Microhabitats Within Venomous Cone Snails Contain Diverse Actinobacteriaᰔ† Olivier Peraud,1 Jason S

Microhabitats within Venomous Cone Snails Contain Diverse Actinobacteriaᰔ† Olivier Peraud,1 Jason S. Biggs,2 Ronald W. Hughen,3 Alan R. Light,3 Gisela P. Concepcion,5 Baldomero M. Olivera,4 and Eric W. Schmidt1,4* Departments of Medicinal Chemistry,1 Anesthesiology,3 and Biology,4 University of Utah, Salt Lake City, Utah 84112; Department of Marine Biology, University of Guam, Mangilao, Guam 969232; and Marine Science Institute, University of the Philippines, Diliman, Quezon City, Philippines 11015

Received 29 May 2009/Accepted 2 September 2009

Actinomycetes can be symbionts in diverse organisms, including both plants and animals. Some actinomycetes beneﬁt their host by producing small molecule secondary metabolites; the resulting symbioses are often developmentally complex. Actinomycetes associated with three cone snails were studied. Cone snails are venomous tropical marine gastropods which have been extensively examined because of their production of peptide-based neurological toxins, but no microbiological studies have been reported on these organisms. A microhabitat approach was used in which dissected tissue from each snail was treated as an individual sample in order to explore bacteria in the tissues separately. Our results revealed a diverse, novel, and highly culturable cone snail-associated actinomycete community, with some isolates showing promising bioactivity in a neurological assay. This suggests that cone snails may represent an underexplored reservoir of novel actinomycetes of potential interest for drug discovery.

Interest in natural products and drug discovery has been our knowledge no study of cone snail microbial communities a major driving force for the study of microbial communities has been reported. Cone snails are part of the larger super- associated with marine invertebrates. Sponges, which have family Conoidea, comprising ϳ20,000 species, making it an provided more bioactive metabolites than any other marine extremely diverse group (25, 34). Given the initial observation invertebrate group (see reference 4 and previous references of cultivable actinomycetes from cone snails, this large group in that series) have been the main focus of these investiga- seemed like a potentially excellent source of new bacterial tions, yielding numerous reports of associated bacteria and natural products and new models of actinomycete symbiosis. complex microbial communities (17, 43). Other examples of The goal of this study was to examine the actinobacteria marine invertebrate-associated microbes explored for their community associated with tropical marine snails of the genus involvement in natural products include bryozoans (9, 41), Conus, using a microhabitat approach by which individual ascidians (37), and shipworms (46). Thus far, there are lit- organs are treated as independent samples, and to assess the erature reports of diverse bacterial taxa involved in natural bioactivities of the isolates obtained by using a neurological product biosynthesis in marine animals (9, 10). Notably less assay. Although similar approaches have previously been used well studied are the symbiotic actinomycetes, for which the in microbial ecology (3, 42), this is a novel approach in drug biology of host-actinomycete associations is just beginning discovery for the identification of bioactive bacteria. We report to be explored in a methodical way. Actinomycetes are here the association between three cone snails, Conus pulica- known to be important symbionts in a number of biological rius, Conus rolani, and Conus tribblei, and their actinobacteria systems, such as plants, insects, and marine invertebrates, as well as the bioactivities of some of these actinobacteria. contributing as nitrogen fixers in plants (39) or utilizing their chemical arsenal for defense purposes (8, 22, 38). An initial investigation of a cone snail yielded a surprisingly MATERIALS AND METHODS high number of actinomycetes (data not shown), prompting Collection and processing of snails. Cone snails were collected in Cebu, this follow-up study on additional samples. Cone snail mollusks Philippines, by professional collectors with appropriate permits. Typically, the belong to the genus Conus, which contains about 500 closely water temperature in Cebu ranges between 26°C and 29°C. The three different related species (11). These mollusks are well known for their snails, C. pulicarius, C. tribblei, and C. rolani, were identified according to their complex neurologically active venoms that they use to immo- shell patterns (Fig. 1). Snails were kept in seawater overnight before being processed. Snails were dissected