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Castro-Falcon, G, Millan-Aguinaga N, Roullier C, Jensen PR, Hughes CC.  2018.  Nitrosopyridine probe to detect polyketide natural products with conjugated alkenes: Discovery of novodaryamide and nocarditriene. Acs Chemical Biology. 13:3097-3106.   10.1021/acschembio.8b00598   AbstractWebsite

An optimized nitroso-based probe that facilitates the discovery of conjugated alkene-containing natural products in unprocessed extracts was developed. It chemoselectively reacts with conjugated olefins via a nitroso-Diels-Alder cyclization to yield derivatives with a distinct chromophore and an isotopically unique bromine atom that can be rapidly identified using liquid chromatography/mass spectrometry and a bioinformatics tool called MeHaloCoA (Marine Halogenated Compound Analysis). The probe is ideally employed when genome-mining techniques identify strains containing polyketide gene clusters with two or more repeating KS-AT-DH-KR-ACP domain sequences, which are required for the biosynthesis of conjugated alkenes. Comparing the reactivity and spectral properties of five brominated arylnitroso reagents with model compounds spiramycin, bufalin, rapamycin, and rifampicin led to the identification of 5-bromo-2-nitrosopyridine as the most suitable probe structure. The utility of the dienophile probe was then demonstrated in bacterial extracts. Tylactone, novodaryamide and daryamide A, piperazimycin A, and the saccharamonopyrones A and B were cleanly labeled in extracts from their respective bacterial producers, in high regioselectivity but with varying degrees of diastereoselectivity. Further application of the method led to the discovery of a new natural product called nocarditriene, containing an unprecedented epoxy-2,3,4,5-tetrahydropyridine structure, from marine-derived Nocardiopsis strain CNY-503.

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.

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.

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.

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.

Jensen, PR, Chavarria KL, Fenical W, Moore BS, Ziemert N.  2014.  Challenges and triumphs to genomics-based natural product discovery. Journal of Industrial Microbiology & Biotechnology. 41:203-209.   10.1007/s10295-013-1353-8   AbstractWebsite

Genome sequencing is rapidly changing the field of natural products research by providing opportunities to assess the biosynthetic potential of strains prior to chemical analysis or biological testing. Ready access to sequence data is driving the development of new bioinformatic tools and methods to identify the products of silent or cryptic pathways. While genome mining has fast become a useful approach to natural product discovery, it has also become clear that identifying pathways of interest is much easier than finding the associated products. This has led to bottlenecks in the discovery process that must be overcome for the potential of genomics-based natural product discovery to be fully realized. In this perspective, we address some of these challenges in the context of our work with the marine actinomycete genus Salinispora, which is proving to be a useful model with which to apply genome mining as an approach to natural product discovery.

Ahmed, L, Jensen PR, Freel KC, Brown R, Jones AL, Kim BY, Goodfellow M.  2013.  Salinispora pacifica sp nov., an actinomycete from marine sediments. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology. 103:1069-1078.   10.1007/s10482-013-9886-4   AbstractWebsite

A polyphasic analysis was carried out to clarify the taxonomic status of four marine actinomycete strains that share a phylogenetic relationship and phenotypic characteristics with the genus Salinispora. These strains formed a distinct lineage within the Salinispora 16S rRNA and gyrB trees and were found to possess a range of phenotypic properties and DNA: DNA hybridization values that distinguished them from the type strains of the two validly named species in this genus, Salinispora tropica (CNB-440(T), ATCC BAA-916(T)) and Salinispora arenicola (CNH-643(T), ATCC BAA-917(T)). The combined genotypic and phenotypic data support this conclusion. It is proposed that the strains be designated as Salinispora pacifica sp. nov., the type strain of which is CNR-114(T) (DSMZ YYYYT = KACC 17160(T)).

Choi, EJ, Beatty DS, Paul LA, Fenical W, Jensen PR.  2013.  Mooreia alkaloidigena gen. nov., sp nov and Catalinimonas alkaloidigena gen. nov., sp nov., alkaloid-producing marine bacteria in the proposed families Mooreiaceae fam. nov and Catalimonadaceae fam. nov in the phylum Bacteroidetes. International Journal of Systematic and Evolutionary Microbiology. 63:1219-1228.   10.1099/ijs.0.043752-0   AbstractWebsite

