Application of Diffusion Growth Chambers for the Cultivation of Marine Sponge-Associated Bacteria

Abstract

Marine sponges contain dense and diverse microbial communities, which are renowned as a source of bioactive metabolites. The biological activities of sponge-microbe natural products span a broad spectrum, from antibacterial and antifungal to antitumor and antiviral applications. However, the potential of sponge-derived compounds has not been fully realized, due largely to the acknowledged “supply issue.” Most bacteria from environmental samples have resisted cultivation on artificial growth media, and cultivation of sponge-associated bacteria has been a major focus in the search for novel marine natural products. One approach to isolate so-called “uncultivable” microorganisms from different environments is the diffusion growth chamber method. Here, we describe the first application of diffusion growth chambers for the isolation of cultivable and previously uncultivated bacteria from sponges. The study was conducted by implanting diffusion growth chambers in the tissue of Rhabdastrella globostellata reef sponges. In total, 255 16S rRNA gene sequences were obtained, with phylogenetic analyses revealing their affiliations with the Alpha- and Gammaproteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes. Fifteen sequences represented previously uncultivated bacteria belonging to the Bacteroidetes and Proteobacteria (Alpha and Gamma classes). Our results indicate that the diffusion growth chamber approach can be successfully applied in a natural, living marine environment such as sponges.

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References

  1. Alain K, Querellou J (2009) Cultivating the uncultured: Limits, advances and future challenges. Extremophiles 13:583–94. doi:10.1007/s00792-009-0261-3

    Article  PubMed  Google Scholar 

  2. Blunt JW, Copp BR, Munro MHG et al (2011) Marine natural products. Nat Prod Rep 28:196–268. doi:10.1039/c005001f

    CAS  Article  PubMed  Google Scholar 

  3. Blunt JW, Copp BR, Keyzers RA et al (2012) Marine natural products. Nat Prod Rep 29:144–222. doi:10.1039/c2np00090c

    CAS  Article  PubMed  Google Scholar 

  4. Blunt JW, Copp BR, Keyzers RA et al (2013) Marine natural products. Nat Prod Rep 30:237–323. doi:10.1039/c2np20112g

    CAS  Article  PubMed  Google Scholar 

  5. Bollmann A, Lewis K, Epstein SS (2007) Incubation of environmental samples in a diffusion chamber increases the diversity of recovered isolates. Appl Environ Microbiol 73:6386–90. doi:10.1128/AEM.01309-07

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  6. Bollmann A, Palumbo AV, Lewis K, Epstein SS (2010) Isolation and physiology of bacteria from contaminated subsurface sediments. Appl Environ Microbiol 76:7413–9. doi:10.1128/AEM.00376-10

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  7. Ebada SS, Lin W, Proksch P (2010) Bioactive sesterterpenes and triterpenes from marine sponges: Occurrence and pharmacological significance. Mar Drugs 8:313–46. doi:10.3390/md8020313

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  8. Fouad M, Edrada RA, Ebel R et al (2006) Cytotoxic isomalabaricane triterpenes from the marine sponge Rhabdastrella globostellata. J Nat Prod 69:211–8. doi:10.1021/np050346t

    CAS  Article  PubMed  Google Scholar 

  9. Gandhimathi R, Arunkumar M, Selvin J et al (2008) Antimicrobial potential of sponge associated marine actinomycetes. J Mycol Médicale/ J Med Mycol 18:16–22. doi:10.1016/j.mycmed.2007.11.001

    Article  Google Scholar 

  10. Gavrish E, Bollmann A, Epstein S, Lewis K (2008) A trap for in situ cultivation of filamentous actinobacteria. J Microbiol Methods 72:257–62. doi:10.1016/j.mimet.2007.12.009

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  11. Hentschel U, Hopke J, Horn M et al (2002) Molecular evidence for a uniform microbial community in sponges from different oceans. Appl Environ Microbiol 68:4431–4440. doi:10.1128/AEM.68.9.4431

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  12. Hentschel U, Usher KM, Taylor MW (2006) Marine sponges as microbial fermenters. FEMS Microbiol Ecol 55:167–77. doi:10.1111/j.1574-6941.2005.00046.x

    CAS  Article  PubMed  Google Scholar 

  13. Hentschel U, Piel J, Degnan SM, Taylor MW (2012) Genomic insights into the marine sponge microbiome. Nat Rev Microbiol 10:641–54. doi:10.1038/nrmicro2839

    CAS  Article  PubMed  Google Scholar 

  14. Huse SM, Welch DM, Morrison HG, Sogin ML (2010) Ironing out the wrinkles in the rare biosphere through improved OTU clustering. Environ Microbiol 12:1889–98. doi:10.1111/j.1462-2920.2010.02193.x

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  15. Joint I, Mühling M, Querellou J (2010) Culturing marine bacteria—an essential prerequisite for biodiscovery. Microb Biotechnol 3:564–75. doi:10.1111/j.1751-7915.2010.00188.x

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  16. Kaeberlein T, Lewis K, Epstein SS (2002) Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment. Science 296:1127–9. doi:10.1126/science.1070633

    CAS  Article  PubMed  Google Scholar 

  17. Kearse M, Moir R, Wilson A et al (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–9. doi:10.1093/bioinformatics/bts199

    PubMed Central  Article  PubMed  Google Scholar 

  18. Koopmans M, Martens D, Wijffels RH (2009) Towards commercial production of sponge medicines. Mar Drugs 7:787–802. doi:10.3390/md7040787

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  19. Lafi FF, Garson MJ, Fuerst JA (2005) Culturable bacterial symbionts isolated from two distinct sponge species (Pseudoceratina clavata and Rhabdastrella globostellata) from the Great Barrier Reef display similar phylogenetic diversity. Microb Ecol 50:213–20. doi:10.1007/s00248-004-0202-8

    CAS  Article  PubMed  Google Scholar 

  20. Lafi FF, Fuerst JA, Fieseler L et al (2009) Widespread distribution of poribacteria in demospongiae. Appl Environ Microbiol 75:5695–9. doi:10.1128/AEM.00035-09

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  21. Lavy A, Keren R, Haber M et al (2013) Implementing sponge physiological and genomic information to enhance the diversity of its culturable associated bacteria. FEMS Microbiol Ecol. doi:10.1111/1574-6941.12240

    PubMed  Google Scholar 

  22. Molinski TF, Dalisay DS, Lievens SL, Saludes JP (2009) Drug development from marine natural products. Nat Rev Drug Discov 8:69–85. doi:10.1038/nrd2487

    CAS  Article  PubMed  Google Scholar 

  23. Nichols D, Cahoon N, Trakhtenberg EM et al (2010) Use of ichip for high-throughput in situ cultivation of “uncultivable” microbial species. Appl Environ Microbiol 76:2445–50. doi:10.1128/AEM.01754-09

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  24. Olson J, McCarthy P (2005) Associated bacterial communities of two deep-water sponges. Aquat Microb Ecol 39:47–55. doi:10.3354/ame039047

    Article  Google Scholar 

  25. Petrosino JF, Highlander S, Luna RA et al (2009) Metagenomic pyrosequencing and microbial identification. Clin Chem 55:856–66. doi:10.1373/clinchem.2008.107565

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  26. Piel J (2009) Metabolites from symbiotic bacteria. Nat Prod Rep 26:338–62. doi:10.1039/b703499g

    CAS  Article  PubMed  Google Scholar 

  27. Piel J, Hui D, Wen G et al (2004) Antitumor polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei. Proc Natl Acad Sci U S A 101:16222–7. doi:10.1073/pnas.0405976101

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  28. Sashidhara KV, White KN, Crews P (2009) A selective account of effective paradigms and significant outcomes in the discovery of inspirational marine natural products. J Nat Prod 72:588–603. doi:10.1021/np800817y

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  29. Schippers KJ, Sipkema D, Osinga R et al (2012) Cultivation of sponges, sponge cells and symbionts: Achievements and future prospects. Adv Mar Biol 62:273–337. doi:10.1016/B978-0-12-394283-8.00006-0

    Article  PubMed  Google Scholar 

  30. Schloss PD, Westcott SL (2011) Assessing and improving methods used in operational taxonomic unit-based approaches for 16S rRNA gene sequence analysis. Appl Environ Microbiol 77:3219–26. doi:10.1128/AEM.02810-10

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  31. Schloss PD, Westcott SL, Ryabin T et al (2009) Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–41. doi:10.1128/AEM.01541-09

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  32. Simister RL, Deines P, Botté ES et al (2012) Sponge-specific clusters revisited: a comprehensive phylogeny of sponge-associated microorganisms. Environ Microbiol 14:517–524. doi:10.1111/j.1462-2920.2011.02664.x

    CAS  Article  PubMed  Google Scholar 

  33. Sipkema D, Franssen MCR, Osinga R et al (2005) Marine sponges as pharmacy. Mar Biotechnol (NY) 7:142–62. doi:10.1007/s10126-004-0405-5

