Accessing chemical diversity from the uncultivated symbionts of small marine animals


Chemistry drives many biological interactions between the microbiota and host animals, yet it is often challenging to identify the chemicals involved. This poses a problem, as such small molecules are excellent sources of potential pharmaceuticals, pretested by nature for animal compatibility. We discovered anti-HIV compounds from small, marine tunicates from the Eastern Fields of Papua New Guinea. Tunicates are a reservoir for new bioactive chemicals, yet their small size often impedes identification or even detection of the chemicals within. We solved this problem by combining chemistry, metagenomics, and synthetic biology to directly identify and synthesize the natural products. We show that these anti-HIV compounds, the divamides, are a novel family of lanthipeptides produced by symbiotic bacteria living in the tunicate. Neighboring animal colonies contain structurally related divamides that differ starkly in their biological properties, suggesting a role for biosynthetic plasticity in a native context wherein biological interactions take place.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Discovery of anti-HIV lanthipeptides, the divamides.
Figure 2: The semi–in vivo synthesis of divamides A and B involves in vivo, chemical, and enzymatic steps.
Figure 3: Cytoprotection assays reveal two biological activities of divamide A.
Figure 4: Segregation of anti-HIV and cytotoxic properties by flow cytometry.
Figure 5: Cinnamycin and divamide both interact with membranes.

Accession codes

Primary accessions

NCBI Reference Sequence


  1. 1

    Blunt, J.W., Copp, B.R., Keyzers, R.A., Munro, M.H. & Prinsep, M.R. Marine natural products. Nat. Prod. Rep. 33, 382–431 (2016).

    CAS  PubMed  Google Scholar 

  2. 2

    Miller, I.J., Vanee, N., Fong, S.S., Lim-Fong, G.E. & Kwan, J.C. Lack of overt genome reduction in the bryostatin-producing bryozoan symbiont “Candidatus Endobugula sertula.”. Appl. Environ. Microbiol. 82, 6573–6583 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Schofield, M.M., Jain, S., Porat, D., Dick, G.J. & Sherman, D.H. Identification and analysis of the bacterial endosymbiont specialized for production of the chemotherapeutic natural product ET-743. Environ. Microbiol. 17, 3964–3975 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Schmidt, E.W. et al. Patellamide A and C biosynthesis by a microcin-like pathway in Prochloron didemni, the cyanobacterial symbiont of Lissoclinum patella. Proc. Natl. Acad. Sci. USA 102, 7315–7320 (2005).

    CAS  PubMed  Google Scholar 

  5. 5

    Donia, M.S. et al. A systematic analysis of biosynthetic gene clusters in the human microbiome reveals a common family of antibiotics. Cell 158, 1402–1414 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Guo, C.J. et al. Discovery of reactive microbiota-derived metabolites that inhibit host proteases. Cell 168, 517–526.e18 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Donia, M.S., Ruffner, D.E., Cao, S. & Schmidt, E.W. Accessing the hidden majority of marine natural products through metagenomics. ChemBioChem 12, 1230–1236 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Lewin, R.A. Prochlorophyta as a proposed new division of algae. Nature 261, 697–698 (1976).

    CAS  PubMed  Google Scholar 

  9. 9

    Donia, M.S. et al. Complex microbiome underlying secondary and primary metabolism in the tunicate-Prochloron symbiosis. Proc. Natl. Acad. Sci. USA 108, E1423–E1432 (2011).

    CAS  PubMed  Google Scholar 

  10. 10

    Arnison, P.G. et al. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 30, 108–160 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Willey, J.M. & van der Donk, W.A. Lantibiotics: peptides of diverse structure and function. Annu. Rev. Microbiol. 61, 477–501 (2007).

    CAS  PubMed  Google Scholar 

  12. 12

    Widdick, D.A. et al. Cloning and engineering of the cinnamycin biosynthetic gene cluster from Streptomyces cinnamoneus cinnamoneus DSM 40005. Proc. Natl. Acad. Sci. USA 100, 4316–4321 (2003).

    CAS  PubMed  Google Scholar 

  13. 13

    Fredenhagen, A. et al. Duramycins B and C, two new lanthionine containing antibiotics as inhibitors of phospholipase A2. Structural revision of duramycin and cinnamycin. J. Antibiot. (Tokyo) 43, 1403–1412 (1990).

