Cellular localisation of secondary metabolites isolated from the Caribbean sponge Plakortis simplex
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.
KeywordsSponge Polyketide Cell Separation Sphingomonas Marine Sponge
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.
- Brusca RC, Brusca GJ (1990) Phylum porifera: The sponges. In: Sinauer AD (ed) Invertebrates. Sinauer Press, MA, USA, pp 181–210Google Scholar
- 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
- 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
- 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
- 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
- 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
- Sambrook J, Russell DW (2001). Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar