Candidatus Nemesobacterales is a sponge-specific clade of the candidate phylum Desulfobacterota adapted to a symbiotic lifestyle.

Members of the candidate phylum Dadabacteria, recently reassigned to the phylum Candidatus Desulfobacterota, are cosmopolitan in the marine environment found both free-living and associated with hosts that are mainly marine sponges. Yet, these microorganisms are poorly characterized, with no cultured representatives and an ambiguous phylogenetic position in the tree of life. Here, we performed genome-centric metagenomics to elucidate their phylogenomic placement and predict the metabolism of the sponge-associated members of this lineage. Rank-based phylogenomics revealed several new species and a novel family (Candidatus Spongomicrobiaceae) within a sponge-specific order, named here Candidatus Nemesobacterales. Metabolic reconstruction suggests that Ca. Nemesobacterales are aerobic heterotrophs, capable of synthesizing most amino acids, vitamins and cofactors and degrading complex carbohydrates. We also report functional divergence between sponge- and seawater-associated metagenome-assembled genomes. Niche-specific adaptations to the sponge holobiont were evident from significantly enriched genes involved in defense mechanisms against foreign DNA and environmental stressors, host-symbiont interactions and secondary metabolite production. Fluorescence in situ hybridization gave a first glimpse of the morphology and lifestyle of a member of Ca. Desulfobacterota. Candidatus Nemesobacterales spp. were found both inside sponge cells centred around sponge nuclei and in the mesohyl of the sponge Geodia barretti. This study sheds light on the enigmatic group Ca. Nemesobacterales and their functional characteristics that reflect a symbiotic lifestyle.

Distribution and diversity of 'Tectomicrobia', a deep-branching uncultivated bacterial lineage harboring rich producers of bioactive metabolites.

Genomic and functional analyses of bacterial sponge symbionts belonging to the uncultivated candidate genus 'Entotheonella' has revealed them as the prolific producers of bioactive compounds previously identified from their invertebrate hosts. These studies also suggested 'Entotheonella' as the first members of a new candidate phylum, 'Tectomicrobia'. Here we analyzed the phylogenetic structure and environmental distribution of this as-yet sparsely populated phylum-like lineage. The data show that 'Entotheonella' and other 'Tectomicrobia' are not restricted to marine habitats but widely distributed among terrestrial locations. The inferred phylogenetic trees suggest several intra-phylum lineages with diverse lifestyles. Of these, the previously described 'Entotheonella' lineage can be more accurately divided into at least three different candidate genera with the terrestrial 'Candidatus Prasianella', the largely terrestrial 'Candidatus Allonella', the 'Candidatus Thalassonella' comprising sponge-associated members, and the more widely distributed 'Candidatus Entotheonella'. Genomic characterization of 'Thalassonella' members from a range of sponge hosts did not suggest a role as providers of natural products, despite high genomic similarity to 'Entotheonella' regarding primary metabolism and implied lifestyle. In contrast, the analysis revealed a correlation between the revised 'Entotheonella' 16S rRNA gene phylogeny and a specific association with sponges and their natural products. This feature might serve as a discovery method to accelerate the identification of new chemically rich 'Entotheonella' variants, and led to the identification of the first 'Entotheonella' symbiont in a non-tetractinellid sponge, Psammocinia sp., indicating a wide host distribution of 'Entotheonella'-based chemical symbiosis.

Omics and imaging combinatorial approach reveals butyrate-induced inflammatory effects in the zebrafish gut.

