Marine Biology

, Volume 151, Issue 4, pp 1365–1373 | Cite as

Cellular localisation of secondary metabolites isolated from the Caribbean sponge Plakortis simplex

  • Marc Laroche
  • Concetta Imperatore
  • Lubomir Grozdanov
  • Valeria Costantino
  • Alfonso Mangoni
  • Ute Hentschel
  • Ernesto FattorussoEmail author
Research Article


The Caribbean sponge, Plakortis simplex, is known to contain a large array of secondary metabolites, including the antimalarial polyketide plakortin, several unusual glycolipids, and some hopanoids, which closely resemble typical bacterial metabolites. The hypothesis that they could be products of bacterial metabolism was tested by localizing specific metabolites in cells using physical separation of sponge cells, bacterial symbionts and supernatant by differential centrifugation. The obtained fractions were analysed separately for the typical P. simplex metabolites by NMR and mass spectrometry, and most of them were shown to be present in the bacterial cells but not in the sponge cells. In addition, PCR screening showed that the biosynthetic pathway for glycosphingolipids was present in the bacterial cells. Isolation of a Sphingomonas strain PS193 from P. simplex and subsequent glycosphingolipid analysis resulted in the detection of a known glycosphingolipid, GSL-1, that did, however, not match the glycosphingolipid profile of P. simplex. Therefore, it is unlikely that Sphingomonas strain PS193 is an abundant member of the microbial community associated with P. simplex. Other glycosphingolipid producing bacteria in P. simplex remain to be identified. In conclusion, this study provides experimental evidence that the glycolipids and hopanoids and possibly also the polyketide plakortin are produced by microbial symbionts rather than the sponge from which the metabolites were originally isolated.


Sponge Polyketide Cell Separation Sphingomonas Marine Sponge 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The authors are very grateful to Prof. J. R. Pawlik, UNCW for inviting them to participate to the third Pawlik expedition during which the material was collected, and to Prof. M. Pansini, University of Genova for the sponge taxonomy determination. Mass and NMR spectra were recorded at the “Centro di Servizi Interdipartimentale di Analisi Strumentale”, Università di Napoli “Federico II”. The assistance of the staff is gratefully acknowledged. This research project is funded by the Italian Government, MIUR PRIN (Italy) and by the CEE “Marie Curie Host Fellowship Contract no. HPMD-CT-2001-101. This study was also supported by grants of the DFG (SFB630 TP A5) and the bmb+f (BiotecMarin: 03F0414E) to U. Hentschel.


