16S rrna

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Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile
Nicole S. Webster, Kate J. Wilson, Linda L. Blackall and Russell T. Hill Appl. Environ. Microbiol. 2001, 67(1):434. DOI: 10.1128/AEM.67.1.434-444.2001.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2001, p. 434–444 0099-2240/01/$04.00 0 DOI: 10.1128/AEM.67.1.434–444.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Vol. 67, No. 1

Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile†
NICOLE S. WEBSTER,1 KATE J. WILSON,1 LINDAL. 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 2000Molecular 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 communitystructure was extremely diverse with representatives of the Actinobacteria, low-G C gram-positive bacteria, the - and -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 casessuggesting 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 16SrRNA 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. The use of molecular approaches for describing microbial diversity has greatly enhanced the knowledge of populationstructure in natural microbial communities. It is widely accepted that culture-based techniques are inadequate for studying bacterial diversity from environmental samples, as many bacteria cannot be cultured using current and traditional techniques (20). Cloning and sequencing of 16S rRNA genes give data that can be used to describe complete microbial community composition and can indicatepossible nutritional requirements and physiological niches of many microbes based on information already available for known phylogenetic relatives (11, 38). This may assist in the experimental manipulation of culture conditions to provide the correct growth environment for targeted bacteria. One of the limitations associated with the construction of 16S rDNA clone libraries from total environmental...
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