    CAS  Google Scholar 

  14. 14

    Knerr, P.J. & van der Donk, W.A. Discovery, biosynthesis, and engineering of lantipeptides. Annu. Rev. Biochem. 81, 479–505 (2012).

    CAS  PubMed  Google Scholar 

  15. 15

    Huo, L., Ökesli, A., Zhao, M. & van der Donk, W.A. Insights into the biosynthesis of duramycin. Appl. Environ. Microbiol. 83, e02698-16 (2017).

    PubMed  PubMed Central  Google Scholar 

  16. 16

    O'Rourke, S., Widdick, D. & Bibb, M. A novel mechanism of immunity controls the onset of cinnamycin biosynthesis in Streptomyces cinnamoneus DSM 40646. J. Ind. Microbiol. Biotechnol. 44, 563–572 (2017).

    CAS  PubMed  Google Scholar 

  17. 17

    Ökesli, A., Cooper, L.E., Fogle, E.J. & van der Donk, W.A. Nine post-translational modifications during the biosynthesis of cinnamycin. J. Am. Chem. Soc. 133, 13753–13760 (2011).

    PubMed  PubMed Central  Google Scholar 

  18. 18

    Cameron, D.M. et al. Thermus thermophilus L11 methyltransferase, PrmA, is dispensable for growth and preferentially modifies free ribosomal protein L11 prior to ribosome assembly. J. Bacteriol. 186, 5819–5825 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Dai, X. et al. Identification of novel α-n-methylation of CENP-B that regulates its binding to the centromeric DNA. J. Proteome Res. 12, 4167–4175 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Dognin, M.J. & Wittmann-Liebold, B. The primary structure of L11, the most heavily methylated protein from Escherichia coli ribosomes. FEBS Lett. 84, 342–346 (1977).

    CAS  PubMed  Google Scholar 

  21. 21

    Zhang, K. et al. Differentiation between peptides containing acetylated or tri-methylated lysines by mass spectrometry: an application for determining lysine 9 acetylation and methylation of histone H3. Proteomics 4, 1–10 (2004).

    CAS  PubMed  Google Scholar 

  22. 22

    Donia, M.S., Fricke, W.F., Ravel, J. & Schmidt, E.W. Variation in tropical reef symbiont metagenomes defined by secondary metabolism. PLoS One 6, e17897 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Tianero, M.D. et al. Metabolic model for diversity-generating biosynthesis. Proc. Natl. Acad. Sci. USA 113, 1772–1777 (2016).

    CAS  PubMed  Google Scholar 

  24. 24

    Pannecouque, C., Daelemans, D. & De Clercq, E. Tetrazolium-based colorimetric assay for the detection of HIV replication inhibitors: revisited 20 years later. Nat. Protoc. 3, 427–434 (2008).

    CAS  PubMed  Google Scholar 

  25. 25

    Märki, F., Hänni, E., Fredenhagen, A. & van Oostrum, J. Mode of action of the lanthionine-containing peptide antibiotics duramycin, duramycin B and C, and cinnamycin as indirect inhibitors of phospholipase A2 . Biochem. Pharmacol. 42, 2027–2035 (1991).

    PubMed  Google Scholar 

  26. 26

    Iwamoto, K. et al. Curvature-dependent recognition of ethanolamine phospholipids by duramycin and cinnamycin. Biophys. J. 93, 1608–1619 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Machaidze, G. & Seelig, J. Specific binding of cinnamycin (Ro 09-0198) to phosphatidylethanolamine. Comparison between micellar and membrane environments. Biochemistry 42, 12570–12576 (2003).

    CAS  PubMed  Google Scholar 

  28. 28

    Choung, S.Y. et al. Hemolytic activity of a cyclic peptide Ro09-0198 isolated from Streptoverticillium. Biochim. Biophys. Acta 940, 171–179 (1988).

    CAS  PubMed  Google Scholar 

  29. 29

    Makino, A. et al. Cinnamycin (Ro 09-0198) promotes cell binding and toxicity by inducing transbilayer lipid movement. J. Biol. Chem. 278, 3204–3209 (2003).

    CAS  PubMed  Google Scholar 

  30. 30

    Richard, A.S. et al. Virion-associated phosphatidylethanolamine promotes TIM1-mediated infection by Ebola, dengue, and West Nile viruses. Proc. Natl. Acad. Sci. USA 112, 14682–14687 (2015).