BACKGROUND: Prebiotic feed additives aim to improve gut health by influencing the microbiota and the gut barrier. Most studies on feed additives concentrate on one or two (monodisciplinary) outcome parameters, such as immunity, growth, microbiota or intestinal architecture. A combinatorial and comprehensive approach to disclose the complex and multifaceted effects of feed additives is needed to understand their underlying mechanisms before making health benefit claims. Here, we used juvenile zebrafish as a model species to study effects of feed additives by integrating gut microbiota composition data and host gut transcriptomics with high-throughput quantitative histological analysis. Zebrafish received either control, sodium butyrate or saponin-supplemented feed. Butyrate-derived components such as butyric acid or sodium butyrate have been widely used in animal feeds due to their immunostimulant properties, thereby supporting intestinal health. Soy saponin is an antinutritional factor from soybean meal that promotes inflammation due to its amphipathic nature. RESULTS: We observed distinct microbial profiles associated with each diet, discovering that butyrate (and saponin to a lesser extent) affected gut microbial composition by reducing the degree of community-structure (co-occurrence network analysis) compared to controls. Analogously, butyrate and saponin supplementation impacted the transcription of numerous canonical pathways compared to control-fed fish. For example, both butyrate and saponin increased the expression of genes associated with immune response and inflammatory response, as well as oxidoreductase activity, compared to controls. Furthermore, butyrate decreased the expression of genes associated with histone modification, mitotic processes and G-coupled receptor activity. High-throughput quantitative histological analysis depicted an increase of eosinophils and rodlet cells in the gut tissue of fish receiving butyrate after one week of feeding and a depletion of mucus-producing cells after 3 weeks of feeding this diet. Combination of all datasets indicated that in juvenile zebrafish, butyrate supplementation increases the immune and the inflammatory response to a greater extent than the established inflammation-inducing anti-nutritional factor saponin. Such comprehensive analysis was supplemented by in vivo imaging of neutrophil and macrophage transgenic reporter zebrafish (mpeg1:mCherry/mpx:eGFPi114) larvae. Upon exposure to butyrate and saponin, these larvae displayed a dose-dependent increase of neutrophils and macrophages in the gut area. CONCLUSION: The omics and imaging combinatorial approach provided an integrated evaluation of the effect of butyrate on fish gut health and unraveled inflammatory-like features not previously reported that question the usage of butyrate supplementation to enhance fish gut health under basal conditions. The zebrafish model, due to its unique advantages, provides researchers with an invaluable tool to investigate effects of feed components on fish gut health throughout life.

A genomic view of trophic and metabolic diversity in clade-specific Lamellodysidea sponge microbiomes.

BACKGROUND: Marine sponges and their microbiomes contribute significantly to carbon and nutrient cycling in global reefs, processing and remineralizing dissolved and particulate organic matter. Lamellodysidea herbacea sponges obtain additional energy from abundant photosynthetic Hormoscilla cyanobacterial symbionts, which also produce polybrominated diphenyl ethers (PBDEs) chemically similar to anthropogenic pollutants of environmental concern. Potential contributions of non-Hormoscilla bacteria to Lamellodysidea microbiome metabolism and the synthesis and degradation of additional secondary metabolites are currently unknown. RESULTS: This study has determined relative abundance, taxonomic novelty, metabolic capacities, and secondary metabolite potential in 21 previously uncharacterized, uncultured Lamellodysidea-associated microbial populations by reconstructing near-complete metagenome-assembled genomes (MAGs) to complement 16S rRNA gene amplicon studies. Microbial community compositions aligned with sponge host subgroup phylogeny in 16 samples from four host clades collected from multiple sites in Guam over a 3-year period, including representatives of Alphaproteobacteria, Gammaproteobacteria, Oligoflexia, and Bacteroidetes as well as Cyanobacteria (Hormoscilla). Unexpectedly, microbiomes from one host clade also included Cyanobacteria from the prolific secondary metabolite-producer genus Prochloron, a common tunicate symbiont. Two novel Alphaproteobacteria MAGs encoded pathways diagnostic for methylotrophic metabolism as well as type III secretion systems, and have been provisionally assigned to a new order, designated Candidatus Methylospongiales. MAGs from other taxonomic groups encoded light-driven energy production pathways using not only chlorophyll, but also bacteriochlorophyll and proteorhodopsin. Diverse heterotrophic capabilities favoring aerobic versus anaerobic conditions included pathways for degrading chitin, eukaryotic extracellular matrix polymers, phosphonates, dimethylsulfoniopropionate, trimethylamine, and benzoate. Genetic evidence identified an aerobic catabolic pathway for halogenated aromatics that may enable endogenous PBDEs to be used as a carbon and energy source. CONCLUSIONS: The reconstruction of high-quality MAGs from all microbial taxa comprising greater than 0.1% of the sponge microbiome enabled species-specific assignment of unique metabolic features that could not have been predicted from taxonomic data alone. This information will promote more representative models of marine invertebrate microbiome contributions to host bioenergetics, the identification of potential new sponge parasites and pathogens based on conserved metabolic and physiological markers, and a better understanding of biosynthetic and degradative pathways for secondary metabolites and halogenated compounds in sponge-associated microbiota. Video Abstract.