    CAS  Article  Google Scholar 

  34. Sipkema D, Schippers K, Maalcke WJ et al (2011) Multiple approaches to enhance the cultivability of bacteria associated with the marine sponge Haliclona (gellius) sp. Appl Environ Microbiol 77:2130–40. doi:10.1128/AEM.01203-10

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  35. Stamatakis A, Ludwig T, Meier H (2005) RAxML-III: a fast program for maximum likelihood-based inference of large phylogenetic trees. Bioinformatics 21:456–463. doi:10.1093/bioinformatics/bti191

    CAS  Article  PubMed  Google Scholar 

  36. Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap algorithm for the RAxML Web servers. Syst Biol 57:758–71. doi:10.1080/10635150802429642

    Article  PubMed  Google Scholar 

  37. Tasdemir D, Mangalindan GC, Concepción GP et al (2002) Bioactive Isomalabaricane Triterpenes from the Marine Sponge Rhabdastrella globostellata. J Nat Prod 65:210–214. doi:10.1021/np0104020

    CAS  Article  PubMed  Google Scholar 

  38. Taylor MW, Radax R, Steger D, Wagner M (2007) Sponge-associated microorganisms: Evolution, ecology, and biotechnological potential. Microbiol Mol Biol Rev 71:295–347. doi:10.1128/MMBR.00040-06

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  39. Wang G (2006) Diversity and biotechnological potential of the sponge-associated microbial consortia. J Ind Microbiol Biotechnol 33:545–51. doi:10.1007/s10295-006-0123-2

    CAS  Article  PubMed  Google Scholar 

  40. Webster NS, Taylor MW (2012) Marine sponges and their microbial symbionts: love and other relationships. Environ Microbiol, 14(2):335–46. doi:10.1111/j.1462-2920.2011.02460.x

  41. Zhang L, An R, Wang J et al (2005) Exploring novel bioactive compounds from marine microbes. Curr Opin Microbiol 8:276–81. doi:10.1016/j.mib.2005.04.008

    CAS  Article  PubMed  Google Scholar 

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Acknowledgments

PJS acknowledges funding by NIH MBRS SCORE grant S06-GM-44796. CT acknowledges the support by a Fedor-Lynen-Fellowship from the Alexander-von-Humboldt Foundation. We thank the University of Guam Marine Laboratory staff for assisting with field work. GS acknowledges the funding for phylogenetic analyses at the University of Auckland in the authors' laboratory by the German Academic Exchange Service (DAAD) short-term fellowship 'Microbial Symbiosis and Diversity in Marine Sponges' from 02/2013 to 06/2013.

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Correspondence to Peter J. Schupp.

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Supplementary Fig. 1

16S rRNA-based phylogeny of R. globostellata-associated Gammaproteobacteria organisms. Details are the same as those provided in Fig. 3 (JPEG 485 kb)

Supplementary Fig. 2

16S rRNA-based phylogeny of R. globostellata-associated Gammaproteobacteria organisms. Details are the same as those provided in Fig. 3 (JPEG 457 kb)

Supplementary Fig. 3

16S rRNA-based phylogeny of R. globostellata-associated Alphaproteobacteria organisms. Details are the same as those provided in Fig. 3 (JPEG 599 kb)

Supplementary Fig. 4

16S rRNA-based phylogeny of R. globostellata-associated Alphaproteobacteria organisms. Details are the same as those provided in Fig. 3 (JPEG 526 kb)

Supplementary Fig. 5

16S rRNA-based phylogeny of R. globostellata-associated Actinobacteria organisms. Details are the same as those provided in Fig. 3 (JPEG 784 kb)

Supplementary Fig. 6

16S rRNA-based phylogeny of R. globostellata-associated Firmicutes organisms. Details are the same as those provided in Fig. 3 (JPEG 466 kb)

Supplementary Table 1

Sequence information for all isolates. Novel cultured strains are highlighted in bold. It includes the GenBank accession number (ACC), the length of the sequence (long = >1199, short ≤1199), the isolate DGC source of the sequence (direct or DGC1 to DGC4), the different media types, and the GenBank blast results with the taxonomic classification from phylum to genus, the maximum identity and the related GenBank accession number. (XLSX 90 kb)

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Steinert, G., Whitfield, S., Taylor, M.W. et al. Application of Diffusion Growth Chambers for the Cultivation of Marine Sponge-Associated Bacteria. Mar Biotechnol 16, 594–603 (2014). https://doi.org/10.1007/s10126-014-9575-y

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Keywords

  • Cultivation-dependent
  • Marine sponge
  • Bacterial symbionts
  • Diffusion growth chamber
  • In vivo cultivation
  • Rhabdastrella globostellata
  • Sponge-specific cluster
  • 16S rRNA