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.
Cultivation-dependent Marine sponge Bacterial symbionts Diffusion growth chamber In vivo cultivation Rhabdastrella globostellataSponge-specific cluster 16S rRNA
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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.
Supplementary Table 1Sequence 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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