Comparative Genomics of Cyanobacterial Symbionts Reveals Distinct, Specialized Metabolism in Tropical Dysideidae Sponges.

Marine sponges are recognized as valuable sources of bioactive metabolites and renowned as petri dishes of the sea, providing specialized niches for many symbiotic microorganisms. Sponges of the family Dysideidae are well documented to be chemically talented, often containing high levels of polyhalogenated compounds, terpenoids, peptides, and other classes of bioactive small molecules. This group of tropical sponges hosts a high abundance of an uncultured filamentous cyanobacterium, Hormoscilla spongeliae Here, we report the comparative genomic analyses of two phylogenetically distinct Hormoscilla populations, which reveal shared deficiencies in essential pathways, hinting at possible reasons for their uncultivable status, as well as differing biosynthetic machinery for the production of specialized metabolites. One symbiont population contains clustered genes for expanded polybrominated diphenylether (PBDE) biosynthesis, while the other instead harbors a unique gene cluster for the biosynthesis of the dysinosin nonribosomal peptides. The hybrid sequencing and assembly approach utilized here allows, for the first time, a comprehensive look into the genomes of these elusive sponge symbionts.IMPORTANCE Natural products provide the inspiration for most clinical drugs. With the rise in antibiotic resistance, it is imperative to discover new sources of chemical diversity. Bacteria living in symbiosis with marine invertebrates have emerged as an untapped source of natural chemistry. While symbiotic bacteria are often recalcitrant to growth in the lab, advances in metagenomic sequencing and assembly now make it possible to access their genetic blueprint. A cell enrichment procedure, combined with a hybrid sequencing and assembly approach, enabled detailed genomic analysis of uncultivated cyanobacterial symbiont populations in two chemically rich tropical marine sponges. These population genomes reveal a wealth of secondary metabolism potential as well as possible reasons for historical difficulties in their cultivation.

Targeted antibiotic discovery through biosynthesis-associated resistance determinants: target directed genome mining.

Intense competition between microbes in the environment has directed the evolution of antibiotic production in bacteria. Humans have harnessed these natural molecules for medicinal purposes, magnifying them from environmental concentrations to industrial scale. This increased exposure to antibiotics has amplified antibiotic resistance across bacteria, spurring a global antimicrobial crisis and a search for antibiotics with new modes of action. Genetic insights into these antibiotic-producing microbes reveal that they have evolved several resistance strategies to avoid self-toxicity, including product modification, substrate transport and binding, and target duplication or modification. Of these mechanisms, target duplication or modification will be highlighted in this review, as it uniquely links an antibiotic to its mode of action. We will further discuss and propose a strategy to mine microbial genomes for these genes and their associated biosynthetic gene clusters to discover novel antibiotics using target directed genome mining.

Diversity and distribution of the bmp gene cluster and its Polybrominated products in the genus Pseudoalteromonas.

The production of pentabromopseudilin and related brominated compounds by Pseudoalteromonas spp. has recently been linked to the bmp biosynthetic gene cluster. This study explored the distribution and evolutionary history of this gene cluster in the genus Pseudoalteromonas. A phylogeny of the genus revealed numerous clades that do not contain type strains, suggesting considerable species level diversity has yet to be described. Comparative genomics revealed four distinct versions of the gene cluster distributed among 19 of the 101 Pseudoalteromonas genomes examined. These were largely localized to the least inclusive clades containing the Pseudoalteromonas luteoviolacea and Pseudoalteromonas phenolica type strains and show clear evidence of gene and gene cluster loss in certain lineages. Bmp gene phylogeny is largely congruent with the Pseudoalteromonas species phylogeny, suggesting vertical inheritance within the genus. However, the gene cluster is found in three different genomic environments suggesting either chromosomal rearrangement or multiple acquisition events. Bmp conservation within certain lineages suggests the encoded products are highly relevant to the ecology of these bacteria.

Metagenomic discovery of polybrominated diphenyl ether biosynthesis by marine sponges.

