View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by University of Queensland eSpace APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2001, p. 434–444 Vol. 67, No. 1 0099-2240/01/$04.00ϩ0 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† 1 1 2 1,3 NICOLE S. WEBSTER, KATE J. WILSON, LINDA L. BLACKALL, AND RUSSELL T. HILL * 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 Downloaded from 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 ␤- and ␥-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomyce- tes, 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 http://aem.asm.org/ microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation condi- tions 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 tion that microbial symbionts play a role in the defense of their diversity has greatly enhanced the knowledge of population host sponge (5, 21, 22, 44). Marine sponges produce a wide structure in natural microbial communities. It is widely ac- array of other natural products and bioactive secondary me- cepted that culture-based techniques are inadequate for study- tabolites (4, 10, 18, 35; for a review of recent reports, see ref- on May 10, 2018 by UQ Library ing bacterial diversity from environmental samples, as many erence 14). In some instances, the origin of these compounds bacteria cannot be cultured using current and traditional tech- has also been shown to be bacteria associated with sponges. niques (20). Cloning and sequencing of 16S rRNA genes give For example, Vibrio spp. associated with the sponge Dysidea sp. data that can be used to describe complete microbial commu- were shown to synthesize cytotoxic and antibacterial tetrabro- nity composition and can indicate possible nutritional require- modiphenyl ethers (13). The diketopiperazines associated with ments and physiological niches of many microbes based on the sponge Tedania ignis were found to be produced by a Micro- information already available for known phylogenetic relatives coccus sp. (43). Recently, the antifungal peptide theopalauamide, (11, 38). This may assist in the experimental manipulation of isolated from the marine sponge Theonella swinhoei, was shown to culture conditions to provide the correct growth environment be contained in a novel ␦-proteobacterial symbiont (42a). for targeted bacteria. One of the limitations associated with the Secondary metabolite production can be assigned to symbio- construction of 16S rDNA clone libraries from total environ- tic microorganisms only when synthesis has been demonstrated mental DNA is that it requires the use of PCR, which pre- in cultures isolated from the host species (15) and it is still cludes quantitative estimates of abundance for each organism. possible that these compounds are simultaneously being pro- This can be overcome to some degree by the use of fluores- duced by the host. In many instances, the limited availability of cence in situ hybridization (FISH) probing, which allows the sponge material may preclude the commercial production of cells to be visualized and semiquantified (30). bioactive compounds of potential pharmaceutical importance The biology of the bacterium-sponge relationship has elic- (34). The isolation of bioactive compounds from symbiotic ited considerable interest among researchers investigating ma- bacteria could overcome these limitations by providing a con- rine organisms as sources of natural products. Antimicrobial sistent yield using large-scale laboratory culture, eliminating compounds have been isolated from sponge-associated bacte- the need to harvest sponges from the natural environment. ria on numerous occasions, and this has prompted the sugges- Rhopaloeides odorabile is a common Great Barrier Reef sponge. R. odorabile possesses an unusual group of C20 diter- penes which show variation in yield with changing environmen- * Corresponding author. Mailing address: Center of Marine Bio- tal parameters, such as depth and light exposure (46). It has technology, Columbus Center Suite 236, 701 East Pratt St., Baltimore, been hypothesized that these fluctuations combined with ob- MD 21202. Phone: (410) 234 8883. Fax: (410) 234 8896. E-mail: hillr served variability in external appearance (pigmentation and @umbi.umd.edu. †Contribution no. 1030 from the Australian Institute of Marine shape) may be due to variation in symbiotic microbial commu- Science; contribution no. 532 from the Center of Marine Biotechnol- nities. A previous study investigated the culturable bacterial ogy. community associated with R. odorabile (48). This study found 434 VOL. 67, 2001 DIVERSITY OF BACTERIA ASSOCIATED WITH A MARINE SPONGE 435 TABLE 1. Sequences of oligonucleotide probes used for FISH rRNA target site Probe Sequence Specificity % Formamideb Reference (position)a EUB338 GCTGCCTCCCGTAGGAGT 16S, 338–355 Bacteria 20 2 BET42a GCCTTCCCACTTCGTTT 23S, 1027–1043 ␤-Proteobacteria 35 29 GAM42a GCCTTCCCACATCGTTT 16S, 1027–1043 ␥-Proteobacteria 35 29 HGC69a TATAGTTACCACCGCCGT 23S, 1901–1918 Actinobacteria 25 39 LGC354a, -b, and -c YSGAAGATTCCCTACTGC 16S, 354–371 Part of low-GϩC gram-positive division 35 31 CF319a TGGTCCGTGTCTCAGTAC 16S, 319–336 Cytophaga-Flavobacterium 35 29 PLA46 GACTTGCATGCCTAATCC 16S, 46–63 Planctomycetes division 30 33 GSB532 TGCCACCCCTGTATC 16S, 532–546 Part of green sulfur division 10 (45°C) 47 NonEUB338 ACTCCTACGGGAGGCAGC 16S, 338–355 Bacteria (negative control probe) 20 2 a E. coli numbering b All hybridization temperatures were 46°C unless otherwise stated. Downloaded from the culturable community to be dominated by a single bacterial PCR products were cloned with a TOPO TA cloning kit according to the strain, designated NW001, which is a member of the ␣ subdi- manufacturer’s instructions (Invitrogen, San Diego, Calif.). Plasmids were Eco Proteobacteria checked for inserts by digestion with RI restriction endonuclease (Promega, vision of the . Strain NW001 has a close associ- Inc., Madison, Wis.). A negative-control cloning reaction was performed in ation with R. odorabile, which is stable over space and time, and which no sponge-extracted DNA was added. hence variations in the population of this bacterial strain are Sequencing and phylogenetic analysis. Plasmid inserts from 70 clones were unlikely to account for the observed variation in diterpene sequenced using the M13 forward and reverse primers, the PRISM Ready Re- production. action kit, and ABI 310 and 373 automated sequencers (PE Applied Biosystems, Foster City, Calif.). Sequences were compared to those in databases using the The present study aimed to investigate the diversity of the Basic Local Alignment Search Tool (BLAST) algorithm (1) to identify known http://aem.asm.org/ total bacterial community within the sponge R. odorabile. 16S sequences with a high degree of similarity. Sequences were examined for the rRNA gene sequence data and FISH with group-specific oli- formation of chimeras using the program CHECK CHIMERA (28). Partial gonucleotide probes were used to provide a culture-indepen- sequences were manually compiled and aligned using Phydit software (8). Evo- dent investigation into community composition. Phylogenetic lutionary trees were generated using the neighbor-joining (41), Fitch-Margoliash (16), and maximum parsimony (26) algorithms in the PHYLIP package (version data on microbial community composition in sponges are of 3.5c; J. Felsenstein, University of Washington, Seattle). Evolutionary distance biotechnological interest since these data will assist in the matrices for the neighbor-joining and Fitch-Margoliash methods were generated rational selection of culture conditions to increase the diversity as described by Jukes and Cantor (25). The robustness of inferred tree topologies of bacteria available for natural product screening. was evaluated after 1,000 bootstrap resamplings of the neighbor-joining