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CASE STUDY High Th roughput Cultivation for Isolation of Novel Marine Oceanography

BY GERARDO TOLEDO, WAYNE GREEN, RICARDO A. GONZALEZ, LEIF CHRISTOFFERSEN, , Volume 2, a quarterly journal of Th 19, Number MIRCEA PODAR, HWAI W. CHANG, THOMAS HEMSCHEIDT, HENRY G. TRAPIDOROSENTHAL, JAY M. SHORT, ROBERT R. BIDIGARE, AND ERIC J. MATHUR

MARINE NATURAL PRODUCTS have arisen from a small number of taxonomic One well-studied example is the synthesis Natural products are organic de- groups that include Streptomyces, Alteromo- of bryostatin-1 by the marine bryozoan, Bugula e Oceanography Society. Copyright e Oceanography 2006 by Th rived from , , or microorganisms, nas, , Vibrio, Agrobacterium, and neritina, which has been directly linked to the and represent the starting point for most of the (Wagner-Döbler et al., 2002; presence of the uncultured bacterium Candida- the anti-infective and anti-cancer drugs on the Burja et al., 2001). Over the past decade, a con- tus Endobugula sertula (Davidson et al., 2001). market today. Until recently, the majority of sensus has developed among marine natural Similarly, Bugula simplex hosts the symbionts natural products has been isolated from ter- products chemists and chemical ecologists, Candidatus Endobugula glebosa, which are restrial sources. During the last two decades, who believe that most novel natural products linked to bryostatin production (Lim and Hay- with the approval of Th however, the rate of discovery of novel com- found in extracts of marine are good, 2004). Such examples demonstrate the pounds has declined signifi cantly, as exempli- synthesized, either in part or in their entirety, promise of marine symbiotic microorganisms

fi ed by the fact that extracts from soil-derived by the symbiotic microbes that are intimately as a viable source for the discovery of novel Permission reserved. Society. All rights is e Oceanography gran actinomycetes have yielded unacceptably high associated with these marine metazoans. small molecules. all correspondence to: Society. Send e Oceanography [email protected] or Th numbers of previously described (Mincer et al., 2002). In addition to the redun- THE EXAMPLE OF MARINE MICROBIAL CULTIVATION dancy and associated issue of de-replication, an TECHNOLOGIES innovation gap has been postulated as a cause Marine sponges provide classic examples of Marine agar, a cultivation medium formulated for the dramatic reduction in small microbial-macrofaunal partnerships that have by Claude ZoBell during the 1940s (ZoBell, novelty. Even today, most microbiologists are been a productive source for the discovery of 1946), has been extensively used for the isola- constrained by the use of traditional cultiva- bioactive compounds (Faulkner, 2002). For ex- tion of marine . However, this medium tion methods, which primarily target previ- ample, Monks et al. (2002) found that extracts contains organic in concentrations ously cultured microbes (“microbial weeds”). of eight out of ten diff erent Brazilian much higher than those found in most natural species exhibited anti-bacterial, anti-tumor, environments; as such, strains isolated using

As a result, most pharmaceutical companies ted to in teaching copy this and research. for use article Repu no longer place an emphasis on natural-prod- or anti-chemotactic activities. According to marine agar are typically fast growing, and not uct discovery as a source of lead compounds chemical ecologists, secondary metabolites always representative of cells that may play e Oceanography Society, PO Box 1931, Rockville, MD 20849-1931, USA. 1931, Rockville, Box PO Society, e Oceanography (Walsh, 2003). are produced as part of a chemical arsenal de- relevant roles in situ. Incubation times for the In contrast, the marine environment is be- signed to deter grazers by imparting toxicity or development of colonies from cells growing in coming increasingly appreciated as a rich and low palatability to the metazoan . Th ese rich media range from one day to one week. untapped reservoir of novel natural products marine -microbial assemblages also Mincer et al. (2002) have addressed the high- (see Fenical, this issue). Bioactive compounds produce toxic compounds to prevent coloniza- carbon-content issue and the relatively short in- are frequently associated with marine inver- tion by other non-benefi cial microbial species. cubation times and have signifi cantly improved tebrates, including sponges, bryozoans, mol- Interestingly, some of the small molecules iso- the techniques for the isolation of novel marine lusks, and (Proksch et al., 2002). More lated from marine metazoans display a striking actinomycetes strains from . Th ey

recently, have been resemblance to prokaryotic-borne metabolites; employed several concentrations of organic reproduction, systemmatic blication, recognized as a productive source of novel it has thus been suggested that the source of carbon from diverse sources, longer incubation secondary metabolites. To date, the majority many of these bioactives is in fact the symbi- times, and the pretreatment of the samples of natural products of marine bacterial origin otic microorganisms (Piel et al., 2004). with a heat shock to enrich for -forming