  1. Althoff K, Schütt C, Steffen R, Batel R, Müller WEG (1998) Evidence for a symbiosis between bacteria of the genus Rhodobacter and the marine sponge Halichondria panicea: harbor also for putatively toxic bacteria? Mar Biol 130:529–53CrossRefGoogle Scholar
  2. Bewley CA, Holland ND, Faulkner DJ (1996) Two classes of metabolites from Theonella swinhoei are localized in distinct populations of bacterial symbionts. Experientia 52:716–722CrossRefGoogle Scholar
  3. Brusca RC, Brusca GJ (1990) Phylum porifera: The sponges. In: Sinauer AD (ed) Invertebrates. Sinauer Press, MA, USA, pp 181–210Google Scholar
  4. Campagnuolo C, Fattorusso E, Taglialatela-Scafati O, Ianaro A, Pisano B (2002) Plakortethers A-G: a new class of cytotoxic plakortin-derived metabolites. Eur J Org Chem 61–69CrossRefGoogle Scholar
  5. Campagnuolo C, Fattorusso E, Romano A, Taglialatela-Scafati O, Basilico S,Taramelli D (2005) Antimalarial polyketide cycloperoxides from the marine sponge Plakortis simplex. Eur J Org Chem 5077–5083 CrossRefGoogle Scholar
  6. Costantino V, Fattorusso E, Mangoni A (1993) Isolation of five-membered glycolipids, crasserides: unique glycerides from the sponge Pseudoceratina crassa. J Org Chem 58:186–191CrossRefGoogle Scholar
  7. Costantino V, Fattorusso E, Mangoni A, Di Rosa M, Ianaro A (1997) A unique prenylated glycosphingolipds with immunosuppressive activity from the marine sponge Plakortis simplex. J Am Chem Soc 119:12465–12470CrossRefGoogle Scholar
  8. Costantino V, Fattorusso E, Mangoni A, Di Rosa M, Ianaro A (1999) Simplexides, novel immunosuppressive glycolipids from the Caribbean sponge Plakortis simplex. Bioorg Med Chem Lett 9:271–276CrossRefGoogle Scholar
  9. Costantino V, Fattorusso E, Imperatore C, Mangoni A (2000) The first 12-methylhopanoid: 12-methylbacteriohopanetrol from the marine sponge Plakortis simplex. Tetrahedron 56:3781–3784CrossRefGoogle Scholar
  10. Costantino V, Fattorusso E, Imperatore C, Mangoni A (2001) A biosynthetically significant new bacteriohopanoid present in large amounts in the Caribbean sponge Plakortis simplex. Tetrahedron 57:4045–4048CrossRefGoogle Scholar
  11. Costantino V, Fattorusso E, Menna M, Taglialatela-Scafati O (2004) Chemical diversity of bioactive marine natural products: an illustrative case study. Curr Med Chem 11:1671–1692CrossRefGoogle Scholar
  12. Fattorusso E, Mangoni A (1997) Progress in the chemistry of organic natural products. In: Hertz W, Kirby GW, Moore RE, Steiglich W, Tamm Ch (eds) Marine glycolipids. Springer, Wien, pp 215–301Google Scholar
  13. Flatt PM, Gautschi JT, Thacker RW, Musafija-Girt M, Crews P, Gerwick W (2005) Identification of the cellular site of polychlorinated peptide biosynthesis in the marine sponge Dysidea (Lamellodysidea) herbacea and symbiotic cyanobacterium Oscillatoria spongeliae by CARD-FISH analysis. Mar Biol 147:761–774CrossRefGoogle Scholar
  14. Flowers AE, Garson MJ, Webb RI, Dumdei EJ, Charan RD (1998) Cellular origin of chlorinated diketopiperazines in the dictyoceratid sponge Dysidea herbacea (Keller). Cell Tissue Res 292:597–607CrossRefGoogle Scholar
  15. Friedrich AB, Hacker J, Fischer I, Proksch P, Hentschel U (2001) Temporal variations of the microbial community associated with the Mediterranean sponge Aplysina aerophoba. FEMS Microbiol Ecol 38: 105–113 CrossRefGoogle Scholar
  16. Garson MJ, Zimmermann MP, Battershill CN, Holden JL, Murphy PT (1994) The distribution of brominated long-chain fatty acids in sponge and symbiont cell types from the tropical marine sponge Amphimedon terpenensis. Lipids 29:509–516CrossRefGoogle Scholar
  17. Giovannoni SJ, Connon SA (2002) High-throughput methods for culturing microorganisms in very-low-nutrient media yield diverse new marine isolates. Appl Environ Microbiol 68:3878–3885CrossRefGoogle Scholar
  18. Hentschel U, Hopke J, Horn M, Friedrich AB, Wagner M, Moore BS (2002) Molecular evidence for a uniform microbial community in sponges from different oceans. Appl Environ Microbiol 68:4431–4440CrossRefGoogle Scholar
  19. Hentschel U, Usher KM, Taylor MW (2006) Marine sponges as microbial fermenters. FEMS Microb Ecol 55:167–177CrossRefGoogle Scholar
  20. Higgs MD, Faulkner DJ (1978) Plakortin, an antibiotic from Plakortishalichondroides. J Org Chem 43:3454–3457CrossRefGoogle Scholar
  21. Ikushiro H, Hayashi H, Kagamiyama H (2001) A water-soluble homodimeric serine palmitoyltransferase from Sphingomonas paucimobilis EY2395 Strain. J Biol Chem 276:18249–18256CrossRefGoogle Scholar
  22. Kawahara K, Moll H, Knirel YA, Seydel U, Zähringer U (2000) Structural analysis of two glycosphingolipids from the lipopolysaccharide-lacking bacterium Sphingomonas capsulata. Eur J Biochem 267:1837–1846CrossRefGoogle Scholar