under sterile conditions, and each tissue (body, bilize their prey, including fish, worms, and other mollusks. hepatopancreas, and venom duct) was rinsed three times with sterile seawater. The venom of cone snails has been extensively studied, but to Body and hepatopancreas were divided in two, with one half used for cultivation and the other half transferred into RNAlater (Ambion) and kept frozen until DNA was extracted for molecular analysis. * Corresponding author. Mailing address: College of Pharmacy, Me- Cultivation of actinobacteria. Cone snail tissue extract was obtained by grind- dicinal Chemistry, 307 Skaggs Hall, 30 S. 2000 E., Rm. 201, Salt Lake ing ca. 5 mm3 of tissue in 2 ml of sterile artificial seawater using a sterile mortar City, Utah 84112-5820. Phone: (801) 585-5234. Fax: (801) 585-9119. and pestle. Three different medium types were used: marine agar 2216 (Difco), E-mail: [email protected]. ISP2 (Difco), and R2A agar (Difco). ISP2 and R2A are media designed for † Supplemental material for this article may be found at http://aem selective growth of actinomycetes and were supplemented with a final concen- .asm.org/. tration of 10 ␮g of nalidixic acid mlϪ1,10␮g of cycloheximide mlϪ1,25␮gof ᰔ Published ahead of print on 11 September 2009. nystatin mlϪ1, and 2% NaCl. Serial dilutions of the extracts were plated on the

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topologies was evaluated after 1,000 bootstrap resamplings of the neighbor- joining data, and only values of Ͼ50% are shown. Rarefaction and statistical analysis. The assemblages of 16S rRNA gene sequences in C. pulicarius and C. tribblei libraries were analyzed by rarefaction analysis using EcoSim (15) to assess the extent to which the diversity of microbial communities was represented by the library. A 99% and 97% clone-to-clone maximum identity cutoff was used for the generation of the rarefaction curves. The phylogenetic tree branches statistical analysis was done with Unifrac (http: //bmf2.colorado.edu/unifrac/index.psp) (27). Using the Unifrac significance algorithm, the reported P value was Յ0.06, which is in the suggestive range, while a P value if Ͼ0.1 would not be significant. FISH. Samples for fluorescence in situ hybridization (FISH) were fixed in 4% paraformaldehyde in artificial seawater and then stored in 70% ethanol prior to embedding and sectioning as described previously (47). Sections were hybridized with fluorescein-labeled eubacterial probe EUB338 (5Ј-GCTGCCTCCCGTAG GAGT-3Ј) (1) and a Cy-3 HG69a actinobacteria probe (5Ј-TATAGTTACCAC CGCCGT-3Ј) (35). Nonsense fluorescein-labeled EUB338 (5Ј-CTCCTACGGG AGGCAGC-3Ј) (1) was used as a negative control. Images were captured by laser scanning confocal microscopy using the FV-300 XY system (Olympus). Final images were merged and artificially colored using FV10-ASW software (Olympus). Extraction of cultivated isolates. Purified actinomycetes were grown in 50 ml of ISP2 or R2A broth for 7 days at 30°C. For each isolate, the culture was centrifuged and the supernatant was incubated for 45 min with HP20SS resin. The resin was then washed three times with Milli-Q water and eluted with methanol. The methanol was dried, and the resulting extract was partitioned FIG. 1. Radial cladogram showing the evolutionary relationships three times between ethyl acetate and water. The organic fraction was trans- between C. rolani, C. pulicarius, and C. tribblei (12, 31; N. Puillandre, ferred to a clean vial and dried using a rotovap. The dried extract was resus- M. Watkins, and B. M. Olivera, unpublished data). The tree was pended in methanol and passed through a C column before being dried by generated using mitochondrial 16S sequences from the Olivera data- 18 rotary evaporation. base. This figure shows that snails used in this study occupy distinct DRG assay. Dorsal root ganglia (DRG) cells from cervical and lumbar regions branches of the Conus radiation. were obtained from mice and used in an assay with bacterial culture extracts. DRG cells were suspended in medium with additives and loaded with Fura-2 AM (Molecular Probes), a fluorescent dye used to measure intracellular calcium different media, and cultures were incubated at 30°C for up to 4 weeks. One levels. Experiments were performed at room temperature (20 to 25°C) in a bacterial colony of each morphotype was selected and subcultured until pure. 24-well plate format using fluorescence microscopy. Individual cells were treated Analysis of snail-associated actinobacteria. DNA from preserved snail organs as single samples, so that the individual responses of diverse neuron subtypes was extracted using the Puregene tissue kit (Qiagen) and quantified using a from the DRG could be examined. After