Naturally produced polybrominated diphenyl ethers (PBDEs) pervade the marine environment and structurally resemble toxic man-made brominated flame retardants. PBDEs bioaccumulate in marine animals and are likely transferred to the human food chain. However, the biogenic basis for PBDE production in one of their most prolific sources, marine sponges of the order Dysideidae, remains unidentified. Here, we report the discovery of PBDE biosynthetic gene clusters within sponge-microbiome-associated cyanobacterial endosymbionts through the use of an unbiased metagenome-mining approach. Using expression of PBDE biosynthetic genes in heterologous cyanobacterial hosts, we correlate the structural diversity of naturally produced PBDEs to modifications within PBDE biosynthetic gene clusters in multiple sponge holobionts. Our results establish the genetic and molecular foundation for the production of PBDEs in one of the most abundant natural sources of these molecules, further setting the stage for a metagenomic-based inventory of other PBDE sources in the marine environment.

Effects of Actinomycete Secondary Metabolites on Sediment Microbial Communities.

Marine sediments harbor complex microbial communities that remain poorly studied relative to other biomes such as seawater. Moreover, bacteria in these communities produce antibiotics and other bioactive secondary metabolites, yet little is known about how these compounds affect microbial community structure. In this study, we used next-generation amplicon sequencing to assess native microbial community composition in shallow tropical marine sediments. The results revealed complex communities comprised of largely uncultured taxa, with considerable spatial heterogeneity and known antibiotic producers comprising only a small fraction of the total diversity. Organic extracts from cultured strains of the sediment-dwelling actinomycete genus Salinispora were then used in mesocosm studies to address how secondary metabolites shape sediment community composition. We identified predatory bacteria and other taxa that were consistently reduced in the extract-treated mesocosms, suggesting that they may be the targets of allelopathic interactions. We tested related taxa for extract sensitivity and found general agreement with the culture-independent results. Conversely, several taxa were enriched in the extract-treated mesocosms, suggesting that some bacteria benefited from the interactions. The results provide evidence that bacterial secondary metabolites can have complex and significant effects on sediment microbial communities. IMPORTANCE: Ocean sediments represent one of Earth's largest and most poorly studied biomes. These habitats are characterized by complex microbial communities where competition for space and nutrients can be intense. This study addressed the hypothesis that secondary metabolites produced by the sediment-inhabiting actinomycete Salinispora arenicola affect community composition and thus mediate interactions among competing microbes. Next-generation amplicon sequencing of mesocosm experiments revealed complex communities that shifted following exposure to S. arenicola extracts. The results reveal that certain predatory bacteria were consistently less abundant following exposure to extracts, suggesting that microbial metabolites mediate competitive interactions. Other taxa increased in relative abundance, suggesting a benefit from the extracts themselves or the resulting changes in the community. This study takes a first step toward assessing the impacts of bacterial metabolites on sediment microbial communities. The results provide insight into how low-abundance organisms may help structure microbial communities in ocean sediments.

Sequencing rare marine actinomycete genomes reveals high density of unique natural product biosynthetic gene clusters.

Traditional natural product discovery methods have nearly exhausted the accessible diversity of microbial chemicals, making new sources and techniques paramount in the search for new molecules. Marine actinomycete bacteria have recently come into the spotlight as fruitful producers of structurally diverse secondary metabolites, and remain relatively untapped. In this study, we sequenced 21 marine-derived actinomycete strains, rarely studied for their secondary metabolite potential and under-represented in current genomic databases. We found that genome size and phylogeny were good predictors of biosynthetic gene cluster diversity, with larger genomes rivalling the well-known marine producers in the Streptomyces and Salinispora genera. Genomes in the Micrococcineae suborder, however, had consistently the lowest number of biosynthetic gene clusters. By networking individual gene clusters into gene cluster families, we were able to computationally estimate the degree of novelty each genus contributed to the current sequence databases. Based on the similarity measures between all actinobacteria in the Joint Genome Institute's Atlas of Biosynthetic gene Clusters database, rare marine genera show a high degree of novelty and diversity, with Corynebacterium, Gordonia, Nocardiopsis, Saccharomonospora and Pseudonocardia genera representing the highest gene cluster diversity. This research validates that rare marine actinomycetes are important candidates for exploration, as they are relatively unstudied, and their relatives are historically rich in secondary metabolites.

Indexing the Pseudomonas specialized metabolome enabled the discovery of poaeamide B and the bananamides.

