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2018
Kim, MC, Machado H, Jang KH, Trzoss L, Jensen PR, Fenical W.  2018.  Integration of genomic data with NMR analysis enables assignment of the full stereostructure of Neaumycin B, a potent inhibitor of glioblastoma from a marine-derived micromonospora. Journal of the American Chemical Society. 140:10775-10784.   10.1021/jacs.8b04848   AbstractWebsite

The microbial metabolites known as the macrolides are some of the most successful natural products used to treat infectious and immune diseases. Describing the structures of these complex metabolites, however, is often extremely difficult due to the presence of multiple stereogenic centers inherent in this class of polyketide-derived metabolites. With the availability of genome sequence data and a better understanding of the molecular genetics of natural product biosynthesis, it is now possible to use bioinformatic approaches in tandem with spectroscopic tools to assign the full stereostructures of these complex metabolites. In our quest to discover and develop new agents for the treatment of cancer, we observed the production of a highly cytotoxic macrolide, neaumycin B, by a marine-derived actinomycete bacterium of the genus Micromonospora. Neaumycin B is a complex polycyclic macrolide possessing 19 asymmetric centers, usually requiring selective degradation, crystallization, derivatization, X-ray diffraction analysis, synthesis, or other time-consuming approaches to assign the complete stereostructure. As an alternative approach, we sequenced the genome of the producing strain and identified the neaumycin gene cluster (neu). By integrating the known stereospecificities of biosynthetic enzymes with comprehensive NMR analysis, the full stereostructure of neaumycin B was confidently assigned. This approach exemplifies how mining gene cluster information while integrating NMR-based structure data can achieve rapid, efficient, and accurate stereostructural assignments for complex macrolides.

Patin, NV, Floros DJ, Hughes CC, Dorrestein PC, Jensen PR.  2018.  The role of inter-species interactions in Salinispora specialized metabolism. Microbiology-Sgm. 164:946-955.   10.1099/mic.0.000679   AbstractWebsite

Bacterial genome sequences consistently contain many more biosynthetic gene clusters encoding specialized metabolites than predicted by the compounds discovered from the respective strains. One hypothesis invoked to explain the cryptic nature of these gene clusters is that standard laboratory conditions do not provide the environmental cues needed to trigger gene expression. A potential source of such cues is other members of the bacterial community, which are logical targets for competitive interactions. In this study, we examined the effects of such interactions on specialized metabolism in the marine actinomycete Salinispora tropica. The results show that antibiotic activities and the concentration of some small molecules increase in the presence of co-occurring bacterial strains relative to monocultures. Some increases in antibiotic activity could be linked to nutrient depletion by the competitor as opposed to the production of a chemical cue. Other increases were correlated with the production of specific compounds by S. tropica. In particular, one interaction with a Vibrio sp. consistently induced antibiotic activity and was associated with parent ions that were unique to this interaction, although the associated compound could not be identified. This study provides insight into the metabolomic complexities of bacterial interactions and baseline information for future genome mining efforts.

Bruns, H, Crusemann M, Letzel AC, Alanjary M, McInerney JO, Jensen PR, Schulz S, Moore BS, Ziemert N.  2018.  Function-related replacement of bacterial siderophore pathways. Isme Journal. 12:320-329.   10.1038/ismej.2017.137   AbstractWebsite

Bacterial genomes are rife with orphan biosynthetic gene clusters (BGCs) associated with secondary metabolism of unrealized natural product molecules. Often up to a tenth of the genome is predicted to code for the biosynthesis of diverse metabolites with mostly unknown structures and functions. This phenomenal diversity of BGCs coupled with their high rates of horizontal transfer raise questions about whether they are really active and beneficial, whether they are neutral and confer no advantage, or whether they are carried in genomes because they are parasitic or addictive. We previously reported that Salinispora bacteria broadly use the desferrioxamine family of siderophores for iron acquisition. Herein we describe a new and unrelated group of peptidic siderophores called salinichelins from a restricted number of Salinispora strains in which the desferrioxamine biosynthesis genes have been lost. We have reconstructed the evolutionary history of these two different siderophore families and show that the acquisition and retention of the new salinichelin siderophores co- occurs with the loss of the more ancient desferrioxamine pathway. This identical event occurred at least three times independently during the evolution of the genus. We surmise that certain BGCs may be extraneous because of their functional redundancy and demonstrate that the relative evolutionary pace of natural pathway replacement shows high selective pressure against retention of functionally superfluous gene clusters.

