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Use of Real-Time qPCR to Quantify Members of the Unculturable Heterotrophic Bacterial Community in a Deep Sea Marine Sponge, Vetulina sp

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Abstract

In this report, real-time quantitative PCR (TaqMan® qPCR) of the small subunit (SSU) 16S-like rRNA molecule, a universal phylogenetic marker, was used to quantify the relative abundance of individual bacterial members of a diverse, yet mostly unculturable, microbial community from a marine sponge. Molecular phylogenetic analyses of bacterial communities derived from Caribbean Lithistid sponges have shown a wide diversity of microbes that included at least six major subdivisions; however, very little overlap was observed between the culturable and unculturable microbial communities. Based on sequence data of three culture-independent Lithistid-derived representative bacteria, we designed probe/primer sets for TaqMan® qPCR to quantitatively characterize selected microbial residents in a Lithistid sponge, Vetulina, metagenome. TaqMan® assays included specificity testing, DNA limit of detection analysis, and quantification of specific microbial rRNA sequences such as Nitrospira-like microbes and Actinobacteria up to 172 million copies per microgram per Lithistid sponge metagenome. By contrast, qPCR amplification with probes designed for common previously cultured sponge-associated bacteria in the genera Rheinheimera and Marinomonas and a representative of the CFB group resulted in only minimal detection of the Rheiheimera in total DNA extracted from the sponge. These data verify that a large portion of the microbial community within Lithistid sponges may consist of currently unculturable microorganisms.

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Acknowledgements

This work was partially funded by NSF Grants 9974984 and DEB 0103668 to JVL and PJM. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. The authors are also grateful to Dr. Julie Olson for helpful discussion on microbial culturing, John Reed for archiving and retrieval of taxonomic specimens, Dr. Eric Brown for helpful comments, Kris Metzger for reprint requests, and UNOLS for funding ship time on the R/V Edwin Link. This manuscript is designated HBOI #1650.

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  1. Molecular Biology Program, MRI Florida Division, 1470 Treeland Blvd., S.E. Palm Bay, FL, 32909, USA

    M. Cassler & R. Winegar

  2. Harbor Branch Oceanographic Institution, 5600 US 1 North, Ft. Pierce, FL, 34946, USA

    A. Ledger, S. A. Pomponi, A. E. Wright, P. J. McCarthy & J. V. Lopez

  3. Historic Bok Sanctuary, 1151 Tower Blvd., Lake Wales, FL, 33853, USA

    C. L. Peterson

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  1. M. Cassler
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Cassler, M., Peterson, C.L., Ledger, A. et al. Use of Real-Time qPCR to Quantify Members of the Unculturable Heterotrophic Bacterial Community in a Deep Sea Marine Sponge, Vetulina sp. Microb Ecol 55, 384–394 (2008). https://doi.org/10.1007/s00248-007-9283-5

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