120 Oceanography Vol. 19, No. 2, June 2006 microorganisms. As a result, novel lineages of UNIVERSITY OF HAWAII sociated microbial cells were separated using a marine actinomycetes have recently been isolat- DIVERSA COLLABORATION Nycodenz cushion and diff erential centrifuga- ed and cultured from diverse marine sediments. Th e Hawaiian archipelago is a geographically tion (Figure 1B). Th e crude pellets of liv- Extracts of some of these cultures have yielded isolated chain of oceanic islands with high bio- ing but uncultured microorganisms were then promising molecules, some of which have diversity and endemism, and is considered to encapsulated in the agarose microcapsules been advanced to the pre-clinical trial phase be an environmental “hot spot” in terms of its (Figure 1C) as previously described (Zengler et by Nereus Pharmaceuticals (more information rich and often unique , , and micro- al., 2002; Zengler et al., 2005). Th e capsules were available at http://www.nereuspharm.com/ bial forms. Consequently, Hawaiian marine transferred into mini fermentation columns overview.shtml). Similarly, Maldonado et al. invertebrates and their associated microbial equipped with a 0.22 µm fi lter at the inlet and (2005) selected carbon sources for the targeted represent a promising resource for an 8 µm fi lter at the outlet to allow free-living isolation of marine actinomyces based on data the discovery of novel bioactive molecules. cells to be washed out of the system while re- generated by culture-independent studies on In 2004, Diversa Corporation signed a bio- taining the microcapsules. the in situ diversity as seen by ribosomal analysis diversity access and collaboration agreement Culture medium was prepared by diluting of the actinomycetes present in the samples. with the University of Hawaii; the goal was to (1/1000) sterile sponge homogenates or sedi- A number of new and innovative techniques access Hawaiian samples to cultivate novel ment extracts with fi lter-sterilized seawater. have been developed in recent years to increase microorganisms associated with marine meta- Th e samples were incubated over a period of the effi ciency of the isolation of novel micro- zoans, sea grasses, and ocean sediments. Th is fi ve weeks and sorted (Figure 1D) with a MoFlo from the marine . Th ese discovery eff ort has been co-funded by the fl ow cytometry system (Cytomation) to array techniques include various modifi cations to Oceans and Human Health Initiative of the individual -containing microcapsules growth media and end-point dilution methods National Oceanographic and Atmospheric (Figure 1E) into 96-well plates containing ma- using microtitre dish plate formats that al- Administration (NOAA) and the Centers for rine broth. Th e growth of most strains is not in- lowed the cultivation of the ubiquitous marine Oceans and Human Health of the National Sci- hibited by high marine broth carbon concentra- bacterial clade SAR11 (Connon and Giovan- ence Foundation (NSF) and the National Insti- tions, and its inclusion allows the generation of noni, 2002; Rappé et al., 2002; Giovannoni et al., tute of Environmental Health Sciences (NIEHS) the relatively large amounts of required 2005). At Diversa Corporation (more informa- of the National Institutes of Health. Th is col- for anti-infective and anti-tumor screening. tion available at http://www.diversa.com/), a laborative research eff ort provides a unique Th e isolates were de-replicated via Fourier- high-throughput cultivation (HTC) technology opportunity to integrate Diversa Corporation’s transformed infrared (FT/IR) . that employs agarose microcapsules to encap- HTC technology with Hawaii’s unique biodiver- Briefl y, dense culture aliquots (3 mL) were spot- sulate single cells directly from environmental sity and expertise in marine natural products ted individually onto aluminum plates and al- samples has been developed. Th e microcapsules chemistry. Th e strategy involves the employ- lowed to air dry. Th e fi lm formed on each plate are then transferred into mini fermentation ment of HTC to isolate novel strains of marine produces a distinct FT/IR spectrum due to the columns for growth and microcolony devel- microbes, which will be screened for the pres- composition of carbohydrate, and pro- opment. Th e column is perfused with culture ence of secondary metabolites exhibiting anti- tein. Th e FT/IR signature essentially produces a medium that contains concentrations tumor and anti-infective bioactivities. Qualifi ed metabolic fi ngerprint of each microbial isolate similar to those seen in the natural environment hits will then be dereplicated and undergo and thus can be utilized to dereplicate strains from which the cells were collected (i.e., fi ltered structure determination at the University of by comparison of historical reference spectra sea water, diluted sponge extracts). One of the Hawaii (Manoa). from known strains. Unique cultures were then advantages of using this approach is the abil- selected, and their 16S small subunit ribosomal ity to enrich for slow-growing species. Th e use HIGH THROUGHPUT sequenced in the case of bacteria or the of fl uorescence-activated cell sorting (FACS) CULTIVATION OF MARINE internal transcribed spacer (ITS) sequenced in enables the discrimination of slow-growing mi- SAMPLES FROM HAWAII the case of fungi for higher-resolution taxo- crobes retained in the microcapsules from fast- Samples of the sponge, Mycale armata (Fig- nomic identifi cation. Cultures of selected mi- growing cells that overgrow and burst the mi- ure 1A), were collected from Kaneohe Bay crobes were then scaled up to a volume of 1 L crocapsule. Th is method has been suggested to (Oahu, Hawaii), along with sediments sampled to generate suffi cient biomass for screening. be suitable for massively parallel cultivation of from various (i.e., rubble, sea As a parallel approach to HTC, environ- microorganisms for natural-product screening grass beds, a ). Th e sponge and sedi- mental samples were plated onto marine agar and drug discovery (Keller and Zengler, 2004). ment samples were homogenized, and the as- (traditional isolation methodology) to com-

