TM6, TM7, LD1, WS2, and GN06) in the data set, in addition to multiple novel orders and families. The discovery of multiple novel phyla within a pilot study of a single ecosystem clearly shows the potential of the approach in identifying novel diversities within the rare biosphere.     

The extent of protist diversity: insights from molecular ecology of freshwater eukaryotes

Classical studies on protist diversity of freshwater environments worldwide have led to the idea that most species of microbial eukaryotes are known. One exemplary case would be constituted by the ciliates, which have been claimed to encompass a few thousands of ubiquitous species, most of them already described. Recently, molecular methods have revealed an unsuspected protist diversity, especially in oceanic as well as some extreme environments, suggesting the occurrence of a hidden diversity of eukaryotic lineages. In order to test if this holds also for freshwater environments, we have carried out a molecular survey of small subunit ribosomal RNA genes in water and sediment samples of two ponds, one oxic and another suboxic, from the same geographic area. Our results show that protist diversity is very high. The majority of phylotypes affiliated within a few well established eukaryotic kingdoms or phyla, including alveolates, cryptophytes, heterokonts, Cercozoa, Centroheliozoa and haptophytes, although a few sequences did not display a clear taxonomic affiliation. The diversity of sequences within groups was very large, particularly that of ciliates, and a number of them were very divergent from known species, which could define new intra-phylum groups. This suggests that, contrary to current ideas, the diversity of freshwater protists is far from being completely described.     

Recent trends in industrial microbiology

The majority of current biotechnological applications are of microbial origin, and it is widely appreciated that the microbial world contains by far the greatest fraction of biodiversity in the biosphere. Because of their biotech impact, numerous efforts are being undertaken worldwide, with an ultimate goal to deliver new usable substances of microbial origin to the marketplace. However, the direct isolation of microbes always revealed that the majority are not amenable to be cultured and no representatives for many major microbial phyla have been thus far characterized. Therefore, the knowledge on new microbes and/or genomic information thereof, or from their communities, will pose an enormous potential to provide industry with novel products and processes based on the use of microbial resources, and contribute to and extend the basic mechanistic knowledge on the functioning of organisms. The present review highlights some examples and advances in the exploration of the genetic reservoir of (un)cultured microbes for industrial applications.     

Metagenomics--the key to the uncultured microbes

It is widely accepted that up to 99.8% of the microbes present in many environments are not readily culturable. 'Metagenome technology' tries to overcome this bottleneck by developing and using culture-independent approaches. From the outset, metagenome-based approaches have led to the accumulation of an increasing number of DNA sequences, but until this time the sequences retrieved have been those of uncultured microbes. These genomic sequences are currently exploited for novel biotechnological and pharmaceutical applications and to increase our knowledge on microbial ecology and physiology of these microbes. Using the metagenome sequences to fully understand how complex microbial communities function and how microbes interact within these niches represents a major challenge for microbiologists today.     

Microbial Pathogens in the Fungal Kingdom

The fungal kingdom is vast, spanning ~1.5 to as many as 5 million species diverse as unicellular yeasts, filamentous fungi, mushrooms, lichens, and both plant and animal pathogens. The fungi are closely aligned with animals in one of the six to eight supergroups of eukaryotes, the opisthokonts. The animal and fungal kingdoms last shared a common ancestor ~1 billion years ago, more recently than other groups of eukaryotes. As a consequence of their close evolutionary history and shared cellular machinery with metazoans, fungi are exceptional models for mammalian biology, but prove more difficult to treat in infected animals. The last common ancestor to the fungal/metazoan lineages is thought to have been unicellular, aquatic, and motile with a posterior flagellum, and certain extant species closely resemble this hypothesized ancestor. Species within the fungal kingdom were traditionally assigned to four phyla, including the basal fungi (Chytridiomycota, Zygomycota) and the more recently derived monophyletic lineage, the dikarya (Ascomycota, Basidiomycota). The fungal tree of life project has revealed that the basal lineages are polyphyletic, and thus there are as many as eight to ten fungal phyla. Fungi that infect vertebrates are found in all of the major lineages, and virulence arose multiple times independently. A sobering recent development involves the species Batrachochytrium dendrobatidis from the basal fungal phylum, the Chytridiomycota, which has emerged to cause global amphibian declines and extinctions. Genomics is revolutionizing our view of the fungal kingdom, and genome sequences for zygomycete pathogens (Rhizopus, Mucor), skin-associated fungi (dermatophytes, Malassezia), and the Candida pathogenic species clade promise to provide insights into the origins of virulence. Here we survey the diversity of fungal pathogens and illustrate key principles revealed by genomics involving sexual reproduction and sex determination, loss of conserved pathways in derived fungal lineages that are retained in basal fungi, and shared and divergent virulence strategies of successful human pathogens, including dimorphic and trimorphic transitions in form. The overarching conclusion is that fungal pathogens of animals have arisen repeatedly and independently throughout the fungal tree of life, and while they share general properties, there are also unique features to the virulence strategies of each successful microbial pathogen.     