2017
Amos, GCA, Awakawa T, Tuttle RN, Letzel AC, Kim MC, Kudo Y, Fenical W, Moore BS, Jensen PR.  2017.  Comparative transcriptomics as a guide to natural product discovery and biosynthetic gene cluster functionality. Proceedings of the National Academy of Sciences of the United States of America. 114:E11121-E11130.   10.1073/pnas.1714381115   AbstractWebsite

Bacterial natural products remain an important source of new medicines. DNA sequencing has revealed that a majority of natural product biosynthetic gene clusters (BGCs) maintained in bacterial genomes have yet to be linked to the small molecules whose biosynthesis they encode. Efforts to discover the products of these orphan BGCs are driving the development of genome mining techniques based on the premise that many are transcriptionally silent during normal laboratory cultivation. Here, we employ comparative transcriptomics to assess BGC expression among four closely related strains of marine bacteria belonging to the genus Salinispora. The results reveal that slightly more than half of the BGCs are expressed at levels that should facilitate product detection. By comparing the expression profiles of similar gene clusters in different strains, we identified regulatory genes whose inactivation appears linked to cluster silencing. The significance of these subtle differences between expressed and silent BGCs could not have been predicted a priori and was only revealed by comparative transcriptomics. Evidence for the conservation of silent clusters among a larger number of strains for which genome sequences are available suggests they may be under different regulatory control from the expressed forms or that silencing may represent an underappreciated mechanism of gene cluster evolution. Coupling gene expression and metabolomics data established a bioinformatic link between the salinipostins and their associated BGC, while genetic manipulation established the genetic basis for this series of compounds, which were previously unknown from Salinispora pacifica.

Machado, H, Tuttle RN, Jensen PR.  2017.  Omics-based natural product discovery and the lexicon of genome mining. Current Opinion in Microbiology. 39:136-142.   10.1016/j.mib.2017.10.025   AbstractWebsite

Genome sequencing and the application of omic techniques are driving many important advances in the field of microbial natural products research. Despite these gains, there remain aspects of the natural product discovery pipeline where our knowledge remains poor. These include the extent to which biosynthetic gene clusters are transcriptionally active in native microbes, the temporal dynamics of transcription, translation, and natural product assembly, as well as the relationships between small molecule production and detection. Here we touch on a number of these concepts in the context of continuing efforts to unlock the natural product potential revealed in genome sequence data and discuss nomenclatural issues that warrant consideration as the field moves forward.

Letzel, AC, Li J, Amos GCA, Millan-Aguinaga N, Ginigini J, Abdelmohsen UR, Gaudencio SP, Ziemert N, Moore BS, Jensen PR.  2017.  Genomic insights into specialized metabolism in the marine actinomycete Salinispora. Environmental Microbiology. 19:3660-3673.   10.1111/1462-2920.13867   AbstractWebsite

Comparative genomics is providing new opportunities to address the diversity and distributions of genes encoding the biosynthesis of specialized metabolites. An analysis of 119 genome sequences representing three closely related species of the marine actinomycete genus Salinispora reveals extraordinary biosynthetic diversity in the form of 176 distinct biosynthetic gene clusters (BGCs) of which only 24 have been linked to their products. Remarkably, more than half of the BGCs were observed in only one or two strains, suggesting they were acquired relatively recently in the evolutionary history of the genus. These acquired gene clusters are concentrated in specific genomic islands, which represent hot spots for BGC acquisition. While most BGCs are stable in terms of their chromosomal position, others migrated to different locations or were exchanged with unrelated gene clusters suggesting a plug and play type model of evolution that provides a mechanism to test the relative fitness effects of specialized metabolites. Transcriptome analyses were used to address the relationships between BGC abundance, chromosomal position and product discovery. The results indicate that recently acquired BGCs can be functional and that complex evolutionary processes shape the micro-diversity of specialized metabolism observed in closely related environmental bacteria.