baseline measurements, the cells were Nanodrop (Thermo Scientific). Bacterial isolates were grown in 50 ml of their treated with KCl solution and then washed. After return to baseline, bacterial medium of isolation and incubated for a week prior to DNA extraction using an extracts, fractions, or pure compounds were applied. This solution was then later UltraClean microbial DNA isolation kit (MoBio). The 16S rRNA genes were replaced with KCl solution. The use of KCl permitted observation of direct, amplified using 100 ng/␮l of DNA with PrimeStar polymerase (Takara) in a modulatory, as well as inhibitory and excitatory effects of the extracts. Full DNA Engine thermocycler (Bio-Rad). Universal bacterial 16S primers 8-27f/ experimental details can be found in the supplemental material section. 1492r (33, 44) were applied to pure cultures and to DNA from whole tissues. To amplify actinomycete sequences from whole tissues, specific primers 243F (19) RESULTS AND DISCUSSION and AB1165R (28) were applied to the universal primer PCR product in a nested PCR strategy. Cycling conditions for the universal primers were a hot start at Three cone snails, C. pulicarius, C. rolani, and C. tribblei, 98°C for 5 min, 30 cycles of 98°C for 30 s, 48°C for 10 s, and 72°C for 45 s, and were dissected freshly after collection, and three tissue a final extension step at 72°C for 5 min. The PCR product was then gel purified on a 1% (wt/vol) agarose gel, and bands of approximately 1,500 bp were excised parts, body, hepatopancreas, and venom duct, were used to and recovered using a QIAQuick gel extraction kit (Qiagen) and eluted in a conduct an analysis of the snail-associated actinomycete 20-␮l volume. PCR amplifications using actinomycete-specific primers 243f/ community. A total of 229 morphologically distinct bacteria AB1165R (28) were carried out under the following cycling conditions: a hot were isolated from C. rolani (n ϭ 121), C. pulicarius (n ϭ start at 98°C for 5 min, 30 cycles of 98°C for 30 s, 55°C for 10 s, and 72°C for 45 s, ϭ and a final extension step at 72°C for 5 min. The PCR product was then gel 121), and C. tribblei (n 105). Morphologically, 96 actino- purified on a 1% (wt/vol) agarose gel, extracted using the QIAQuick gel extrac- mycetes were identified: 47 from C. rolani, 28 from C. puli- tion kit (Qiagen), and eluted in 20 ␮l of water before being used for cloning. carius, and 21 from C. tribblei. At the end of the isolation Purified PCR products were cloned with a TOPO-XL cloning kit (Invitrogen) and purification process, 57 unique actinomycete strains according to the manufacturer’s instructions. A total of 1,017 sequences were remained viable (see Table S1 in the supplemental mate- obtained, 960 from clone library sequencing (480 from universal primer-generated libraries and 480 from actinobacteria primer-generated libraries) and 57 rial). Most isolates were recovered from the hepatopancreas from isolates. (n ϭ 27) and the body (n ϭ 18), while the venom duct Sequencing and phylogenetic analysis. Sequencing was performed using an yielded 11 isolates. Phylogenetic analysis of the isolates us- ABI 3730 automated sequencer (PE Applied Biosystems, Foster City, CA) with ing the 16S rRNA gene showed that 16 different genera (Fig. M13 forward and reverse primers. Partial sequences were compared to those in the GenBank database with the Basic Local Alignment Search Tool algorithm to 2; see also Fig. S2 in the supplemental material) were identify known closely related sequences. Sequences were examined for the present, representing 11 families. Four isolates had 97% or formation of chimeras using the program CHECK_CHIMERA (6). Sequences less maximum identity to their closest relative in GenBank. were manually compiled and aligned using Phydit software (5). Evolutionary The snails had in common four actinomycete genera: Strep- trees were generated using the neighbor-joining (36), Fitch-Margoliash (14), and tomyces, Microbacterium, Gordonia, and Brevibacterium. maximum parsimony (24) algorithms in the PHYLIP package (13). Evolutionary distance matrices for the neighbor-joining and Fitch-Margoliash methods were The snail-associated actinomycete community was then ex- generated as described by Jukes and Cantor (21). The robustness of inferred tree amined using a non-culture-based approach that entailed PCR 6822 VOL. 75, 2009 ACTINOBACTERIA IN VENOMOUS CONE SNAIL MICROHABITATS 6823 amplification and cloning of 16S rRNA genes obtained from statistically suggestive (P Յ 0.06). However, the sample size cone snail tissues. An initial amplification of 16S rRNA genes was not large enough to provide statistically significant results using the bacterial universal primer pair 8-27F and 1492r (P Յ 