Oceanography Vol. 19, No. 2, June 2006 121 Figure 1. High-throughput cultivation of the sponge Mycale armata: (A) freshly collected specimen; (B) separation of bacterial cells by density gradient centrifugation (bacterial band indicated with an arrow); (C) encapsulation of single bacterial cells and visualization by fl uorescence after SybrGreen™ (scale bar = 20 µm); (D) Flow cytogram of microcapsules after 5 weeks of incubation; and (E) bacterial colony growing within microcapsule (scale bar = 20 µm).

pare with the set of strains isolated using HTC teria classes, one to the Bacteroidetes, and three and used for cytotoxity and anti-infective activ- technology. Similarly, for fungal strains, the to the Bacilli group. Novel strains were defi ned ity screening. samples were plated directly onto R2A medium as those showing ≤ 98 percent nucleotide se- Th e cytotoxicity testing was performed with with penicillin and streptomycin (50 µg mL-1), quence identity to the closest relative in public three cell lines that were selected by the Na- respectively, to prevent bacterial growth and databases. Strains with identical gene sequenc- tional Cancer Institute (NCI) for pre-screening, incubated for 30 days at 30°C. es were considered redundant, and only one prior to consideration of extracts for evalua- was advanced for screening. tion in the more comprehensive NCI panel of BIOACTIVE MOLECULE DISCOVERY On the basis of this criterion, 42 percent 60 cell lines. Th e pre-screen cell lines are MCF-7 To identify the selected strains, DNA was of the HTC strains were novel as compared to (breast), SF268 (central nervous system) and isolated from each culture followed by PCR 7 percent using the traditional agar plate isola- H460 (small-cell lung). Th e assay was conduct- (Polymerase Chain Reaction) amplifi cation and tion technique (Table 1). All of the strains were ed at a standard screening concentration of of a 500 bp small subunit ribosomal then grown in 1 L of marine broth at 30°C and 25 µg mL-1 crude extract, in triplicate in 96 well RNA gene fragment (16S rDNA). Sequence data 70 rpm of constant agitation for a period of format using MTT (yellow tetrazolium salt) as were used for construction 48 hours. Th e cultures were then pelleted by an indicator of cell viability. using the neighbor-joining method (Figure 2). centrifugation and yielded ~ 3 g wet weight of Th e anti-infective screen was carried out Phylogenetic analysis revealed that most strains cellular material for extract preparation and by assaying for the growth inhibition of three belonged to the gamma and alpha Proteobac- screening. Methanolic extracts were prepared target species: Escherichia coli, Staphylococcus

122 Oceanography Vol. 19, No. 2, June 2006 CMMED 223 CMMED 182 CMMED 230 CMMED 222 CMMED 218 Sulfitobacter sp. BIO-7

CMMED 069 Sulfitobacter pontiacus alph CMMED 227 sp. JL-132 a CMMED 225 Ruegeria algocolus ATCC 51440 pr Roseobacter sp. RED85 ote