Gallagher, KA, Wanger G, Henderson J, Llorente M, Hughes CC, Jensen PR.  2017.  Ecological implications of hypoxia-triggered shifts in secondary metabolism. Environmental Microbiology. 19:2182-2191.   10.1111/1462-2920.13700   AbstractWebsite

Members of the actinomycete genus Streptomyces are non-motile, filamentous bacteria that are well-known for the production of biomedically relevant secondary metabolites. While considered obligate aerobes, little is known about how these bacteria respond to periods of reduced oxygen availability in their natural habitats, which include soils and ocean sediments. Here, we provide evidence that the marine streptomycete strain CNQ-525 can reduce MnO2 via a diffusible mechanism. We investigated the effects of hypoxia on secondary metabolite production and observed a shift away from the antibiotic napyradiomycin towards 8-aminoflaviolin, an intermediate in the napyradiomycin biosynthetic pathway. We purified 8-amino-flaviolin and demonstrated that it is reversibly redox-active (midpoint potential -474.5 mV), indicating that it has the potential to function as an endogenous extracellular electron shuttle. This study provides evidence that environmentally triggered changes in secondary metabolite production may provide clues to the ecological functions of specific compounds, and that Gram-positive bacteria considered to be obligate aerobes may play previously unrecognized roles in biogeochemical cycling through mechanisms that include extracellular electron shuttling.

Millan-Aguinaga, N, Chavarria KL, Ugalde JA, Letzel AC, Rouse GW, Jensen PR.  2017.  Phylogenomic Insight into Salinispora (Bacteria, Actinobacteria) Species Designations. Scientific Reports. 7   10.1038/s41598-017-02845-3   AbstractWebsite

Bacteria represent the most genetically diverse kingdom of life. While great progress has been made in describing this diversity, it remains difficult to identify the phylogenetic and ecological characteristics that delineate groups of bacteria that possess species-like properties. One major challenge associated with species delineations is that not all shared genes have the same evolutionary history, and thus the choice of loci can have a major impact on phylogenetic reconstruction. Sequencing the genomes of large numbers of closely related strains provides new opportunities to distinguish ancestral from acquired alleles and assess the effects of recombination on phylogenetic inference. Here we analyzed the genomes of 119 strains of the marine actinomycete genus Salinispora, which is currently comprised of three named species that share 99% 16S rRNA gene sequence identity. While 63% of the core genome showed evidence of recombination, this had no effect on species-level phylogenomic resolution. Recombination did however blur intra-species relationships and biogeographic resolution. The genome-wide average nucleotide identity provided a new perspective on Salinispora diversity, revealing as many as seven new species. Patterns of orthologous group distributions reveal a genetic basis to delineation the candidate taxa and insight into the levels of genetic cohesion associated with bacterial species.

Asolkar, RN, Singh A, Jensen PR, Aalbersberg W, Carte BK, Feussner KD, Subramani R, DiPasquale A, Rheingold AL, Fenical W.  2017.  Marinocyanins, cytotoxic bromo-phenazinone meroterpenoids from a marine bacterium from the streptomycete Glade MAR4. Tetrahedron. 73:2234-2241.   10.1016/j.tet.2017.03.003   AbstractWebsite

Six cytotoxic and antimicrobial metabolites of a new bromo-phenazinone class, the marinocyanins A-F (1-6), were isolated together with the known bacterial metabolites 2-bromo-1-hydroxyphenazine (7), lavanducyanin (8, WS-9659A) and its chlorinated analog WS-9659B (9). These metabolites were purified by bioassay-guided fractionation of the extracts of our MAR4 marine actinomycete strains CNS-284 and CNY-960. The structures of the new compounds were determined by detailed spectroscopic methods and marinocyanin A (1) was confirmed by crystallographic methods. The marinocyanins represent the first bromo-phenazinones with an N-isoprenoid substituent in the skeleton. Marinocyanins A-F show strong to weak cytotoxicity against HCT-116 human colon carcinoma and possess modest antimicrobial activities against Staphylococcus aureus and amphotericin-resistant Candida albicans. (C) 2017 Elsevier Ltd. All rights reserved.