0.05). yielded only a limited number of actinomycete sequences, During the microbiological and molecular analyses of the while the majority of the clone libraries (480 clones total) were snails that yielded over a thousand 16S rRNA gene sequences, dominated by clones closely related to common marine inver- only actinomycetes were observed and no other actinobacteria. tebrate-associated bacteria, such as Vibrio, Photobacterium, Since the specific primers used were aimed at the actinobac- and Staphylococcus species. By using a set of specific acti- teria group, this indicates that the samples are strongly domi- nobacteria primers, 243F (19) and AB1165R (27), in a nated by the actinomycetes group. nested PCR approach, the number of actinobacteria se- The reasons for the association between cone snails and quences in the clone libraries (480 clones total) increased actinomycetes remain unclear. From the clone library phylo- substantially and accounted for 97% of the clone library genetic analysis, it was observed that each snail and each tissue bacterial sequences. contain members of similar subfamilies. However, at the spe- All of the actinobacterial sequences belonged to the Actino- cies level, differences are evident. These differences are statis- mycetales order. Relatively complete data were obtained for tically suggestive of true differences between the groups. It is the body and the hepatopancreas, while more limited data possible that the bacterial groups common to all snails repre- were available for the venom ducts. Thus, hepatopancreas (C. sent a core of symbionts, performing similar tasks required by pulicarius and C. tribblei) and body (C. pulicarius, C. tribblei, all host snails, while other bacterial groups specific to their host and C. rolani) analyses are presented here. One 96-well plate snail, i.e., Streptomycetaceae for C. tribblei and Pseudonocardi- of clones was analyzed for each tissue type, resulting in 103 aceae for C. rolani, have been selected to fit the biology of this unique actinomycete sequences. Rarefaction analysis using a host. In order to test this hypothesis, more snails should be 97% and 99% identity threshold level (see Fig. S1 in the sampled, including multiple replicates of the same species. supplemental material) showed that the libraries were appro- Results obtained from cultivation compare favorably to priately sampled at the 97% level. At the 99% level more those from other marine invertebrates, such as sponges, which clones would need to be sequenced to provide a better bacte- are often considered microbial fermenters (18). For example, rial diversity representation. Zhang et al. (47) identified 63 different actinomycete morpho- The molecular analysis of the 16S rRNA gene actinobacteria types isolated from five different sponges, with Streptomyces clone libraries from the body of C. pulicarius, C. rolani, and C. accounting for 90% of the morphotypes. In this study, of the 96 tribblei and the hepatopancreas from C. pulicarius and C. trib- actinomycete morphotypes identified and 57 actinomycetes pu- blei showed representatives of 10 actinomycete families (Fig. 2; rified, Streptomyces accounted for ca. 38% of the morphotypes. see also Fig. S2 in the supplemental material). The body clone The analysis of cone snail-associated actinomycete com- libraries contain members of the Streptomycetaceae (C. trib- munities from clone libraries and cultivation yielded a total blei), Sporichthyaceae (C. rolani), Microbacteriaceae (C. pulica- of 12 actinomycete families. This diversity rivals and/or sur- rius), Nocardioidaceae (C. pulicarius), Pseudonocardiaceae (C. passes that seen in other marine invertebrates, such as rolani), Dietziaceae (C. pulicarius, C. rolani, and C. tribblei), sponges (45). The actinomycetes obtained are novel and, Propionibacteriaceae (C. tribblei), and Corynebacteriaceae (C. based on 16S rRNA gene identity level, include three new pulicarius and C. tribblei). The hepatopancreas libraries of C. Streptomyces species. tribblei and C. pulicarius contained members of Streptomyceta- FISH analysis of C. pulicarius. FISH analysis using the ac- ceae (C. tribblei), Micrococcaceae (C. tribblei), Intrasporangi- tinobacteria probe HGC69a and a scrambled probe as a con- aceae (C. tribblei), Nocardioidaceae (C. pulicarius and C. trib- trol revealed that living actinobacteria are present in both the blei), Dietziaceae (C. tribblei), and Corynebacteriaceae (C. hepatopancreas and the body tissues (Fig. 3). In the hepato- pulicarius). No clone sequences were identical to those of iso- pancreas, actinobacteria are present in moderate numbers with lated actinomycetes, although there were representatives of no clear distribution pattern. In the