PCR Primers for Metazoan Nuclear 18S and 28S Ribosomal DNA Sequences

Background

Metagenetic analyses, which amplify and sequence target marker DNA regions from environmental samples, are increasingly employed to assess the biodiversity of communities of small organisms. Using this approach, our understanding of microbial diversity has expanded greatly. In contrast, only a few studies using this approach to characterize metazoan diversity have been reported, despite the fact that many metazoan species are small and difficult to identify or are undescribed. One of the reasons for this discrepancy is the availability of universal primers for the target taxa. In microbial studies, analysis of the 16S ribosomal DNA is standard. In contrast, the best gene for metazoan metagenetics is less clear. In the present study, we have designed primers that amplify the nuclear 18S and 28S ribosomal DNA sequences of most metazoan species with the goal of providing effective approaches for metagenetic analyses of metazoan diversity in environmental samples, with a particular emphasis on marine biodiversity.

Methodology/Principal Findings

Conserved regions suitable for designing PCR primers were identified using 14,503 and 1,072 metazoan sequences of the nuclear 18S and 28S rDNA regions, respectively. The sequence similarity of both these newly designed and the previously reported primers to the target regions of these primers were compared for each phylum to determine the expected amplification efficacy. The nucleotide diversity of the flanking regions of the primers was also estimated for genera or higher taxonomic groups of 11 phyla to determine the variable regions within the genes.

Conclusions/Significance

The identified nuclear ribosomal DNA primers (five primer pairs for 18S and eleven for 28S) and the results of the nucleotide diversity analyses provide options for primer combinations for metazoan metagenetic analyses. Additionally, advantages and disadvantages of not only the 18S and 28S ribosomal DNA, but also other marker regions as targets for metazoan metagenetic analyses, are discussed.     

New subgroup of Bacteroidetes and diverse microorganisms in Tibetan plateau glacial ice provide a biological record of environmental conditions

We recovered microorganisms from five ice core samples from three glaciers (Puruogangri, Malan, and Dunde) located in the Tibetan Plateau in China and analyzed their small subunit rRNA gene sequences. Most of the bacterial sequences were unknown previously; the most closely related known sequences were from bacteria of the Proteobacteria, Bacteroidetes , and Actinobacteria phyla. Chlorophyta, Streptophyta, Ciliophora , and fungal groups were represented among the 18S rRNA gene sequences that we obtained. The most abundantly represented glacial bacteria were Bacteroidetes , and Chlamydomonas was the predominant eukaryote. Comparative analysis showed that the Bacteroidetes sequences obtained from this study were highly similar to one another but most were only distantly related to previously characterized Bacteroidetes (<92% identity). We propose that our Bacteroidetes sequences represent two novel subgroups: one at the family level and one at the genus level. The unique ice environment and the high abundance of Bacteroidetes , combined with the coexistence of a high abundance of psychrophilic Chlamydomonas , strongly suggests that there is a viable ecosystem on the surface of Tibetan glaciers. Comparisons of microbial community structures in the five ice samples showed distinct differences, likely due to environmental differences in the locations in which the samples were obtained.     