Agarwal, V, Blanton JM, Podell S, Taton A, Schorn MA, Busch J, Lin Z, Schmidt EW, Jensen PR, Paul VJ, Biggs JS, Golden JW, Allen EE, Moore BS.  2017.  Metagenomic discovery of polybrominated diphenyl ether biosynthesis by marine sponges. Nat Chem Biol. advance online publication: Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.   10.1038/nchembio.2330   Abstract

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.

Crusemann, M, O'Neill EC, Larson CB, Melnik AV, Floros DJ, da Silva RR, Jensen PR, Dorrestein PC, Moore BS.  2017.  Prioritizing natural product diversity in a collection of 146 bacterial strains based on growth and extraction protocols. Journal of Natural Products. 80:588-597.   10.1021/acsjnatprod.6b00722   AbstractWebsite

In order to expedite the rapid and efficient discovery and isolation of novel specialized metabolites, while minimizing the waste of resources on rediscovery of known compounds, it is crucial to develop efficient approaches for strain prioritization, rapid dereplication, and the assessment of favored cultivation and extraction conditions. Herein we interrogated bacterial strains by systematically evaluating cultivation and extraction parameters with LC-MS/MS analysis and subsequent dereplication through the Global Natural Product Social Molecular Networking (GNPS) platform. The developed method is fast, requiring minimal time and sample material, and is compatible with high throughput extract analysis, thereby streamlining strain prioritization and evaluation of culturing parameters. With this approach, we analyzed 146 marine Salinispora and Streptomyces strains that were grown and extracted using multiple different protocols. In total, 603 samples were analyzed, generating approximately 1.8 million mass spectra. We constructed a comprehensive molecular network and identified 15 molecular families of diverse natural products and their analogues. The size and breadth of this network shows statistically supported trends in molecular diversity when comparing growth and extraction conditions. The network provides an extensive survey of the biosynthetic capacity of the strain collection and a method to compare strains based on the variety and novelty of their metabolites. This approach allows us to quickly identify patterns in metabolite production that can be linked to taxonomy, culture conditions, and extraction methods, as well as informing the most valuable growth and extraction conditions.

Patin, NV, Schorn M, Aguinaldo K, Lincecum T, Moore BS, Jensen PR.  2017.  Effects of actinomycete secondary metabolites on sediment microbial communities. Applied and Environmental Microbiology. 83   10.1128/aem.02676-16   Abstract

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 sedimentdwelling 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.

2016
Jensen, PR.  2016.  Natural products and the gene cluster revolution. Trends in Microbiology. 24:968-977.   10.1016/j.tim.2016.07.006   AbstractWebsite

Genome sequencing has created unprecedented opportunities for natural product discovery and new insight into the diversity and distributions of natural-product biosynthetic gene clusters (BGCs). These gene collectives are highly evolved for horizontal exchange, thus providing immediate opportunities to test the effects of small molecules on fitness. The marine actinomycete genus Salinispora maintains extraordinary levels of BGC diversity and has become a useful model for studies of secondary metabolism. Most Salinispora BGCs are observed infrequently, resulting in high population-level diversity while conforming to constraints associated with maximum genome size. Comparative genomics is providing a mechanism to assess secondary metabolism in the context of evolution and evidence that some products represent ecotype-defining traits while others appear selectively neutral.