body, actinobacteria many groups. formed dense clusters near the body wall, inside large oval Although the purpose of this study was to use the micro- cells, while other bacteria could be seen in other parts of the habitat approach to obtain bioactive actinomycetes for drug tissue. These large oval cells open toward the outside and are discovery, a few other features were evident in the uncultivated similar in shape and position to mucocytes that produce the analysis. It was observed that sequences of clones from given mucus lubricating the head and foot of the mollusk (Y. Kantor, snails and tissues appeared to cluster according to their snail of personal communication). This analysis confirmed that acti- origin. A statistical analysis of these clusters using Unifrac nobacteria are associated with the snail and are actively divid- showed that the observed clustering was not random and was ing. Snails have been known to use mucus to cover their shells

FIG. 2. Neighbor-joining phylogenetic tree of 16S rRNA gene sequences from uncultivated and cultivated actinobacteria. Dots indicate cultivated isolates, while capital letters indicate sequences obtained from the body of C. pulicarius (CPB), C. rolani (CRB), and C. tribblei (CTB) and hepatopancreas of C. tribblei (CTHP) and C. pulicarius (CPHP). Colors indicate source snails: black (C. tribblei), blue (C. pulicarius), and red (C. rolani). Reference strains from GenBank are marked by an asterisk. f and p indicate branches that were also found using the Fitch-Margoliash or maximum parsimony methods, respectively. The numbers at the nodes are percentages indicating the levels of bootstrap support, based on a neighbor-joining analysis of 1,000 resampled data sets. Only values of Ͼ50% are shown. Bar, 0.01 substitutions per nucleotide position. All actinobacteria identiﬁed fall into the actinomycete group. 6824 PERAUD ET AL. APPL.ENVIRON.MICROBIOL.

FIG. 3. Epifluorescence micrograph section of Conus pulicarius tissue visualized by FISH. (A) Body region hybridized with fluorescein-labeled non-EUB338. (B) Body region hybridized with fluorescein-labeled EUB338 and Cy3-labeled HGC69a. (C) Hepatopancreas region hybridized with fluorescein-labeled non-EUB338. (D) Hepatopancreas region hybridized with fluorescein-labeled EUB338 and Cy3-labeled HGC69a.

(20), and mucus has been reported to contain bioactive com- performance liquid chromatography fractions containing small pounds (23), indicating one of many potential roles for these bioactive molecules have been obtained. In addition, six of the bacteria. Moreover, since actinobacterial sequences in the isolates exhibited antifungal activity during isolation. Together, samples were derived solely from actinomycetales, it is proba- these results indicate that the cone snail microhabitats are ble that these identified bacteria fall within this group. suitable sources for drug discovery studies. Dorsal root ganglion assay. This neurological assay consists Significance. The need for new bioactive compounds has led of mouse neurons from DRG (26, 29). Since the DRG contains to the pursuit of actinomycetes from novel habitats, including many types of neurons, responses on many different channel remote tropical forests on land (2) and coral reefs or benthic and receptor types can be observed in real time by microscopy. sediments at sea (32). It has been increasingly common to In this particular assay, freshly obtained DRG cells were examine animals (7, 16, 30) or plants (40) for symbiotic acti- treated with a fluorophore that binds to cellular calcium. Using nomycetes and to study the biology of these relationships. The fluorescence microscopy, the calcium response of DRG cells to microhabitat approach can bridge both drug discovery and chemicals and extracts is followed in real time. The protocol is microbial ecology studies. By looking at individual organs and optimized to observe effects that are immediate and are highly tissue types as independent samples, it is possible to simulta- biased toward direct interactions with ion channels or with neously explore the natural product components while study- receptors that are closely coupled to ion channels. Using this ing details of the host-symbiont relationship. DRG assay, we tested a total of 87 extracts from cone snail In this study, a relatively large number of novel actinomy- isolates (actinomycetes and nonactinomycetes), which yielded cetes were obtained by using the microhabitat approach. From four leads, CT8 (Fig. 4), CP32, CP41, and CR60. Although the a drug discovery point of view, the novel actinomycete strains active compounds remain to be elucidated, results from the are attractive, as they are likely to contain new compounds. DRG assay suggest that the compounds act directly on chan- Additionally, the remarkable accessibility to the snail-associ- nels or receptors. The data support either a decrease in Kϩ ated actinomycete community is quite unique and will help channel activation, and/or an increase in Naϩ channel activa- circumvent the supply issue which so often plagues natural tion. The leads are potent molecules on which structural elu- products. If this diversity truly scales with the 20,000 species in cidations are ongoing and for which individual active high- the Conoidean superfamily, this diversity is potentially quite VOL. 75, 2009 ACTINOBACTERIA IN VENOMOUS CONE SNAIL MICROHABITATS 6825