Bacterial strains CNX-216(T) and CNU-914(T) were isolated from marine sediment samples collected from Palmyra Atoll and off Catalina Island, respectively. Both strains were Gram-negative and aerobic and produce deep-orange to pink colonies and alkaloid secondary metabolites. Cells of strain CNX-216(T) were short, non-motile rods, whereas cells of strain CNU-914(T) were short, curved rods with gliding motility. The DNA G + C contents of CNX-216(T) and CNU-914(T) were respectively 57.7 and 44.4 mol%. Strains CNX-216(T) and CNU-914(T) contained MK-7 as the predominant menaquinone and iso-C-15:0 and C-(16:1)omega 5c as the major fatty acids. Phylogenetic analyses revealed that both strains belong to the order Cytophagales in the phylum Bacteroicletes. Strain CNX-216(T) exhibited low 16S rRNA gene sequence identity (87.1 %) to the nearest type strain, Cesiribacter roseus 311(T), and formed a well-supported lineage that is outside all currently described families in the order Cytophagales. Strain CNU-914(T) shared 97.6% 16S rRNA gene sequence identity with 'Porifericola rhodea' N5EA6-3A2B and, together with 'Tunicatimonas pelagia' N5DB8-4 and four uncharacterized marine bacteria isolated as part of this study, formed a lineage that is clearly distinguished from other families in the order Cytophagales. Based on our polyphasic taxonomic characterization, we propose that strains CNX-216(T) and CNU-914(T) represent novel genera and species, for which we propose the names Mooreia alkaloidigena gen. nov., sp. nov. (type strain CNX-216(T) =DSM 25187(T) =KCCM 90102(T)) and Catalinimonas alkaloidigena gen. nov., sp. nov. (type strain CNU-914(T) =DSM 25186(T) =KCCM 90101(T)) within the new families Mooreiaceae fam. nov. and Catalimonadaceae fam. nov.

Jensen, PR.  2010.  Linking species concepts to natural product discovery in the post-genomic era. Journal of Industrial Microbiology & Biotechnology. 37:219-224.   10.1007/s10295-009-0683-z   AbstractWebsite

A widely accepted species concept for bacteria has yet to be established. As a result, species designations are inconsistently applied and tied to what can be considered arbitrary metrics. Increasing access to DNA sequence data and clear evidence that bacterial genomes are dynamic entities that include large numbers of horizontally acquired genes have added a new level of insight to the ongoing species concept debate. Despite uncertainties over how to apply species concepts to bacteria, there is clear evidence that sequence-based approaches can be used to resolve cohesive groups that maintain the properties of species. This cohesion is clearly evidenced in the genus Salinispora, where three species have been discerned despite very close relationships based on 16S rRNA sequence analysis. The major phenotypic differences among the three species are associated with secondary metabolite production, which occurs in species-specific patterns. These patterns are maintained on a global basis and provide evidence that secondary metabolites have important ecological functions. These patterns also suggest that an effective strategy for natural product discovery is to target the cultivation of new Salinispora taxa. Alternatively, bioinformatic analyses of biosynthetic genes provide opportunities to predict secondary metabolite novelty and reduce the redundant isolation of well-known metabolites. Although much remains to be learned about the evolutionary relationships among bacteria and how fundamental units of diversity can be resolved, genus and species descriptions remain the most effective method of scientific communication.

Nam, SJ, Gaudencio SP, Kauffman CA, Jensen PR, Kondratyuk TP, Marler LE, Pezzuto JM, Fenical W.  2010.  Fijiolides A and B, Inhibitors of TNF-alpha-Induced NF kappa B Activation, from a Marine-Derived Sediment Bacterium of the Genus Nocardiopsis. Journal of Natural Products. 73:1080-1086.   10.1021/np100087c   AbstractWebsite

Fijiolide A, a potent inhibitor of TNF-alpha-induced NF kappa B activation, along with fijiolide B. were isolated from a marine-derived bacterium of the genus Nocardiopsis. The planar structures of fijiolides A (1) and B (2) were elucidated by interpretation of 2D NMR spectroscopic data, while the absolute configurations of these compounds were defined by interpretation of circular dichroism and 2D NMR data combined with application of the advanced Mosher's method. Fijiolides A and B are related to several recently isolated chloroaromatic compounds, which appear to be the Bergman cyclization products of enediyne precursors. Fijiolide A reduced TNF-alpha-induced NE kappa B activation by 70.3%, with an IC(50) value of 0.57 mu M. Fijiolide B demonstrated less inhibition, only 46.5%, without dose dependence. The same pattern was also observed with quinone reductase (QR) activity: fijiolide A was found to induce quinone reductase-1 (QR1) with an induction ratio of 3.5 at a concentration of 20 mu g/mL (28.4 mu M). The concentration required to double the activity was 1.8 mu M. Fijiolide B did not affect QR1 activity, indicating the importance of the nitrogen substitution pattern for biological activity. On the basis of these data, fijiolide A is viewed as a promising lead for more advanced anticancer testing.