Schorn, MA, Alanjary MM, Aguinaldo K, Korobeynikov A, Podell S, Patin N, Lincecum T, Jensen PR, Ziemert N, Moore BS.  2016.  Sequencing rare marine actinomycete genomes reveals high density of unique natural product biosynthetic gene clusters. Microbiology-Sgm. 162:2075-2086.   10.1099/mic.0.000386   AbstractWebsite

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.

Floros, DJ, Jensen PR, Dorrestein PC, Koyama N.  2016.  A metabolomics guided exploration of marine natural product chemical space. Metabolomics. 12   10.1007/s11306-016-1087-5   AbstractWebsite

Introduction Natural products from culture collections have enormous impact in advancing discovery programs for metabolites of biotechnological importance. These discovery efforts rely on the metabolomic characterization of strain collections. Objective Many emerging approaches compare metabolomic profiles of such collections, but few enable the analysis and prioritization of thousands of samples from diverse organisms while delivering chemistry specific read outs. Method In this work we utilize untargeted LC-MS/MS based metabolomics together with molecular networking to inventory the chemistries associated with 1000 marine microorganisms. Result This approach annotated 76 molecular families (a spectral match rate of 28 %), including clinically and biotechnologically important molecules such as valinomycin, actinomycin D, and desferrioxamine E. Targeting a molecular family produced primarily by one microorganism led to the isolation and structure elucidation of two new molecules designated maridric acids A and B. Conclusion Molecular networking guided exploration of large culture collections allows for rapid dereplication of know molecules and can highlight producers of uniques metabolites. These methods, together with large culture collections and growing databases, allow for data driven strain prioritization with a focus on novel chemistries.

Wang, MX, Carver JJ, Phelan VV, Sanchez LM, Garg N, Peng Y, Nguyen DD, Watrous J, Kapono CA, Luzzatto-Knaan T et al..  2016.  Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking. Nature Biotechnology. 34:828-837.   10.1038/nbt.3597   AbstractWebsite

The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry (MS) techniques are well-suited to high-throughput characterization of NP, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social Molecular Networking (GNPS; http://gnps.ucsd.edu), an open-access knowledge base for community-wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS, crowdsourced curation of freely available community-wide reference MS libraries will underpin improved annotations. Data-driven social-networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of 'living data' through continuous reanalysis of deposited data.

Patin, NV, Duncan KR, Dorrestein PC, Jensen PR.  2016.  Competitive strategies differentiate closely related species of marine actinobacteria. Isme Journal. 10:478-490.   10.1038/ismej.2015.128   AbstractWebsite

Although competition, niche partitioning, and spatial isolation have been used to describe the ecology and evolution of macro-organisms, it is less clear to what extent these principles account for the extraordinary levels of bacterial diversity observed in nature. Ecological interactions among bacteria are particularly challenging to address due to methodological limitations and uncertainties over how to recognize fundamental units of diversity and link them to the functional traits and evolutionary processes that led to their divergence. Here we show that two closely related marine actinomycete species can be differentiated based on competitive strategies. Using a direct challenge assay to investigate inhibitory interactions with members of the bacterial community, we observed a temporal difference in the onset of inhibition. The majority of inhibitory activity exhibited by Salinispora arenicola occurred early in its growth cycle and was linked to antibiotic production. In contrast, most inhibition by Salinispora tropica occurred later in the growth cycle and was more commonly linked to nutrient depletion or other sources. Comparative genomics support these differences, with S. arenicola containing nearly twice the number of secondary metabolite biosynthetic gene clusters as S. tropica, indicating a greater potential for secondary metabolite production. In contrast, S. tropica is enriched in gene clusters associated with the acquisition of growth-limiting nutrients such as iron. Coupled with differences in growth rates, the results reveal that S. arenicola uses interference competition at the expense of growth, whereas S. tropica preferentially employs a strategy of exploitation competition. The results support the ecological divergence of two co-occurring and closely related species of marine bacteria by providing evidence they have evolved fundamentally different strategies to compete in marine sediments.

