SRA045798.1/ABAZUT16SADKSKSa
Bacterial natural product biosynthetic diversity in geographically distinct soil microbiomes.
Molecular phylogenetic analyses based on 16S rRNA gene sequences indicate that environmental samples often contain thousands of unique bacterial species, only a small fraction of which have been cultured using traditional fermentation techniques.  Contrary to the century old tenant in microbiology that “everything is everywhere; but the environment selects,”  recent molecular phylogenetic based biogeography studies have found that ecologically similar yet geographically distant environments can contain related but distinct consortia of bacterial species.  Extrapolations from these and other studies have led to predictions that there may be as many as 10xe7 to 10xe9 unique bacterial species on earth.  As bacteria are already considered one of the world’s richest sources of bioactive natural products, such predictions may be of profound importance to the natural products community.  However, whether this newly recognized bacterial species diversity corresponds to a modest increase in secondary metabolite biosynthetic diversity or to a radical increase in the number of unexplored biosynthetic systems is currently unknown.  It is possible that there exists a relatively small global collection of secondary metabolite gene clusters that is largely conserved from one location to the next, or conversely, that secondary metabolite gene clusters are to some extent geographically and environmentally constrained, resulting in largely orthogonal collections of gene clusters from within geographically-distinct microbiomes.    Here we seek to address this issue by determining whether the collections of biosynthetic genes present in ecologically similar but geographically distinct soil microbiomes differ from soil to soil or are conserved across geographic boundaries.  The number and diversity of novel natural products that remains to be examined for potentially useful bioactivities is likely to depend heavily on which of these two possibilities is dominant throughout the biosphere.    To compare the biosynthetic potentials of different soil microbiomes, environmental DNA (eDNA) samples extracted directly from three geographically distinct arid regions of the American Southwest [the Sonoran desert of Arizona (AZ), the Anza-Borrego section of the Sonoran desert of California (AB) and the Great Basin desert of Utah (UT)] were used to construct three independent eDNA libraries.  Each library contains at least 100,000 bacterial genome equivalents or 350-400 GB of DNA and is predicted to provide sufficient sequence coverage to capture the major constituents of their respective soil metagenomes.  Degenerate primers designed to recognize conserved sequences found in gene clusters encoding three common families of natural products (nonribosomal peptides, type I polyketides and type II polyketides) were used to PCR amplify secondary metabolite specific gene sequences from each cloned metagenome.  The resulting amplicons, corresponding to nonribosomal peptide synthetase (NRPS) adenylation domain (AD), type I polyketide synthase (PKSI) ketosynthase domain (KS) and type II polyketide synthases (PKSII) ketosynthase alpha (KSa) gene fragments were then deep sequenced using 454 GS-FLX technology and bioinformatically compared.  While the individual soil microbiomes explored in this study contain largely orthogonal sets of biosynthetic gene sequences, the collections of genes found within each soil sample do not appear to have radically different evolutionary origins.  Clustering KSa sequences from all three metagenomes at 15% divergence resulted in 509 OTUs.  A ClustalW derived phylogenetic tree of a representative sequence from each of these OTUs shows that these sequences do not group into metagenome specific clades but are instead distributed throughout the tree, suggesting that sequences from different soils share a diverse set of common ancestors.  AD and KS sequences do show the same general trend, where sequences from different metagenomes distribute evenly throughout the combined phylogenetic trees.  Soils contain highly diverse collections of bacteria, which has made them very attractive starting points for both culture-dependent and culture-independent small molecule discovery efforts.  We chose to investigate secondary metabolism in geographically distinct yet ecologically similar soils based on the belief that ecologically similar soils would have the highest likelihood of containing related sets of biosynthetic gene sequences.  We found that even ecologically similar metagenomes contain almost completely distinct collections of conserved biosynthetic gene sequences.  Although there is unlikely to be a simple correlation between gene sequence diversity and the diversity of metabolites encoded by gene clusters in which these genes reside, our data suggests that not only do these microbiomes contain largely orthogonal sets of biosynthetic genes but that most sequences in one soil metagenome are so distantly related to sequences in another metagenome that they are unlikely to be found in gene clusters that encode the same metabolites.  If this holds true for other soil types, the as yet untapped small molecule biosynthetic diversity present in the Earth's biosphere is likely to be much larger than previously predicted.
The number of bacterial species estimated to exist in the Earth's biosphere has increased dramatically in recent years.  This newly recognized species diversity has raised the possibility that bacterial natural product diversity has also been underestimated by previous studies.  Here we use comparative analysis of conserved secondary metabolite biosynthetic genes derived from three geographically distinct arid soils to show that ecologically similar microbiomes contain largely orthogonal collections of secondary metabolite biosynthetic gene sequences, suggesting that the unexplored pool of bacterial biosynthetic diversity present in nature is likely much larger than previously predicted.