Strangman, WK, Kwon HC, Broide D, Jensen PR, Fenical W.  2009.  Potent Inhibitors of Pro-Inflammatory Cytokine Production Produced by a Marine-Derived Bacterium. Journal of Medicinal Chemistry. 52:2317-2327.   10.1021/jm801110j   AbstractWebsite

Cytokines produced through the antigen presenting cell (APC)-T-cell interaction play a key role in the activation of the allergic asthmatic response. Evaluating small molecules that inhibit the production of these pro-inflammatory proteins is therefore important for the discovery of novel chemical structures with potential antiasthma activity. We adapted a mouse splerrocyte cytokine assay to screen a library of 2,500 marine microbial extracts for their ability to inhibit T(H)2 cytokine release and identified potent activity in a marine-derived strain CNQ431, identified as a Streptomyces species. Bioactivity guided fractionation of the organic extract of this strain led to the isolation of ten new 9-membered bis-lactones, splenocins A-J (1-10). The new compounds display potent biological activities, comparable to that of the corticosteroid dexamethasone, with IC(50) values from 2 to 50 nM in the splenocyte cytokine assay. This study provides the foundation for the optimization of these potent anti-inflammatory compounds for development in the treatment of asthma.

Hughes, CC, MacMillan JB, Gaudencio SR, Jensen PR, Fenical W.  2009.  The Ammosamides: Structures of Cell Cycle Modulators from a Marine-Derived Streptomyces Species. Angewandte Chemie-International Edition. 48:725-727.   10.1002/anie.200804890   Website
Jensen, PR, Lauro FM.  2008.  An assessment of actinobacterial diversity in the marine environment. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology. 94:51-62.   10.1007/s10482-008-9239-x   AbstractWebsite

The 16S rRNA gene sequence diversity within the Phylum Actinobacteria was assessed from four sources: PCR-generated V6 sequence tags derived from seawater samples, metagenomic data from the Global Ocean Sampling (GOS) expedition, marine-derived sequences maintained in the Ribosomal Database Project (RDP), and select cultured strains for which sequence data is not yet available in the RDP. This meta-analysis revealed remarkable levels of phylogenetic diversity and confirms the existence of major, deeply rooted, and as of yet uncharacterized lineages within the phylum. A dramatic incongruence among cultured strains and those detected using culture-independent techniques was also revealed. Redundancy among the actinobacteria detected using culture-independent techniques suggests that greater sequence coverage or improved DNA extraction efficiencies may be required to detect the rare phylotypes that can be readily cultured from marine samples. Conversely, new strategies need to be developed for the cultivation of frequently observed but yet to be cultured marine actinobacteria.

Jiang, ZD, Jensen PR, Fenical W.  1997.  Actinoflavoside, a novel flavonoid-like glycoside produced by a marine bacterium of the genus Streptomyces. Tetrahedron Letters. 38:5065-5068.   10.1016/s0040-4039(97)01127-1   AbstractWebsite

Actinoflavoside (1), a novel glycoside composed of a rare 2, 3, 6-trideoxy-3-amino-ribopyranoside (ristosamine) aminosugar and an aglycone reminiscent of the plant flavonoids, was isolated from the culture broth of a marine Streptomyces sp. The structure of actinoflavoside was established by spectroscopic analysis and by chemical degradation. (C) 1997 Elsevier Science Ltd.

Jensen, PR, Fenical W.  1996.  Marine bacterial diversity as a resource for novel microbial products. Journal of Industrial Microbiology & Biotechnology. 17:346-351.   10.1007/bf01574765   AbstractWebsite

Marine bacteria are an important and relatively unexplored resource for novel microbial products. In this review, we discuss a number of issues relevant to the industrial potential of marine microorganisms including how marine and terrestrial bacteria differ, both physiologically and taxonomically, and what constitute reasonable expectations of the biosynthetic capabilities of marine bacteria relative to terrestrial bacteria and to marine macroorganisms. Also discussed is the concept that bacterial associations with marine plants and animals, which range from casual encounters to obligate symbioses, provide unique opportunities for bacterial adaptation. It is proposed that some of these adaptations would not be selected for in the absence of environmental parameters associated with the host, and that these adaptations can include the biosynthesis of unique metabolic products.