2015
Ruckert, C, Leipoldt F, Zeyhle P, Fenical W, Jensen PR, Kalinowski J, Heide L, Kaysser L.  2015.  Complete genome sequence of Streptomyces sp CNQ-509, a prolific producer of meroterpenoid chemistry. Journal of Biotechnology. 216:140-141.   10.1016/j.jbiotec.2015.08.009   AbstractWebsite

Streptomyces sp. CNQ-509 is a marine actinomycete belonging to the MAR4 streptomycete lineage. MAR4 strains have been linked to the production of diverse and otherwise rare meroterpenoid compounds. The genome sequence of Streptomyces sp. CNQ-509 was found to contain 29 putative gene clusters for the biosynthesis of secondary metabolites, some of them potentially involved in the formation of meroterpenoid molecules. (C) 2015 Elsevier B.V. All rights reserved.

Tang, XY, Li J, Millan-Aguinaga N, Zhang JJ, O'Neill EC, Ugalde JA, Jensen PR, Mantovani SM, Moore BS.  2015.  Identification of thiotetronic acid antibiotic biosynthetic pathways by target-directed genome mining. Acs Chemical Biology. 10:2841-2849.   10.1021/acschembio.5b00658   AbstractWebsite

Recent genome sequencing efforts have led to the rapid accumulation of uncharacterized or "orphaned" secondary metabolic biosynthesis gene clusters (BGCs) in public databases. This increase in DNA-sequenced big data has given rise to significant challenges in the applied field of natural product genome mining, including (i) how to prioritize the characterization of orphan BGCs and (ii) how to rapidly connect genes to biosynthesized small molecules. Here, we show that by correlating putative antibiotic resistance genes that encode target-modified proteins with orphan BGCs, we predict the biological function of pathway specific small molecules before they have been revealed in a process we call target-directed genome mining. By querying the pan-genome of 86 Salinispora bacterial genomes for duplicated house-keeping genes colocalized with natural product BGCs, we prioritized an orphan polyketide synthase-nonribosomal peptide synthetase hybrid BGC (tlm) with a putative fatty acid synthase resistance gene. We employed a new synthetic double-stranded DNA-mediated cloning strategy based on transformation-associated recombination to efficiently capture tlm and the related Mu BGCs directly from genomic DNA and to heterologously express them in Streptomyces hosts. We show the production of a group of unusual thiotetronic acid natural products, including the well-known fatty acid synthase inhibitor thiolactomycin that was first described over 30 years ago, yet never at the genetic level in regards to biosynthesis and autoresistance. This finding not only validates the target-directed genome mining strategy for the discovery of antibiotic producing gene clusters without a priori knowledge of the molecule synthesized but also paves the way for the investigation of novel: enzymology involved in thiotetronic, acid natural product biosynthesis.

Gallagher, KA, Jensen PR.  2015.  Genomic insights into the evolution of hybrid isoprenoid biosynthetic gene clusters in the MAR4 marine streptomycete clade. Bmc Genomics. 16   10.1186/s12864-015-2110-3   AbstractWebsite

Background: Considerable advances have been made in our understanding of the molecular genetics of secondary metabolite biosynthesis. Coupled with increased access to genome sequence data, new insight can be gained into the diversity and distributions of secondary metabolite biosynthetic gene clusters and the evolutionary processes that generate them. Here we examine the distribution of gene clusters predicted to encode the biosynthesis of a structurally diverse class of molecules called hybrid isoprenoids (HIs) in the genus Streptomyces. These compounds are derived from a mixed biosynthetic origin that is characterized by the incorporation of a terpene moiety onto a variety of chemical scaffolds and include many potent antibiotic and cytotoxic agents. Results: One hundred and twenty Streptomyces genomes were searched for HI biosynthetic gene clusters using ABBA prenyltransferases (PTases) as queries. These enzymes are responsible for a key step in HI biosynthesis. The strains included 12 that belong to the 'MAR4' clade, a largely marine-derived lineage linked to the production of diverse HI secondary metabolites. We found ABBA PTase homologs in all of the MAR4 genomes, which averaged five copies per strain, compared with 21 % of the non-MAR4 genomes, which averaged one copy per strain. Phylogenetic analyses suggest that MAR4 PTase diversity has arisen by a combination of horizontal gene transfer and gene duplication. Furthermore, there is evidence that HI gene cluster diversity is generated by the horizontal exchange of orthologous PTases among clusters. Many putative HI gene clusters have not been linked to their secondary metabolic products, suggesting that MAR4 strains will yield additional new compounds in this structure class. Finally, we confirm that the mevalonate pathway is not always present in genomes that contain HI gene clusters and thus is not a reliable query for identifying strains with the potential to produce HI secondary metabolites. Conclusions: We found that marine-derived MAR4 streptomycetes possess a relatively high genetic potential for HI biosynthesis. The combination of horizontal gene transfer, duplication, and rearrangement indicate that complex evolutionary processes account for the high level of HI gene cluster diversity in these bacteria, the products of which may provide a yet to be defined adaptation to the marine environment.

Choi, EJ, Nam SJ, Paul L, Beatty D, Kauffman CA, Jensen PR, Fenical W.  2015.  Previously uncultured marine bacteria linked to novel alkaloid production. Chemistry & Biology. 22:1270-1279.   10.1016/j.chembiol.2015.07.014   AbstractWebsite

Low-nutrient media and long incubation times facilitated the cultivation of 20 taxonomically diverse Gram-negative marine bacteria within the phyla Bacteroidetes and Proteobacteria. These strains comprise as many as three new families and include members of clades that had only been observed using culture-independent techniques. Chemical studies of the type strains representing two new families within the order Cytophagales led to the isolation of nine new alkaloid secondary metabolites that can be grouped into four distinct structure classes, including azepinones, aziridines, quinolones, and pyrazinones. Several of these compounds possess antibacterial properties and appear, on structural grounds, to be produced by amino acid-based biosynthetic pathways. Our results demonstrate that relatively simple cultivation techniques can lead to the isolation of new bacterial taxa that are capable of the production of alkaloid secondary metabolites with antibacterial activities. These findings support continued investment in cultivation techniques as a method for natural product discovery.

Nam, SJ, Kauffman CA, Jensen PR, Moore CE, Rheingold AL, Fenical W.  2015.  Actinobenzoquinoline and actinophenanthrolines a-c, unprecedented alkaloids from a marine actinobacterium. Organic Letters. 17:3240-3243.   10.1021/acs.orglett.5b01387   AbstractWebsite

Chemical investigation of a marine actinomycete within the family Streptomycetaceae (our strain CNQ-149) has led to the isolation of the unprecedented alkaloids, actinobenzoquinoline (1) and actinophenanthrolines A-C (2-4). The chemical structures of 1-4 were assigned by interpretation of NMR spectroscopic data, and their absolute configurations were assigned by X-ray analysis. Actinobenzoquinoline possesses a 5-methyloxazolidin-4-one moiety and a dihydrobento[h],quinoline core structure, while actinophenanthrolines A-C are composed of hydroxypropanamide-substituted 1,7-phenanthroline core skeletons.

Duncan, KR, Crusemann M, Lechner A, Sarkar A, Li J, Ziemert N, Wang MX, Bandeira N, Moore BS, Dorrestein PC, Jensen PR.  2015.  Molecular networking and pattern-based genome mining improves discovery of biosynthetic gene clusters and their products from Salinispora species. Chemistry & Biology. 22:460-471.   10.1016/j.chembiol.2015.03.010   AbstractWebsite

Genome sequencing has revealed that bacteria contain many more biosynthetic gene clusters than predicted based on the number of secondary metabolites discovered to date. While this biosynthetic reservoir has fostered interest in new tools for natural product discovery, there remains a gap between gene cluster detection and compound discovery. Here we apply molecular networking and the new concept of pattern-based genome mining to 35 Salinispora strains, including 30 for which draft genome sequences were either available or obtained for this study. The results provide a method to simultaneously compare large numbers of complex microbial extracts, which facilitated the identification of media components, known compounds and their derivatives, and new compounds that could be prioritized for structure elucidation. These efforts revealed considerable metabolite diversity and led to several molecular family-gene cluster pairings, of which the quinomycin-type depsipeptide retimycin A was characterized and linked to gene cluster NRPS40 using pattern-based bioinformatic approaches.