Pseudomonads are cosmopolitan microorganisms able to produce a wide array of specialized metabolites. These molecules allow Pseudomonas to scavenge nutrients, sense population density and enhance or inhibit growth of competing microorganisms. However, these valuable metabolites are typically characterized one-molecule-one-microbe at a time, instead of being inventoried in large numbers. To index and map the diversity of molecules detected from these organisms, 260 strains of ecologically diverse origins were subjected to mass-spectrometry-based molecular networking. Molecular networking not only enables dereplication of molecules, but also sheds light on their structural relationships. Moreover, it accelerates the discovery of new molecules. Here, by indexing the Pseudomonas specialized metabolome, we report the molecular-networking-based discovery of four molecules and their evolutionary relationships: a poaeamide analogue and a molecular subfamily of cyclic lipopeptides, bananamides 1, 2 and 3. Analysis of their biosynthetic gene cluster shows that it constitutes a distinct evolutionary branch of the Pseudomonas cyclic lipopeptides. Through analysis of an additional 370 extracts of wheat-associated Pseudomonas, we demonstrate how the detailed knowledge from our reference index can be efficiently propagated to annotate complex metabolomic data from other studies, akin to the way in which newly generated genomic information can be compared to data from public databases.

Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase-thioesterase enzyme pair.

Halogenated pyrroles (halopyrroles) are common chemical moieties found in bioactive bacterial natural products. The halopyrrole moieties of mono- and dihalopyrrole-containing compounds arise from a conserved mechanism in which a proline-derived pyrrolyl group bound to a carrier protein is first halogenated and then elaborated by peptidic or polyketide extensions. This paradigm is broken during the marine pseudoalteromonad bacterial biosynthesis of the coral larval settlement cue tetrabromopyrrole (1), which arises from the substitution of the proline-derived carboxylate by a bromine atom. To understand the molecular basis for decarboxylative bromination in the biosynthesis of 1, we sequenced two Pseudoalteromonas genomes and identified a conserved four-gene locus encoding the enzymes involved in its complete biosynthesis. Through total in vitro reconstitution of the biosynthesis of 1 using purified enzymes and biochemical interrogation of individual biochemical steps, we show that all four bromine atoms in 1 are installed by the action of a single flavin-dependent halogenase: Bmp2. Tetrabromination of the pyrrole induces a thioesterase-mediated offloading reaction from the carrier protein and activates the biosynthetic intermediate for decarboxylation. Insights into the tetrabrominating activity of Bmp2 were obtained from the high-resolution crystal structure of the halogenase contrasted against structurally homologous halogenase Mpy16 that forms only a dihalogenated pyrrole in marinopyrrole biosynthesis. Structure-guided mutagenesis of the proposed substrate-binding pocket of Bmp2 led to a reduction in the degree of halogenation catalyzed. Our study provides a biogenetic basis for the biosynthesis of 1 and sets a firm foundation for querying the biosynthetic potential for the production of 1 in marine (meta)genomes.

A Bacterial Quorum-Sensing Precursor Induces Mortality in the Marine Coccolithophore, Emiliania huxleyi.

Interactions between phytoplankton and bacteria play a central role in mediating biogeochemical cycling and food web structure in the ocean. However, deciphering the chemical drivers of these interspecies interactions remains challenging. Here, we report the isolation of 2-heptyl-4-quinolone (HHQ), released by Pseudoalteromonas piscicida, a marine gamma-proteobacteria previously reported to induce phytoplankton mortality through a hitherto unknown algicidal mechanism. HHQ functions as both an antibiotic and a bacterial signaling molecule in cell-cell communication in clinical infection models. Co-culture of the bloom-forming coccolithophore, Emiliania huxleyi with both live P. piscicida and cell-free filtrates caused a significant decrease in algal growth. Investigations of the P. piscicida exometabolome revealed HHQ, at nanomolar concentrations, induced mortality in three strains of E. huxleyi. Mortality of E. huxleyi in response to HHQ occurred slowly, implying static growth rather than a singular loss event (e.g., rapid cell lysis). In contrast, the marine chlorophyte, Dunaliella tertiolecta and diatom, Phaeodactylum tricornutum were unaffected by HHQ exposures. These results suggest that HHQ mediates the type of inter-domain interactions that cause shifts in phytoplankton population dynamics. These chemically mediated interactions, and other like it, ultimately influence large-scale oceanographic processes.

