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CASE STUDY High Th roughput Cultivation for Isolation of Novel Marine

BY GERARDO TOLEDO, WAYNE GREEN, RICARDO A. GONZOLEZ, LEIF CHRISTOFFERSEN, MIRCEA PODA, 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 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 have 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- however, the rate of discovery of novel com- found in extracts of marine are wood, 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 actinomycetes have yielded unacceptably high associated with these marine metazoans. small molecules. 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 cultivation bioactive compounds (Faulkner, 2002). For ex- tion of marine . However, this medium methods, which primarily cultivate only 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 As a result, most pharmaceutical companies species exhibited anti-bacterial, anti-tumor, environments; as such, strains isolated using 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 (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 recognized 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 (see Fenical, this issue). Bioactive compounds produce toxic compounds to prevent coloniza- high-carbon-content issue and the relatively are frequently associated with marine inver- tion by other non-benefi cial microbial species. short incubation times and have signifi cantly tebrates, including sponges, bryozoans, mol- Interestingly, some of the small molecules iso- improved the techniques for the isolation of lusks, and (Proksch et al., 2002). More lated from marine metazoans display a striking novel marine actinomycetes strains from sedi- recently, have been resemblance to prokaryotic-borne metabolites; ments. Th ey employed several concentrations recognized as a productive source of novel it has thus been suggested that the source of of organic carbon from diverse sources, longer secondary metabolites. To date, the majority many of these bioactives is in fact the symbi- incubation times, and the pretreatment of the of natural products of marine bacterial origin otic microorganisms (Piel et al., 2004). samples with a heat shock to enrich for -

100 Oceanography Vol. 19, No. 2, June 2006 forming microorganisms. As a result, novel UNIVERSITY OF HAWAII sociated microbial cells were separated using a lineages of marine actinomycetes have recently DIVERSA COLLABORATION Nycodenz cushion and diff erential centrifuga- been isolated and cultured from diverse marine Th e Hawaiian archipelago is a geographically tion (Figure 1B). Th e crude pellets of liv- . Extracts of some of these cultures isolated chain of oceanic islands with high bio- ing but uncultured microorganisms were then have yielded promising molecules, some of diversity and endemism, and is considered to encapsulated in the agarose microcapsules which have been advanced to pre-clinical trials 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 analy- diversity access and collaboration agreement Culture medium was prepared by diluting sis 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 microor- zoans, sea grasses, and ocean sediments. Th is fi ve weeks and sorted (Figure 1D) with a MoFlo ganisms from the marine . Th ese tech- discovery eff ort has been co-funded by the fl ow cytometry system (Cytomation) to array niques include various modifi cations to growth Oceans and Human Health Initiative of the individual -containing microcapsules media and end-point dilution methods using National Oceanographic and Atmospheric (Figure 1E) into 96-well plates containing ma- microtitre dish plate formats that allowed the Administration (NOAA) and the Centers for rine broth. Th e growth of most strains is not in- cultivation of the ubiquitous marine bacterial Ocean and Human Health of the National Sci- hibited by high marine broth carbon concentra- clade SAR11 (Connon and Giovannoni, 2002; ence Foundation (NSF) and the National Insti- tions, and its inclusion allows the generation of Rappe et al., 2002; Giovannoni et al., 2005). At tute of Environmental Health Sciences (NIEHS) the relatively large amounts of required Diversa Corporation (more information avail- of the National Institutes of Health. Th is col- for anti-infective and anti-tumor screening. able at http://www.diversa.com/), we have de- laborative research eff ort provides a unique Th e isolates were de-replicated via Fourier- veloped a high-throughput cultivation (HTC) opportunity to integrate Diversa Corporation’s transformed infrared (FT/IR) . technology that employs agarose microcapsules HTC technology with Hawaii’s unique biodiver- Briefl y, dense culture aliquots (3 mL) were spot- to encapsulate single cells directly from envi- sity and expertise in marine natural products ted individually onto aluminum plates and al- ronmental samples. Th e microcapsules are then chemistry. Th e strategy involves the employ- lowed to air dry. Th e fi lm formed on each plate transferred into mini fermentation columns ment of HTC to isolate novel strains of marine produces a distinct FT/IR spectrum due to the for growth and microcolony development. microbes, which will be screened for the pres- composition of carbohydrate, and pro- Th e column is perfused with culture medium ence of secondary metabolites exhibiting anti- tein. Th e FT/IR signature essentially produces a that contains concentrations similar tumor and anti-infective bioactivities. Qualifi ed metabolic fi ngerprint of each microbial isolate to those seen in the natural environment from hits will then be dereplicated and undergo and thus can be utilized to dereplicate strains which the cells were collected (i.e., fi ltered sea structure determination at the University of by comparison of historical reference spectra water, diluted sponge extracts). One of the ad- Hawaii (Manoa). from known strains. Unique cultures were then vantages of using this approach is the ability to selected, and their 16S small subunit ribosomal enrich for slow-growing species. Th e use of fl uo- HIGH THROUGHPUT sequenced in the case of bacteria or the rescence-activated cell sorting (FACS) enables CULTIVATION OF MARINE internal transcribed spacer (ITS) sequenced in the discrimination of slow-growing microbes SAMPLES FROM HAWAII the case of fungi for higher-resolution taxo- retained in the microcapsules from fast growing Samples of the sponge, Mycale armata (Fig- nomic identifi cation. Cultures of selected mi- cells that overgrow and burst the microcapsule. ure 1A), were collected from Kaneohe Bay crobes were then scaled up to a volume of 1 L Th is method has been suggested to be suitable (Oahu, Hawaii), along with sediments sampled to generate suffi cient biomass for screening. for massively parallel cultivation of microorgan- from various (i.e., rubble, sea As a parallel approach to HTC, environ- isms for natural-product screening and drug grass beds, a ). Th e sponge and sedi- mental samples were plated onto marine agar discovery (Keller and Zengler, 2004). ment samples were homogenized, and the as- (traditional isolation methodology) to com-

Oceanography Vol. 19, No. 2, June 2006 101 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 of 60 cell lines. Th e pre-screen cell lines are MCF-7 To identify the selected strains, DNA was the HTC strains were novel as compared to 7 (breast), SF268 (central nervous system) and isolated from each culture followed by PCR 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 25 of a 500 bp small subunit ribosomal then grown in 1 L of marine broth at 30°C and µg mL-1 crude extract, in triplicate in 96 well RNA gene fragment (16S rRNA). 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

102 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 isolated 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 103 Table1. 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

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

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