Xanthine Dehydrogenase (XDH): Episodic Evolution of a ``Neutral' Protein

We investigated the evolution of xanthine dehydrogenase (Xdh) in 34 species from the three multicellular kingdoms, including one plant, two fungi, and three animal phyla, two classes of vertebrates, four orders of mammals, and two orders of insects. We adopted a model-based maximum-likelihood framework of inference. After accounting for among-site rate variation and heterogeneous nucleotide composition of the sequences using the discrete gamma distribution, and using nonhomogeneous nonstationary representations of the substitution process, the rate of amino acid replacement is 30.4 x 10(-10)/site/year when Drosophila species are compared but only approximately 18 x 10(-10)/site/year when comparisons are made between mammal orders, between insect orders, or between different animal phyla and approximately 11 x 10(-10)/site/year when comparisons are made between birds and mammals, between fungi, or between the three multicellular kingdoms. To account for these observations, the rate of amino acid replacement must have been eight or more times higher in some lineages and at some times than in others. Spastic evolution of Xdh appears to be related to the particularities of the genomes in which the locus is embedded.     

Phage-bacteria relationships and CRISPR elements revealed by a metagenomic survey of the rumen microbiome

Viruses are the most abundant biological entities on the planet and play an important role in balancing microbes within an ecosystem and facilitating horizontal gene transfer. Although bacteriophages are abundant in rumen environments, little is known about the types of viruses present or their interaction with the rumen microbiome. We undertook random pyrosequencing of virus-enriched metagenomes (viromes) isolated from bovine rumen fluid and analysed the resulting data using comparative metagenomics. A high level of diversity was observed with up to 28,000 different viral genotypes obtained from each environment. The majority (~78%) of sequences did not match any previously described virus. Prophages outnumbered lytic phages approximately 2:1 with the most abundant bacteriophage and prophage types being associated with members of the dominant rumen phyla (Firmicutes and Proteobacteria). Metabolic profiling based on SEED subsystems revealed an enrichment of sequences with putative functional roles in DNA and protein metabolism, but a surprisingly low proportion of sequences assigned to carbohydrate and amino acid metabolism. We expanded our analysis to include previously described metagenomic data and 14 reference genomes. Clustered regularly interspaced short palindromic repeats (CRISPR) were detected in most of the microbial genomes, suggesting previous interactions between viral and microbial communities.     

Recombinant environmental libraries provide access to microbial diversity for drug discovery from natural products

To further explore possible avenues for accessing microbial biodiversity for drug discovery from natural products, we constructed and screened a 5,000-clone "shotgun" environmental DNA library by using an Escherichia coli-Streptomyces lividans shuttle cosmid vector and DNA inserts from microbes derived directly (without cultivation) from soil. The library was analyzed by several means to assess diversity, genetic content, and expression of heterologous genes in both expression hosts. We found that the phylogenetic content of the DNA library was extremely diverse, representing mostly microorganisms that have not been described previously. The library was screened by PCR for sequences similar to parts of type I polyketide synthase genes and tested for the expression of new molecules by screening of live colonies and cell extracts. The results revealed new polyketide synthase genes in at least eight clones. In addition, at least five additional clones were confirmed by high-pressure liquid chromatography analysis and/or biological activity to produce heterologous molecules. These data reinforce the idea that exploiting previously unknown or uncultivated microorganisms for the discovery of novel natural products has potential value and, most importantly, suggest a strategy for developing this technology into a realistic and effective drug discovery tool.     

Biosynthetic potential of uncultured Antarctic soil bacteria revealed through long-read metagenomic sequencing

The growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as potential sources of new antimicrobials. PCR amplicon analyses and short-read sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes, indicating their potential for producing novel and useful compounds. However, recovering full-length BGC sequences from uncultivated bacteria remains a challenge due to the technological restraints of short-read sequencing, thus making assessment of BGC diversity difficult. Here, long-read sequencing and genome mining were used to recover >1400 mostly full-length BGCs that demonstrate the rich diversity of BGCs from uncultivated lineages present in soil from Mars Oasis, Antarctica. A large number of highly divergent BGCs were not only found in the phyla Acidobacteriota, Verrucomicrobiota and Gemmatimonadota but also in the actinobacterial classes Acidimicrobiia and Thermoleophilia and the gammaproteobacterial order UBA7966. The latter furthermore contained a potential novel family of RiPPs. Our findings underline the biosynthetic potential of underexplored phyla as well as unexplored lineages within seemingly well-studied producer phyla. They also showcase long-read metagenomic sequencing as a promising way to access the untapped genetic reservoir of specialised metabolite gene clusters of the uncultured majority of microbes.Subject terms: Metagenomics, Antibiotics, Microbial ecology, DNA sequencing     

The dynamic nature of eukaryotic genomes

Analyses of diverse eukaryotes reveal that genomes are dynamic, sometimes dramatically so. In numerous lineages across the eukaryotic tree of life, DNA content varies within individuals throughout life cycles and among individuals within species. Discovery of examples of genome dynamism is accelerating as genome sequences are completed from diverse eukaryotes. Though much is known about genomes in animals, fungi, and plants, these lineages represent only 3 of the 60-200 lineages of eukaryotes. Here, we discuss diverse genomic strategies in exemplar eukaryotic lineages, including numerous microbial eukaryotes, to reveal dramatic variation that challenges established views of genome evolution. For example, in the life cycle of some members of the "radiolaria," ploidy increases from haploid (N) to approximately 1,000N, whereas intrapopulation variability of the enteric parasite Entamoeba ranges from 4N to 40N. Variation has also been found within our own species, with substantial differences in both gene content and chromosome lengths between individuals. Data on the dynamic nature of genomes shift the perception of the genome from being fixed and characteristic of a species (typological) to plastic due to variation within and between species.     

Genetic Diversity of Microbial Eukaryotes in Anoxic Sediment of the Saline Meromictic Lake Namako-ike (Japan): On the Detection of Anaerobic or Anoxic-tolerant Lineages of Eukaryotes

Available sequence data on eukaryotic small-subunit ribosomal DNA (SSU rDNA) directly retrieved from various environments have increased recently, and the diversity of microbial eukaryotes (protists) has been shown to be much greater than previously expected. However, the molecular information accumulated to date does still not thoroughly reveal ecological distribution patterns of microbial eukaryotes. In the ongoing challenge to detect anaerobic or anoxic-tolerant lineages of eukaryotes, we directly extracted DNA from the anoxic sediment of a saline meromictic lake, constructed genetic libraries of PCR-amplified SSU rDNA, and performed phylogenetic analyses with the cloned SSU rDNA sequences. Although a few sequences could not be confidently assigned to any major eukaryotic groups in the analyses and are debatable regarding their taxonomic positions, most sequences obtained have affiliations with known major lineages of eukaryotes (Cercozoa, Alveolata, Stramenopiles, and Opisthokonta). Among these sequences, some branched with lineages predominantly composed of uncultured environmental clones retrieved from other anoxic environments, while others were closely related to those of eukaryotic parasites (e.g. Phytomyxea of Cercozoa, Gregarinea of Alveolata, and Ichthyosporea of Opisthokonta).     

Allopatric divergence, secondary contact, and genetic isolation in wild yeast populations

In plants and animals, new biological species clearly have arisen as a byproduct of genetic divergence in allopatry. However, our understanding of the processes that generate new microbial species remains limited [1] despite the large contribution of microbes to the world's biodiversity. A recent hypothesis claims that microbes lack biogeographical divergence because their population sizes are large and their migration rates are presumably high [2, 3]. In recapitulating the classic microbial-ecology dictum that "everything is everywhere, and the environment selects"[4, 5], this hypothesis casts doubt on whether geographic divergence promotes speciation in microbes. To date, its predictions have been tested primarily with data from eubacteria and archaebacteria [6-8]. However, this hypothesis's most important implication is in sexual eukaryotic microbes, where migration and genetic admixture are specifically predicted to inhibit allopatric divergence and speciation [9]. Here, we use nuclear-sequence data from globally distributed natural populations of the yeast Saccharomyces paradoxus to investigate the role of geography in generating diversity in sexual eukaryotic microbes. We show that these populations have undergone allopatric divergence and then secondary contact without genetic admixture. Our data thus support the occurrence of evolutionary processes necessary for allopatric speciation in sexual microbes.     

Mining genomes and 'metagenomes' for novel catalysts

Advances in the field of genomics and 'metagenomics' have dramatically revised our view of microbial biodiversity and its potential for biotechnological applications. Considering the estimation that >99% of microorganisms in most environments are not amenable to culturing, very little is known about their genomes, genes and encoded enzymatic activities. The isolation, archiving and analysis of environmental DNA (or so-called 'metagenomes') has enabled us to mine microbial diversity, allowing us to access their genomes, identify protein coding sequences and even to reconstruct biochemical pathways, providing insights into the properties and functions of these organisms. The generation and analysis of (meta)genomic libraries is thus a powerful approach to harvest and archive environmental genetic resources. It will enable us to identify which organisms are present, what they do, and how their genetic information can be beneficial to mankind.     

Bacterial diversity in human subgingival plaque

The purpose of this study was to determine the bacterial diversity in the human subgingival plaque by using culture-independent molecular methods as part of an ongoing effort to obtain full 16S rRNA sequences for all cultivable and not-yet-cultivated species of human oral bacteria. Subgingival plaque was analyzed from healthy subjects and subjects with refractory periodontitis, adult periodontitis, human immunodeficiency virus periodontitis, and acute necrotizing ulcerative gingivitis. 16S ribosomal DNA (rDNA) bacterial genes from DNA isolated from subgingival plaque samples were PCR amplified with all-bacterial or selective primers and cloned into Escherichia coli. The sequences of cloned 16S rDNA inserts were used to determine species identity or closest relatives by comparison with sequences of known species. A total of 2,522 clones were analyzed. Nearly complete sequences of approximately 1,500 bases were obtained for putative new species. About 60% of the clones fell into 132 known species, 70 of which were identified from multiple subjects. About 40% of the clones were novel phylotypes. Of the 215 novel phylotypes, 75 were identified from multiple subjects. Known putative periodontal pathogens such as Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola were identified from multiple subjects, but typically as a minor component of the plaque as seen in cultivable studies. Several phylotypes fell into two recently described phyla previously associated with extreme natural environments, for which there are no cultivable species. A number of species or phylotypes were found only in subjects with disease, and a few were found only in healthy subjects. The organisms identified only from diseased sites deserve further study as potential pathogens. Based on the sequence data in this study, the predominant subgingival microbial community consisted of 347 species or phylotypes that fall into 9 bacterial phyla. Based on the 347 species seen in our sample of 2,522 clones, we estimate that there are 68 additional unseen species, for a total estimate of 415 species in the subgingival plaque. When organisms found on other oral surfaces such as the cheek, tongue, and teeth are added to this number, the best estimate of the total species diversity in the oral cavity is approximately 500 species, as previously proposed.     

Recombinant Environmental Libraries Provide Access to Microbial Diversity for Drug Discovery from Natural Products

To further explore possible avenues for accessing microbial biodiversity for drug discovery from natural products, we constructed and screened a 5,000-clone “shotgun” environmental DNA library by using an Escherichia coli-Streptomyces lividans shuttle cosmid vector and DNA inserts from microbes derived directly (without cultivation) from soil. The library was analyzed by several means to assess diversity, genetic content, and expression of heterologous genes in both expression hosts. We found that the phylogenetic content of the DNA library was extremely diverse, representing mostly microorganisms that have not been described previously. The library was screened by PCR for sequences similar to parts of type I polyketide synthase genes and tested for the expression of new molecules by screening of live colonies and cell extracts. The results revealed new polyketide synthase genes in at least eight clones. In addition, at least five additional clones were confirmed by high-pressure liquid chromatography analysis and/or biological activity to produce heterologous molecules. These data reinforce the idea that exploiting previously unknown or uncultivated microorganisms for the discovery of novel natural products has potential value and, most importantly, suggest a strategy for developing this technology into a realistic and effective drug discovery tool.