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Applied and Environmental Microbiology, January 2001, p. 434-444, Vol. 67, No. 1
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.1.434-444.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile†

Nicole S. Webster,1 Kate J. Wilson,1 Linda L. Blackall,2 and Russell T. Hill1,3,*

Australian Institute of Marine Science, Townsville, Queensland, Australia 48101; Department of Microbiology and Parasitology, University of Queensland, St. Lucia, Queensland, Australia 40672; and Centre of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 212023

Received 20 June 2000/Accepted 19 September 2000

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the beta - and gamma -subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.

* Corresponding author. Mailing address: Center of Marine Biotechnology, Columbus Center Suite 236, 701 East Pratt St., Baltimore, MD 21202. Phone: (410) 234 8883. Fax: (410) 234 8896. E-mail: hillr{at}umbi.umd.edu.

dagger Contribution no. 1030 from the Australian Institute of Marine Science; contribution no. 532 from the Center of Marine Biotechnology.

Applied and Environmental Microbiology, January 2001, p. 434-444, Vol. 67, No. 1
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.1.434-444.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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