Biosynthesis of polybrominated aromatic organic compounds by marine bacteria.

Polybrominated diphenyl ethers (PBDEs) and polybrominated bipyrroles are natural products that bioaccumulate in the marine food chain. PBDEs have attracted widespread attention because of their persistence in the environment and potential toxicity to humans. However, the natural origins of PBDE biosynthesis are not known. Here we report marine bacteria as producers of PBDEs and establish a genetic and molecular foundation for their production that unifies paradigms for the elaboration of bromophenols and bromopyrroles abundant in marine biota. We provide biochemical evidence of marine brominases revealing decarboxylative-halogenation enzymology previously unknown among halogenating enzymes. Biosynthetic motifs discovered in our study were used to mine sequence databases to discover unrealized marine bacterial producers of organobromine compounds.

Genetic basis for the biosynthesis of the pharmaceutically important class of epoxyketone proteasome inhibitors.

The epoxyketone proteasome inhibitors are an established class of therapeutic agents for the treatment of cancer. Their unique α',ß'-epoxyketone pharmacophore allows binding to the catalytic ß-subunits of the proteasome with extraordinary specificity. Here, we report the characterization of the first gene clusters for the biosynthesis of natural peptidyl-epoxyketones. The clusters for epoxomicin, the lead compound for the anticancer drug Kyprolis, and for eponemycin were identified in the actinobacterial producer strains ATCC 53904 and Streptomyces hygroscopicus ATCC 53709, respectively, using a modified protocol for Ion Torrent PGM genome sequencing. Both gene clusters code for a hybrid nonribosomal peptide synthetase/polyketide synthase multifunctional enzyme complex and homologous redox enzymes. Epoxomicin and eponemycin were heterologously produced in Streptomyces albus J1046 via whole pathway expression. Moreover, we employed mass spectral molecular networking for a new comparative metabolomics approach in a heterologous system and discovered a number of putative epoxyketone derivatives. With this study, we have definitively linked epoxyketone proteasome inhibitors and their biosynthesis genes for the first time in any organism, which will now allow for their detailed biochemical investigation.

An integrated semiconductor device enabling non-optical genome sequencing.

The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.

Multiple, novel biologically active endophytic actinomycetes isolated from upper Amazonian rainforests.

Microbial biodiversity provides an increasingly important source of medically and industrially useful compounds. We have isolated 14 actinomycete species from a collection of approximately 300 plant stem samples from the upper Amazonian rainforest in Peru. All of the cultured isolates produce substances with inhibitory activity directed at a range of potential fungal and bacterial pathogens. For some organisms, this activity is very broad in spectrum while other organisms show specific activity against a limited number of organisms. Two of these organisms preferentially inhibit bacterial test organisms over eukaryotic organisms. rDNA sequence analysis indicates that these organisms are not equivalent to any other cultured deposits in GenBank. Our results provide evidence of the untapped biodiversity in the form of biologically active microbes present within the tissues of higher plants.

Bioactive endophytes warrant intensified exploration and conservation.

BACKGROUND: A key argument in favor of conserving biodiversity is that as yet undiscovered biodiversity will yield products of great use to humans. However, the link between undiscovered biodiversity and useful products is largely conjectural. Here we provide direct evidence from bioassays of endophytes isolated from tropical plants and bioinformatic analyses that novel biology will indeed yield novel chemistry of potential value. METHODOLOGY/PRINCIPAL FINDINGS: We isolated and cultured 135 endophytic fungi and bacteria from plants collected in Peru. nrDNAs were compared to samples deposited in GenBank to ascertain the genetic novelty of cultured specimens. Ten endophytes were found to be as much as 15-30% different than any sequence in GenBank. Phylogenetic trees, using the most similar sequences in GenBank, were constructed for each endophyte to measure phylogenetic distance. Assays were also conducted on each cultured endophyte to record bioactivity, of which 65 were found to be bioactive. CONCLUSIONS/SIGNIFICANCE: The novelty of our contribution is that we have combined bioinformatic analyses that document the diversity found in environmental samples with culturing and bioassays. These results highlight the hidden hyperdiversity of endophytic fungi and the urgent need to explore and conserve hidden microbial diversity. This study also showcases how undergraduate students can obtain data of great scientific significance.