    CAS  PubMed  Google Scholar 

  31. 31

    Tabata, T. et al. Zika virus targets different primary human placental cells, suggesting two routes for vertical transmission. Cell Host Microbe 20, 155–166 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Wakamiya, T., Fukase, K., Naruse, N., Konishi, M. & Shiba, T. Lanthiopeptin, a new peptide effective against Herpes simplex virus: structural determination and comparison with Ro 09–0198, an immunopotentiating peptide. Tetrahedr. Lett. 29, 4771–4772 (1988).

    CAS  Google Scholar 

  33. 33

    Yates, K.R. et al. Duramycin exhibits antiproliferative properties and induces apoptosis in tumour cells. Blood Coagul. Fibrinolysis 23, 396–401 (2012).

    CAS  PubMed  Google Scholar 

  34. 34

    Zhao, M. Lantibiotics as probes for phosphatidylethanolamine. Amino Acids 41, 1071–1079 (2011).

    CAS  PubMed  Google Scholar 

  35. 35

    Medema, M.H. & Fischbach, M.A. Computational approaches to natural product discovery. Nat. Chem. Biol. 11, 639–648 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Chu, J. et al. Discovery of MRSA active antibiotics using primary sequence from the human microbiome. Nat. Chem. Biol. 12, 1004–1006 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Challis, G.L. & Ravel, J. Coelichelin, a new peptide siderophore encoded by the Streptomyces coelicolor genome: structure prediction from the sequence of its non-ribosomal peptide synthetase. FEMS Microbiol. Lett. 187, 111–114 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Skinnider, M.A. et al. Genomes to natural products PRediction Informatics for Secondary Metabolomes (PRISM). Nucleic Acids Res. 43, 9645–9662 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Blin, K. et al. antiSMASH 4.0-improvements in chemistry prediction and gene cluster boundary identification. Nucleic Acids Res. 45, W36–W41 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Li, M. et al. TIM-family proteins inhibit HIV-1 release. Proc. Natl. Acad. Sci. USA 111, E3699–E3707 (2014).

    CAS  PubMed  Google Scholar 

  41. 41

    Kondratowicz, A.S. et al. T-cell immunoglobulin and mucin domain 1 (TIM-1) is a receptor for Zaire Ebolavirus and Lake Victoria Marburgvirus. Proc. Natl. Acad. Sci. USA 108, 8426–8431 (2011).

    CAS  PubMed  Google Scholar 

  42. 42

    Meertens, L. et al. The TIM and TAM families of phosphatidylserine receptors mediate dengue virus entry. Cell Host Microbe 12, 544–557 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Firn, R.D. & Jones, C.G. A Darwinian view of metabolism: molecular properties determine fitness. J. Exp. Bot. 60, 719–726 (2009).

    CAS  PubMed  Google Scholar 

  44. 44

    Kliebenstein, D.J. A role for gene duplication and natural variation of gene expression in the evolution of metabolism. PLoS One 3, e1838 (2008).

    PubMed  PubMed Central  Google Scholar 

  45. 45

    Lin, Z., Torres, J.P., Tianero, M.D., Kwan, J.C. & Schmidt, E.W. Origin of chemical diversity in Prochloron-tunicate symbiosis. Appl. Environ. Microbiol. 82, 3450–3460 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Li, B. et al. Catalytic promiscuity in the biosynthesis of cyclic peptide secondary metabolites in planktonic marine cyanobacteria. Proc. Natl. Acad. Sci. USA 107, 10430–10435 (2010).

    CAS  PubMed  Google Scholar 

  47. 47

    Sardar, D., Pierce, E., McIntosh, J.A. & Schmidt, E.W. Recognition sequences and substrate evolution in cyanobactin biosynthesis. ACS Synth. Biol. 4, 167–176 (2015).

    CAS  PubMed  Google Scholar 

  48. 48

    Cubillos-Ruiz, A., Berta-Thompson, J.W., Becker, J.W., van der Donk, W.A. & Chisholm, S.W. Evolutionary radiation of lanthipeptides in marine cyanobacteria. Proc. Natl. Acad. Sci. USA 114, E5424–E5433 (2017).

    CAS  PubMed  Google Scholar 

  49. 49

    Hirose, M., Nozawa, Y. & Hirose, E. Genetic isolation among morphotypes in the photosymbiotic didemnid Didemnum molle (Ascidiacea, Tunicata) from the Ryukyus and Taiwan. Zool. Sci. 27, 959–964 (2010).