CMMED 187 ob CMMED 185 a

CMMED 228 cte CMMED 220 r

Rhodobacteraceae bacterium No. 63 i CMMED 191 a Pseudovibrio denitrificans CMMED 183 CMMED 189 Caulobacter leidyi Rhodovibrio sodomensis Rhodospirillaceae bacterium No. 71 CMMED 215 CMMED 217 CMMED 221 CMMED 074 CMMED 182 CMMED 224 CMMED 226 CMMED 219 CMMED 186 CMMED 213 CMMED 229 g Pseudoalteromonas sp. ws17 amm CMMED 216

CMMED 073 a

CMMED 190 p r

Shewanella sp. GSWBW-H27M o CMMED 071 te CMMED 072 ob

Vibrio fischeri a c CMMED 067 Psychrobacter glacincola te r i

CMMED 065 Psychrobacter pacificensis a CMMED 078 CMMED 075 CMMED 068 CMMED 181 CMMED 214 CMMED 212 CMMED 188 CMMED 066Stenotrophomonas maltophilia

Bacteroidetes bacterium PM13 Bacteroidetes CMMED 192

CMMED 075 CMMED 077 Bacilli CMMED 070 Planococcus citreus

0.05

Figure 2. Neighbor-joining phylogenetic tree constructed from a 500 bp gene fragment derived from 16S sequencing of the bacterial isolates listed in Table 1. Some of the strains showed 100 percent sequence identity to the cultivated isolates and therefore share the same position in the phylogenetic tree (anti-infective hits are shown in yellow and anti-tumor hits are shown in blue).

Oceanography Vol. 19, No. 2, June 2006 123 Table 1. Summary of sequence identity and screening data for the bacterial and fungal isolates.

CMMED Culture Closest Match %ID NCBI CMMED Culture Closest Match %ID NCBI BAY SPONGE MYCALE ARMATA HTC Bacteria HTC Bacteria CMMED 224 Pseudoalteromonas sp. KM-Y92-001 99 CMMED 228 Rhodobacteraceae bacterium JC2049 98 CMMED 219 Pseudoalteromonas sp. 100 CMMED 188 Marinomonas protea 98 CMMED 220 Rhodobacteraceae bacterium No. 63 99 CMMED 189 Caulobacter leidyi 99 CMMED 225 Ruegeria sp. AS-36 98 CMMED 229 Pseudoalteromonas sp. 100 CMMED 221 Rhodospirillaceae bacterium 99 CMMED 190 Shewanella sp. GWS-BW-H27M 99 CMMED 222 Sulfi tobacter pontiacus 98 CMMED 191 Pseudovibrio denitrifi cans 100 CMMED 223 Sulfi tobacter sp. BIO-7 98 CMMED 192 Bacteroidetes bacterium PM13 99 Agar Plate Bacteria Agar Plate Bacteria CMMED 066 Stenotrophomonas sp. 100 CMMED 071 Alteromonadaceae bacterium P3 99 CMMED 067 Psychrobacter glacincola 100 CMMED 075 Agrobacterium agile 99 SEA GRASS SEDIMENT CMMED 076 Bacillus acetylicum 99 HTC Bacteria CMMED 077 Bacillus sp. 99 CMMED 212 Marinomonas sp. 6-2 99 CMMED 078 Pseudomonas sp. 99 CMMED 226 Pseudoalteromonas sp. KM-Y92-001 100 CMMED 065 Psychrobacter pacifi censis 100 CMMED 213 Pseudoalteromonas sp. 100 CMMED 069 Sulfi tobacter pontiacus 100 CMMED 214 Marinomonas sp. 6-2 99 CORAL SEDIMENT CMMED 227 Roseobacter sp. JL-132 99 HTC Bacteria CMMED 215 Rhodospirillaceae bacterium No. 71 98 CMMED 181 Marinomonas sp. 100 CMMED 216 Pseudoalteromonas sp. A28 98 CMMED 182 Sulfi tobacter pontiacus 98 CMMED 217 Rhodospirillaceae bacterium 98 CMMED 183 Caulobacter leidyi 99 CMMED 218 Sulfi tobacter pontiacus 98 CMMED 184 Pseudoalteromonas sp. KM-Y92-001 100 Agar Plate Bacteria CMMED 185 Roseobacter sp. RED85 98 CMMED 072 Shewanella frigidimarina 99 CMMED 186 Pseudoalteromonas sp 100 CMMED 073 Shewanella denitrifi cans 97 CMMED 187 Roseovarius sp. 2S5-2 98 CMMED 074 Pseudoalteromonas sp. 100 CMMED 230 Roseobacter sp. LA7 98 CMMED 070 Planococcus citreus 100 Agar Plate Bacteria Agar Plate Fungi CMMED 068 Marinomonas sp. 100 CMMED 231 Trichoderma aureoviride 97 CMMED 232 Phialophora mustea 98 Anti-infective CMMED 233 Exophiala pisciphila 98 Anti-cancer CMMED 234 Hypomyces aurantius 93 CMMED 235 Aspergillus elegans 96 CMMED 193 Mycospharaella macrospora 99 CMMED 194 Fusarium sp. 98 CMMED 195 Eupenicillum javanicum 99 CMMED 196 Fusarium sp. 98

124 Oceanography Vol. 19, No. 2, June 2006 aureus, and Candida albicans. Th e screening Amy Apprill and Mr. Jay Wheeler with sample the seas—Current status and microbiological im- plications. Applied and Biotechnology results from bacterial strains indicated that collection and the Molecular Diversity and Se- 59:25–34. nine hits were obtained from HTC strains and quencing Departments at Diversa Corporation. Rappé, M.S., S.A. Connon, K.L. Vergin, and S.J. Giovanno- ni. 2002. Cultivation of the ubiquitous SAR11 marine one from agar plate isolates. Among those ten, Th is research was supported by NOAA OHHI clade. 418:630–633. fi ve were novel strains (Table 1), indicating award number NA04OAR4600206, NSF COHH Wagner-Döbler, I., W. Beil, S. Lang, M. Meiners, and H. the potential of discovering isolates that pro- award number OCE04-32479, and NIEHS Laatsch. 2002. Integrated approach to explore the potential of marine microorganisms for the produc- duce novel secondary metabolites. Two fungal COHH award number P50 ES012740. tion of bioactive metabolites. Advances in Biochemi- strains exhibited anti-tumor and one anti-infec- cal Engineering and Biotechnology 74:207–238. Walsh, C. 2003. Where will new antibiotics come from? tive activity (Table 1). REFERENCES Nature Reviews Microbiology 1:65–70. Bull, A.T., J.E. Stach, A.C. Ward, and M. Goodfellow. 2005. Zengler, K., G. Toledo, M. Rappé, J. Elkins, E.J. Mathur, Marine : Perspectives, challenges, fu- J.M. Short, and M. Keller. 2002. Cultivating the uncul- CONCLUDING REMARKS ture directions. 87:65–79. tured. Proceedings of the National Academy of Sci- Th e use of improved cultivation approaches for Burja, A.M., B. Banaigs, E. Abou-Mansour, J.G. Burgess, ences [USA] 99:15,681–15,686. and P.C. Wright. 2001. Marine cyanobacteria-a Zengler, K., M. Walcher, G. Clark, I. Haller, G. Toledo, T. the discovery of marine natural products from prolifi c source of natural products. Tetrahedron Holland, E.J. Mathur, G. Woodnutt, J.M. Short, and M. marine microbes is of paramount importance 57:9,347–9,377. Keller. 2005. High-throughput cultivation of microor- Connon, S.A., and S.J. Giovannoni. 2002. High-through- ganisms using microcapsules. Methods in Enzymology for the development of new pharmaceuticals put methods for culturing microorganisms in 397:124–130. (Bull et al., 2005). In this case study, we have very-low-nutrient media yield diverse new marine ZoBell, C. 1946. Marine microbiology, a monograph on isolates. Applied and Environmental Microbiology hydrobacteriology. Chronica Botanica Company, shown that HTC technology can be used to 68:3,878–3,885. Waltham, MA, 240 pp. isolate novel microbial strains of microorgan- Davidson, S.K., S.W. Allen, G.E. Lim, C.M. Anderson, and M.G. Haygood. 2001. Evidence for the biosynthesis isms from Hawaiian marine environments, some of bryostatins by the bacterial symbiont “Candi- GERARDO TOLEDO ([email protected]) is of which produce metabolites that possess anti- datus Endobugula sertula” of the bryozoan Bugula Staff Scientist II, Molecular Diversity, Diversa Cor- neritina. Applied and Environmental Microbiology infective or cytotoxic activities. Dereplication 67:4,531–4,537. poration, San Diego, CA, USA. WAYNE GREEN is analyses are currently underway to ensure that Faulkner, D.J. 2002. Marine natural products. Natural Senior Research Associate, Molecular Diversity, Product Reports 19:1–48. only activities generated by novel bioactive mol- Diversa Corporation, San Diego, CA, USA. Giovannoni, S.J., H.J. Tripp, S. Givan, M. Podar, K.L. Ver- RICARDO A. GONZALEZ is Intern, Molecular ecules are advanced for further investigation. gin, D. Baptista, L. Bibbs, J. Eads, T.H. Richardson, M. Noordewier, M.S. Rappé, J.M. Short, J.C. Carrington, Diversity, Diversa Corporation, San Diego, CA, We are currently expanding the applicabil- and E.J. Mathur. 2005. streamlining in a USA, and Graduate Student, CICESE, Ensenada, ity of HTC to the exploration of marine eco- cosmopolitan oceanic bacterium. Science 19:1,242– Mexico. LEIF CHRISTOFFERSEN is Associate, E.O. 1,245. systems by enabling the use of diverse media Wilson Biodiversity Foundation. MIRCEA PODAR Keller, M., and K. Zengler. 2004. Tapping into microbial at very high dilutions; mimicking the natural diversity. Nature Reviews Microbiology 2(2):141–150. is Staff Scientist II, Bioinformatics, Diversa environment’s concentrations and diversity of Review. Corporation, San Diego, CA, USA. HWAI W. Lim, G.E., and M.G. Haygood. 2004. “Candidatus Endo- CHANG is Director, Molecular Diversity, Diversa carbon sources may enhance the growth and bugula glebosa,” a specifi c bacterial symbiont of the Corporation, San Diego, CA, USA. THOMAS development of heretofore uncultured species. marine bryozoan Bugula simplex. Applied and Envi- ronmental Microbiology 70:4,921–4,929. HEMSCHEIDT is Associate Professor, Department In addition, we are applying HTC technology Maldonado, L.A., J.E. Stach, W. Pathom-aree, A.C. Ward, of Chemistry, University of Hawaii, Honolulu, to other Hawaiian biotopes, including decay- A.T. Bull, and M. Goodfellow. 2005. Diversity of HI, USA. HENRY G. TRAPIDO-ROSENTHAL is cultivable actinobacteria in geographically wide- ing algal material and a broader array of marine spread marine sediments. Antonie Van Leeuwenhoek Associate Researcher, School of Ocean and metazoans and other invertebrates, as well as 87:11–18. Science and Technology, University of Hawaii, Mincer, T.J., P.R. Jensen, C.A. Kauff man, and W. Fenical. Honolulu, HI, USA. JAY M. SHORT is Principal, cultivating anaerobic microbial fl ora associated 2002. Widespread and persistent populations of a Microbial Solutions Inc., and Founder, Presi- with many diverse marine biotopes. Th e con- major new marine actinomycete taxon in ocean sediments. Applied and Environmental Microbiology dent and Chairman of E.O. Wilson Biodiversity tinued development of HTC technology shows 68:5,005–5,011. Foundation, and Founder, Diversa Corporation, promise for increasing the number of microbial Monks, N.R., C. Lerner, A.T. Henriques, F.M. Farias, D.D.S. San Diego, CA, USA. ROBERT R. BIDIGARE is Schapoval, E.S. Suyenaga, A.B. Da Rocha, G. Schwars- Director, Center for Marine Microbial cultures that can be probed for molecules with mann, and B. Mothes. 2002. Anticancer, antichemo- valuable biomedical applications. tactic and antimicrobial activities of marine sponges and Diversity, University of Hawaii, Honolulu, HI, collected off the coast of Santa Catarina, southern USA, and Principal, Microbial Solutions Inc., and Brazil. Journal of Experimental and Founder and Delegate, E. O. Wilson Biodiver- ACKNOWLEDGEMENTS Ecology 281:1–12. sity Foundation. ERIC J. MATHUR is Principal, Piel, J., D. Hui, G. Wen, D. Butzke, M. Platzer, N. Fusetani, Samples were collected under the auspices of and S. Matsunaga. 2004. Antitumor polyketide bio- Microbial Solutions Inc., and Consultant, J. Craig Scientifi c Collection Permit 2005-25, issued by synthesis by an uncultivated bacterial symbiont of Venter Institute and Synthetic Inc., the marine sponge Th eonella swinhoei. Proceedings of the Hawaii Department of Land and Natural and Delegate, E.O. Wilson Biodiversity Founda- the National Academy of Sciences [USA] 101:16,222– tion, and Founder, Diversa Corporation, San Resources, of Aquatic Resources. We 16,227. Diego, CA, USA. gratefully acknowledge the assistance of Ms. Proksch, P., R.A. Edrada, and R. Ebel. 2002. Drugs from

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