  23. Kobayashi E, Motoki K, Uchida T, Fukushima H, Koezuka Y (1995) KRN7000, a novel immunomodulator, and its antitumor activities. Oncol Res 7:529–34PubMedGoogle Scholar
  24. Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp 115–175Google Scholar
  25. Lang S, Wagner-Döbler I, Beil W, Meiners M, Laatsch H (2002) Integrated approach to explore the potential of marine microorganisms for the production of bioactive metabolites. Adv Biochem Eng Biotechnol 74:207–238PubMedGoogle Scholar
  26. Lee J-S, Shin YK, Yoon J-H, Takeuchi M, Pyun Y-R, Park Y-H (2001). Sphingomonas aquatilis sp. nov., Sphingomonas koreensis sp. nov. and Sphingomonas taejonensis sp. novel yellow-pigmented bacteria isolated from natural mineral water. IJSEM 51:1491–1498PubMedGoogle Scholar
  27. Leys NMEJ, Ryngaert A, Bastiaens L, Verstraete W, Top EM, Springael D (2004) occurrence and phylogenetic diversity of sphingomonas strains in soils contaminated with polycyclic aromatic hydrocarbons. Appl Environ Microbiol 70:1944–1955CrossRefGoogle Scholar
  28. Martin JF, Demain AL (1980) Control of antibiotic biosynthesis. Microbiol Rev 44:230–251PubMedPubMedCentralGoogle Scholar
  29. Merrill AH (2002). De novo sphingolipid biosynthesis: a necessary, but dangerous, pathway. J Biol Chem 227:25843–25846CrossRefGoogle Scholar
  30. Pawlik JR (1993) Marine invertebrate chemical defenses. Chem Rev 93:1911–1922CrossRefGoogle Scholar
  31. Piel J (2004) Metabolites from symbiotic bacteria. Nat Prod Rep 21:519–538CrossRefGoogle Scholar
  32. Piel J, Hui D, Fusetani N, Matsunaga S (2004) Targeting modular polyketide synthase with iteratively acting acyltransferase from metagenomes of uncultured bacterial consortia. Environ Microbiol 6:921–927CrossRefGoogle Scholar
  33. Piel J, Hui D, Wen G, Butzke D, Platzer M, Fusetani N, Matsunaga S (2004b) Antitumor polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei. Proc Natl Acad Sci USA 101:16222–16227CrossRefGoogle Scholar
  34. Pomponi SA, Willoughby R (1994) Sponge cell culture for production of bioactive metabolites. In: Van Soest RWM, Van Kempen TMG, Braekman JC (eds) Sponges in time and space. Balkema, Rotterdam, pp 395–400Google Scholar
  35. Richelle-Maurer E, Braekman JC, De Kluijver MJ, Gomez R, Van de Vyver G, Van Soest RWM, Devijver C (2001) Cellular localization of (2R, 3R, 7Z)-2-aminotetradec-7-ene-1, 3-diol, a potent antimicrobial metabolite produced by the Caribbean sponge Haliclona vansoesti. Cell Tissue Res 306:157–165CrossRefGoogle Scholar
  36. Salomon CE, Deerinck T, Ellisman MH, Faulkner DJ (2001) The cellular localization in the Palauan sponge Oceanapia sagittaria. Marine Biol. 139:313–319CrossRefGoogle Scholar
  37. Sambrook J, Russell DW (2001). Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  38. Schütze J, Skorokhod A, Müller IM, Müller WEG (2001). Molecular evolution of the metazoan extracellular matrix: cloning and expression of structural proteins from the demosponges Suberites domuncula and Geodia cydonium. J Mol Evol 53:402–415CrossRefGoogle Scholar
  39. Towle MJ, Salvato KA, Budrow J et al (2001) In vitro and in vivo anticancer activities of synthetic macrocyclic ketone analogues of halichondrin B. Cancer Res 61:1013–1021PubMedGoogle Scholar
  40. Vacelet J, Donadey C (1977) Electron microscope study of the association between some sponges and bacteria. J Exp Mar Ecol 30:301–314CrossRefGoogle Scholar
  41. Vanbroekhoven K, Ryngaert A, Bastiaens P, Wattiau P, Vancanneyt M, Swings J, De Mot R, Springael D (2004) Streptomycin as a selective agent to facilitate recovery and isolation of introduced and indigenous Sphingomonas from environmental samples. Environ Microbiol 6:1123–36CrossRefGoogle Scholar
  42. Unson MD, Faulkner DJ (1993) Cyanobacterial symbiont biosynthesis of chlorinated metabolites from Dysidea herbacea (Porifera). Experientia 49:349–353CrossRefGoogle Scholar
  43. Uriz MJ, Turon X, Galera J, Tur JM (1996) New light on the cell location of avarol within the sponge Dysidea avara. Cell Tissue Res 285:519–527CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Marc Laroche
    • 1
  • Concetta Imperatore
    • 1
  • Lubomir Grozdanov
    • 2
  • Valeria Costantino
    • 1
  • Alfonso Mangoni
    • 1
  • Ute Hentschel
    • 2
  • Ernesto Fattorusso
    • 1
    Email author
  1. 1.Dipartimento di Chimica delle Sostanze NaturaliUniversità di Napoli Federico IINapoliItaly
  2. 2.Research Center for Infectious DiseasesUniversity of WürzburgWürzburgGermany

Personalised recommendations