    PubMed  Google Scholar 

  50. 50

    Burton, I.W., Quilliam, M.A. & Walter, J.A. Quantitative 1H NMR with external standards: use in preparation of calibration solutions for algal toxins and other natural products. Anal. Chem. 77, 3123–3131 (2005).

    CAS  PubMed  Google Scholar 

  51. 51

    Sokolov, E.P. An improved method for DNA isolation from mucopolysaccharide-rich molluscan tissues. J. Molluscan Stud. 66, 573–575 (2000).

    Google Scholar 

  52. 52

    Tianero, M.D. et al. Species specificity of symbiosis and secondary metabolism in ascidians. ISME J. 9, 615–628 (2015).

    PubMed  Google Scholar 

  53. 53

    Zerbino, D.R. & Birney, E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18, 821–829 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Buhr, F. et al. Synonymous codons direct cotranslational folding toward different protein conformations. Mol. Cell 61, 341–351 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    He, Z. et al. Isolation and identification of a Paenibacillus polymyxa strain that coproduces a novel lantibiotic and polymyxin. Appl. Environ. Microbiol. 73, 168–178 (2007).

    CAS  PubMed  Google Scholar 

  56. 56

    Kawulka, K.E. et al. Structure of subtilosin A, a cyclic antimicrobial peptide from Bacillus subtilis with unusual sulfur to α-carbon cross-links: formation and reduction of α-thio-α-amino acid derivatives. Biochemistry 43, 3385–3395 (2004).

    CAS  PubMed  Google Scholar 

  57. 57

    Chen, H., Boyle, T.J., Malim, M.H., Cullen, B.R. & Lyerly, H.K. Derivation of a biologically contained replication system for human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 89, 7678–7682 (1992).

    CAS  PubMed  Google Scholar 

  58. 58

    Kiser, R., Makovsky, S., Terpening, S.J., Laing, N. & Clanton, D.J. Assessment of a cytoprotection assay for the discovery and evaluation of anti-human immunodeficiency virus compounds utilizing a genetically-impaired virus. J. Virol. Methods 58, 99–109 (1996).

    CAS  PubMed  Google Scholar 

  59. 59

    Hou, S., Johnson, S.E. & Zhao, M. A One-step staining probe for phosphatidylethanolamine. ChemBioChem 16, 1955–1960 (2015).

    CAS  PubMed  Google Scholar 

Download references


This work was funded by the National Institutes of Health (NIH) through Fogarty International Center Grant ICBG U01TW006671, R01 GM102602, and R01 GM107557, as well as the American Federation for Pharmaceutical Education (AFPE) Pre-Doctoral Fellowship and the American Chemical Society (ACS) Division of Medicinal Chemistry Pre-Doctoral Fellowship. Funding for the Varian INOVA 600 and 500 MHz NMR spectrometers was provided by NIH Grant RR06262. We thank the lab of J.C. Vederas (University of Alberta) for providing us with methyllanthionine GC–MS standards and M.S. Donia for providing suggestions in the preparation of the manuscript.

Author information




T.E.S., E.W.S., C.M.I., and L.R.B. designed the research. T.E.S. and E.W.S. wrote the paper. T.E.S. purified and characterized compounds from both tunicates and E. coli, constructed pDiv-2, pDiv-3, and pRSFDuet-DivMT expression vectors, performed all in vitro synthetic steps in divamide production, and designed Figures. C.D.P. and T.E.S. performed biological assays. E.P. constructed the pDiv expression vector. Z.P.H. aided in the purification of compounds from E. coli. J.K. sequenced and assembled the E11-036 cluster. M.M.Z. assembled the partial E11-037 cluster. M.K.H. collected animal material. T.P.W. and T.E.S. collected 900 MHz NMR spectra and the 500 MHz 13C NMR spectrum for compound 1. T.K.M. obtained permits for tunicate collection. T.S.B. provided access to the University of Wisconsin National Magnetic Resonance Facility at Madison (NMRFAM) facility. L.R.B. and C.M.I. provided technical and conceptual assistance.

Corresponding author

Correspondence to Eric W Schmidt.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–9 and Supplementary Figures 1–5 (PDF 3957 kb)

Life Sciences Reporting Summary (PDF 266 kb)

Supplementary Note 1

Chemical characterization of the divamides (PDF 22622 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Smith, T., Pond, C., Pierce, E. et al. Accessing chemical diversity from the uncultivated symbionts of small marine animals. Nat Chem Biol 14, 179–185 (2018).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing