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Tuttle, RN, Demko AM, Patin NV, Kapono CA, Donia MS, Dorrestein P, Jensen PR.  2019.  Detection of natural products and their producers in ocean sediments. Applied and Environmental Microbiology. 85   10.1128/aem.02830-18   AbstractWebsite

Thousands of natural products have been identified from cultured microorganisms, yet evidence of their production in the environment has proven elusive. Technological advances in mass spectrometry, combined with public data-bases, now make it possible to address this disparity by detecting compounds directly from environmental samples. Here, we used adsorbent resins, tandem mass spectrometry, and next-generation sequencing to assess the metabolome of marine sediments and its relationship to bacterial community structure. We identified natural products previously reported from cultured bacteria, providing evidence they are produced in situ, and compounds of anthropogenic origin, suggesting this approach can be used as an indicator of environmental impact. The bacterial metabolite staurosporine was quantified and shown to reach physiologically relevant concentrations, indicating that it may influence sediment community structure. Staurosporine concentrations were correlated with the relative abundance of the staurosporine-producing bacterial genus Salinispora and production confirmed in strains cultured from the same location, providing a link between compound and candidate producer. Metagenomic analyses revealed numerous biosynthetic gene clusters related to indolocarbazole biosynthesis, providing evidence for noncanonical sources of staurosporine and a path forward to assess the relationships between natural products and the organisms that produce them. Untargeted environmental metabolomics circumvents the need for laboratory cultivation and represents a promising approach to understanding the functional roles of natural products in shaping microbial community structure in marine sediments. IMPORTANCE Natural products are readily isolated from cultured bacteria and exploited for useful purposes, including drug discovery. However, these compounds are rarely detected in the environments from which the bacteria are obtained, thus limiting our understanding of their ecological significance. Here, we used environmental metabolomics to directly assess chemical diversity in marine sediments. We identified numerous metabolites and, in one case, isolated strains of bacteria capable of producing one of the compounds detected. Coupling environmental metabolomics with community and metagenomic analyses provides opportunities to link compounds and producers and begin to assess the complex interactions mediated by specialized metabolites in marine sediments.

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.

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.

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.

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.

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;, 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.

Bucarey, SA, Penn K, Paul L, Fenical W, Jensen PR.  2012.  Genetic Complementation of the Obligate Marine Actinobacterium Salinispora tropica with the Large Mechanosensitive Channel Gene mscL Rescues Cells from Osmotic Downshock. Applied and Environmental Microbiology. 78:4175-4182.   10.1128/aem.00577-12   AbstractWebsite

Marine actinomycetes in the genus Salinispora fail to grow when seawater is replaced with deionized (DI) water in complex growth media. While bioinformatic analyses have led to the identification of a number of candidate marine adaptation genes, there is currently no experimental evidence to support the genetic basis for the osmotic requirements associated with this taxon. One hypothesis is that the lineage-specific loss of mscL is responsible for the failure of strains to grow in media prepared with DI water. The mscL gene encodes a conserved transmembrane protein that reduces turgor pressure under conditions of acute osmotic downshock. In the present study, the mscL gene from a Micromonospora strain capable of growth on media prepared with DI water was transformed into S. tropica strain CNB-440. The single-copy, chromosomal genetic complementation yielded a recombinant Salinispora mscL(+) strain that demonstrated an increased capacity to survive osmotic downshock. The enhanced survival of the S. tropica transformant provides experimental evidence that the loss of mscL is associated with the failure of Salinispora spp. to grow in low-osmotic-strength media.

Nam, SJ, Kauffman CA, Jensen PR, Fenical W.  2011.  Isolation and characterization of actinoramides A-C, highly modified peptides from a marine Streptomyces sp. Tetrahedron. 67:6707-6712.   10.1016/j.tet.2011.04.051   AbstractWebsite

Reported herein is the isolation and structure elucidation of three highly modified peptides, actinoramides A-C (1-3), which are produced by a marine bacterium closely related to the genus Streptomyces. The planar structures of the actinoramides, which are composed of the unusual amino acids 2-amino-4-ureidobutanoic acid and 4-amino-3-hydroxy-2-methyl-5-phenylpentanoic acid, were assigned by chemical transformations and by interpretation of spectroscopic data, while the absolute configuration of these new peptides were defined by application of the advanced Marfey's and Mosher's methods. (C) 2011 Elsevier Ltd. All rights reserved.

Freel, KC, Nam SJ, Fenical W, Jensen PR.  2011.  Evolution of Secondary Metabolite Genes in Three Closely Related Marine Actinomycete Species. Applied and Environmental Microbiology. 77:7261-7270.   10.1128/aem.05943-11   AbstractWebsite

The marine actinomycete genus Salinispora is composed of three closely related species. These bacteria are a rich source of secondary metabolites, which are produced in species-specific patterns. This study examines the distribution and phylogenetic relationships of genes involved in the biosynthesis of secondary metabolites in the salinosporamide and staurosporine classes, which have been reported for S. tropica and S. arenicola, respectively. The focus is on "Salinispora pacifica," the most recently discovered and phylogenetically diverse member of the genus. Of 61 S. pacifica strains examined, 15 tested positive for a ketosynthase (KS) domain linked to the biosynthesis of salinosporamide K, a new compound in the salinosporamide series. Compound production was confirmed in two strains, and the domain phylogeny supports vertical inheritance from a common ancestor shared with S. tropica, which produces related compounds in the salinosporamide series. There was no evidence for interspecies recombination among salA KS sequences, providing further support for the geographic isolation of these two salinosporamide-producing lineages. In addition, staurosporine production is reported for the first time for S. pacifica, with 24 of 61 strains testing positive for staD, a key gene involved in the biosynthesis of this compound. High levels of recombination were observed between staD alleles in S. pacifica and the cooccurring yet more distantly related S. arenicola, which produces a similar series of staurosporines. The distributions and phylogenies of the biosynthetic genes examined provide insight into the complex processes driving the evolution of secondary metabolism among closely related bacterial species.

Kwon, HC, Espindola A, Park JS, Prieto-Davo A, Rose M, Jensen PR, Fenical W.  2010.  Nitropyrrolins A-E, Cytotoxic Farnesyl-alpha-nitropyrroles from a Marine-Derived Bacterium within the Actinomycete Family Streptomycetaceae. Journal of Natural Products. 73:2047-2052.   10.1021/np1006229   AbstractWebsite

Five new farnesyl-alpha-nitropyrroles, nitropyrrolins A-E (1-5), were isolated from the saline culture of the marine actinomycete strain CNQ-509. This strain belongs to the "MAR4" group of marine actinomycetes, which have been demonstrated to be a rich source of hybrid isoprenoid secondary metabolites. The structures of the nitropyrrolins are composed of alpha-nitropyrroles with functionalized farnesyl groups at the C-4 position. These compounds are the first examples of naturally occurring terpenyl-alpha-nitropyrroles. Chemical modifications, including one-step acetonide formation from an epoxide, and application of the modified Mosher method provided the full stereostructures and absolute configurations of these compounds. Several of the nitropyrrolins, nitropyrrolin D in particular, are cytotoxic toward HCT-116 human colon carcinoma cells, but show weak to little antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA).

Prieto-Davo, A, Fenical W, Jensen PR.  2008.  Comparative actinomycete diversity in marine sediments. Aquatic Microbial Ecology. 52:1-11.   10.3354/ame01211   AbstractWebsite

The diversity of cultured actinomycete bacteria was compared between near- and offshore marine sediments. Strains were tested for the effects of seawater on growth and analyzed for 16S rRNA gene sequence diversity. In total, 623 strains representing 6 families in the order Actinomycetales were cultured. These strains were binned into 16 to 63 operational taxonomic units (OTUs) over a range of 97 to 100 % sequence identity. The majority of the OTUs were closely related (> 98 % sequence identity) to strains previously reported from non-marine sources; indicating that most are not restricted to the sea. However, new OTUs averaged 96.6 % sequence identity with previously cultured strains and ca. one-third of the OTUs were marine-specific, suggesting that sediment communities include considerable actinomycete diversity that does not occur on land. Marine specificity did not increase at the off-shore sites, indicating high levels of terrestrial influence out to 125 km from shore. The requirement of seawater for growth was observed among < 6%. of the strains, while all members of 9 OTUs possessed this trait, revealing a high degree of marine adaptation among some lineages. Statistical analyses predicted greater OTU diversity at the off-shore sites and provided a rationale for expanded exploration of deep-sea samples. A change in community composition was observed, with the number of Micromonospora OTUs increasing in the off-shore samples. UniFrac (see statistics support a difference in community composition between near- and off-shore locations. Overall, 123 of 176 strains had distinct 16S rRNA gene sequences, indicating a high level of actinomycete diversity in marine sediments.

Oh, DC, Strangman WK, Kauffman CA, Jensen PR, Fenical W.  2007.  Thalassospiramides A and B, immunosuppressive peptides from the marine bacterium Thalassospira sp. Organic Letters. 9:1525-1528.   10.1021/ol070294u   AbstractWebsite

[GRAPHICS] Two new cyclic peptides, thalassospiramides A and B (1 and 2), were isolated from a new member of the marine alpha-proteobacterium Thalassospira. The thalassospiramides, the structures of which were assigned by combined spectral and chemical methods, bear unusual gamma-amino acids and show immunosuppressive activity in an interleukin-5 production inhibition assay (IC50 = 5 mu M for thalassospiramide B).

Cho, JY, Kwon HC, Williams PG, Jensen PR, Fenical W.  2006.  Azamerone, a terpenoid phthalazinone from a marine-derived bacterium related to the genus Streptomyces (actinomycetales). Organic Letters. 8:2471-2474.   10.1021/ol060630r   AbstractWebsite

A novel meroterpenoid, azamerone, was isolated from the saline culture of a new marine-derived bacterium related to the genus Streptomyces. Azamerone is composed of an unprecedented chloropyranophthalazinone core with a 3-chloro-6-hydroxy-2,2,6-trimethylcyclohexylmethyl side chain. The structure was rigorously determined by NMR spectroscopy and X-ray crystallography. A possible biosynthetic origin of this unusual ring system is proposed.

Buchanan, GO, Williams PG, Feling RH, Kauffman CA, Jensen PR, Fenical W.  2005.  Sporolides A and B: Structurally unprecedented halogenated macrolides from the marine actinomycete Salinispora tropica. Organic Letters. 7:2731-2734.   10.1021/ol050901i   AbstractWebsite

Analysis of the fermentation broth of a strain of the marine actinomycete Salinispora tropica has led to the isolation of two unprecedented macrolides, sporolides A (1) and B (2). The structures and absolute stereochemistries of both metabolites were elucidated using a combination of NMR spectroscopy and X-ray crystallography.

Jensen, PR, Gontang E, Mafnas C, Mincer TJ, Fenical W.  2005.  Culturable marine actinomycete diversity from tropical Pacific Ocean sediments. Environmental Microbiology. 7:1039-1048.   10.1111/j.1462-2920.2005.00785.x   AbstractWebsite

Actinomycetes were cultivated using a variety of media and selective isolation techniques from 275 marine samples collected around the island of Guam. In total, 6425 actinomycete colonies were observed and 983 (15%) of these, representing the range of morphological diversity observed from each sample, were obtained in pure culture. The majority of the strains isolated (58%) required seawater for growth indicating a high degree of marine adaptation. The dominant actinomycete recovered (568 strains) belonged to the seawater-requiring marine taxon 'Salinospora', a new genus within the family Micromonosporaceae. A formal description of this taxon has been accepted for publication (Maldonado et al., 2005) and includes a revision of the generic epithet to Salinispora gen. nov. Members of two major new clades related to Streptomyces spp., tentatively called MAR2 and MAR3, were cultivated and appear to represent new genera within the Streptomycetaceae. In total, five new marine phylotypes, including two within the Thermomonosporaceae that appear to represent new taxa, were obtained in culture. These results support the existence of taxonomically diverse populations of phylogenetically distinct actinomycetes residing in the marine environment. These bacteria can be readily cultured using low nutrient media and represent an unexplored resource for pharmaceutical drug discovery.

Feling, RH, Buchanan GO, Mincer TJ, Kauffman CA, Jensen PR, Fenical W.  2003.  Salinosporamide A: A highly cytotoxic proteasome inhibitor from a novel microbial source, a marine bacterium of the new genus Salinospora. Angewandte Chemie-International Edition. 42:355-+.   10.1002/anie.200390115   Website