2014
Wietz, M, Millan-Aguinaga N, Jensen PR.  2014.  CRISPR-Cas systems in the marine actinomycete Salinispora: linkages with phage defense, microdiversity and biogeography. Bmc Genomics. 15   10.1186/1471-2164-15-936   AbstractWebsite

Background: Prokaryotic CRISPR-Cas systems confer resistance to viral infection and thus mediate bacteria-phage interactions. However, the distribution and functional diversity of CRISPRs among environmental bacteria remains largely unknown. Here, comparative genomics of 75 Salinispora strains provided insight into the diversity and distribution of CRISPR-Cas systems in a cosmopolitan marine actinomycete genus. Results: CRISPRs were found in all Salinispora strains, with the majority containing multiple loci and different Cas array subtypes. Of the six subtypes identified, three have not been previously described. A lower prophage frequency in S. arenicola was associated with a higher fraction of spacers matching Salinispora prophages compared to S. tropica, suggesting differing defensive capacities between Salinispora species. The occurrence of related prophages in strains from distant locations, as well as spacers matching those prophages inserted throughout spacer arrays, indicate recurring encounters with widely distributed phages over time. Linkages of CRISPR features with Salinispora microdiversity pointed to subclade-specific contacts with mobile genetic elements (MGEs). This included lineage-specific spacer deletions or insertions, which may reflect weak selective pressures to maintain immunity or distinct temporal interactions with MGEs, respectively. Biogeographic patterns in spacer and prophage distributions support the concept that Salinispora spp. encounter localized MGEs. Moreover, the presence of spacers matching housekeeping genes suggests that CRISPRs may have functions outside of viral defense. Conclusions: This study provides a comprehensive examination of CRISPR-Cas systems in a broadly distributed group of environmental bacteria. The ubiquity and diversity of CRISPRs in Salinispora suggests that CRISPR-mediated interactions with MGEs represent a major force in the ecology and evolution of this cosmopolitan marine actinomycete genus.

Ziemert, N, Lechner A, Wietz M, Millan-Aguinaga N, Chavarria KL, Jensen PR.  2014.  Diversity and evolution of secondary metabolism in the marine actinomycete genus Salinispora. Proceedings of the National Academy of Sciences of the United States of America. 111:E1130-E1139.   10.1073/pnas.1324161111   AbstractWebsite

Access to genome sequence data has challenged traditional natural product discovery paradigms by revealing that the products of most bacterial biosynthetic pathways have yet to be discovered. Despite the insight afforded by this technology, little is known about the diversity and distributions of natural product biosynthetic pathways among bacteria and how they evolve to generate structural diversity. Here we analyze genome sequence data derived from 75 strains of the marine actinomycete genus Salinispora for pathways associated with polyketide and nonribosomal peptide biosynthesis, the products of which account for some of today's most important medicines. The results reveal high levels of diversity, with a total of 124 pathways identified and 229 predicted with continued sequencing. Recent horizontal gene transfer accounts for the majority of pathways, which occur in only one or two strains. Acquired pathways are incorporated into genomic islands and are commonly exchanged within and between species. Acquisition and transfer events largely involve complete pathways, which subsequently evolve by gene gain, loss, and duplication followed by divergence. The exchange of similar pathway types at the precise chromosomal locations in different strains suggests that the mechanisms of integration include pathway-level homologous recombination. Despite extensive horizontal gene transfer there is clear evidence of species-level vertical inheritance, supporting the concept that secondary metabolites represent functional traits that help define Salinispora species. The plasticity of the Salinispora secondary metabolome provides an effective mechanism to maximize population-level secondary metabolite diversity while limiting the number of pathways maintained within any individual genome.