FIG. 4. Dorsal root ganglion assay of CT8 extract. The initial KCl injection and first wash provided the control. Once the cells return to their original state, a series of injections follow, starting with injection of the CT8 extract. In panel C, it can be seen that some cells respond with an influx of calcium directly upon injection of the extract, while others attenuate the response until a second KCl injection (B and C). Other samples show no response to the compound (A). large. At least some of these actinomycetes are actively living 2. Balick, M. J., E. Elisabethsky, and S. A. Laird. 1996. Medicinal resources of and dividing within the snail, as evidenced by FISH. the tropical forest: biodiversity and its importance to human health. Colum- bia University Press, New York, NY. Cone snails have shown to be an unexpectedly rich and 3. Birkby, K. M., and T. F. Preece. 1987. The ligules of green leaves of cock’s- diverse reservoir for novel actinomycetes. The promising bio- foot grass, Dactylis glomerata L., are a micro-habitat for bacteria and fungi. activity from snail-associated actinomycetes and important rate J. Appl. Microbiol. 63:505. 4. Blunt, J. W., B. R. Copp, W. P. Hu, M. H. Munro, P. T. Northcote, and M. R. of cultivation make cone snails animals of interest for drug Prinsep. 2009. Marine natural products. Nat. Prod. Rep. 26:170–244. discovery, while their biology may make them a good model for 5. Chun, J. 1995. Computer-assisted classification and identification of actino- studying symbiosis. mycetes. Ph.D. thesis. University of Maryland, College Park. 6. Cole, J. R., B. Chai, T. L. Marsh, R. J. Farris, Q. Wang, S. A. Kulam, S. Chandra, D. M. McGarrell, T. M. Schmidt, G. M. Garrity, and J. M. Tiedje. ACKNOWLEDGMENTS 2003. The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. We thank the government of the Philippines and the people of Cebu Nucleic Acids Res. 31:442–443. for opportunities to work on these specimens. All field work was 7. Currie, C. R., M. Poulsen, J. Mendenhall, J. J. Boomsma, and J. Billen. performed with appropriate regional and national permits (to G. Con- 2006. Coevolved crypts and exocrine glands support mutualistic bacteria in cepcion and B. Olivera), including prior informed consent permission fungus-growing ants. Science 311:81–83. from local communities. Yuri I. Kantor (Severtzov Institute of Ecology 8. Currie, C. R., J. A. Scott, R. C. Summerbell, and D. Malloch. 1999. Fungus- and Evolution of the Russian Academy of Sciences, Moscow, Russia) growing ants use antibiotic-producing bacteria to control garden parasites. provided key information regarding snail anatomy. Mary Anne Astilla Nature 398:701–704. and Jasmin Jao (University of the Phillippines Marine Science Insti- 9. Davidson, S. K., S. W. Allen, G. E. Lim, C. M. Anderson, and M. G. Haygood. tute) helped in processing of samples. We thank Margo Haygood for 2001. Evidence for the biosynthesis of bryostatins by the bacterial symbiont her comments. “Candidatus Endobugula sertula” of the bryozoan Bugula neritina. Appl. This work was supported by a Synergy Grant from the University Environ. Microbiol. 67:4531–4537. of Utah, NIH GM071425, and NIH GM48677. NIH ICBG 10. De Rosa, S., A. Milone, A. Kujumgiev, K. Stefanov, I. Nechev, and S. Popov. 2000. Metabolites from a marine bacterium Pseudomonas/Alteromonas, as- U01TW008163 supported the analysis of samples. sociated with the sponge Dysidea fragilis. Comp. Biochem. Physiol. B 126: 391–396. REFERENCES 11. Duda, T. F., W. Korn, A. J. Kohn, and S. R. Palumbi. 2001. Origins of diverse 1. Amann, R. I., L. Krumholz, and D. A. Stahl. 1990. Fluorescent-oligonucle- feeding ecologies within Conus, a genus of venomous marine gastropods. otide probing of whole cells for determinative, phylogenetic, and environ- Biol. J. Linnean Soc. 73:391–409. mental studies in microbiology. J. Bacteriol. 172:762–770. 12. Espiritu, D. J. D., M. Watkins, V. Dia-Monje, G. E. Cartier, L. J. Cruz, and 6826 PERAUD ET AL. APPL.ENVIRON.MICROBIOL.

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