Comprehensive Investigation of Marine Actinobacteria Associated with the Sponge Halichondria panicea

Imke Schneemann, Kerstin Nagel, Inga Kajahn, Antje Labes, Jutta Wiese and Johannes F. Imhoff Appl. Environ. Microbiol. 2010, 76(11):3702. DOI: 10.1128/AEM.00780-10. Published Ahead of Print 9 April 2010.

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by scuba diving. By means of these results. * Corresponding author. panicea (85. which exhibit a broad range of bioactivities such as inhibition of enzyme activities and cell division and antiviral. MATERIALS AND METHODS Collection of sponges. Nocardiopsis. Cytophaga/Flavobacteria. In addition. The extracts from most of the cultures showed biological activities. Vol. 47. though representing only 3 to 20% of the sponge-associated bacterial community (41. anti-inflammatory. a sponge species living in coastal habitats worldwide (9). Actinobacteria. 122). Gammaproteobacteria.g. antimicrobial. So far. e.asm. 47. secondary metabolite profiling. 126). Imhoff* Kieler Wirkstoff-Zentrum (KiWiZ) at the Leibniz Institute of Marine Sciences (IFM-GEOMAR).00 doi:10. Among these. Germany. Although the majority of secondary metabolite-producing Actinobacteria originate from terrestrial habitats (101). especially concerning the relationships of the abundance of biosynthesis gene fragments to the number and diversity of produced secondary metabolites. Mailing address: Kieler Wirkstoff-Zentrum (KiWiZ) at the Leibniz Institute of Marine Sciences (IFM-GEOMAR). Am Kiel-Kanal 44. only a few substances were isolated from Actinobacteria associated with H. Members of this phylum account for approximately half of the bioactive secondary metabolites that have so far been discovered in bacteria (64). Among these. recent studies of marine Actinobacteria have revealed many new chemical entities and bioactive metabolites (13. 110. their biosynthetic potential for secondary metabolite production. ᰔ Published ahead of print on 9 April 2010. American Society for Microbiology. 24106 Kiel.4. 3702 . Previous work demonstrated a phylogenetically diverse array of bacterial groups present in this sponge: representatives of Alphaproteobacteria. and 88 so far unidentified compounds were detected. the presence of PKS and NRPS genes is a good indicator for the selection of strains to isolate new natural products. 50. p. Antje Labes. † Supplemental material for this article may be found at http://aem . 122 different substances were identified. and Planctomycetales were identified by means of a genetic approach (47. antitumor. panicea. Secondary metabolite profiles of 46 actinobacterial strains were evaluated. 123). and cardiovascular properties (77). We focused on Actinobacteria associated with Halichondria panicea Pallas (Porifera. a comprehensive investigation was performed with regard to phylogenetic strain identification. Halichondriida. 11 Comprehensive Investigation of Marine Actinobacteria Associated with the Sponge Halichondria paniceaᰔ† Imke Schneemann. Micrococcus.4Ј-trichloro-2Ј-hydroxydiphenylether and acyl-1-(acyl-6Ј-mannobiosyl)-3-glycerol produced by Micrococcus luteus (17). panicea and discuss the biological roles of identified small molecules in the sponge-associated community. By combining data about the phylogenetic characterization of the Actinobacteria associated with H..APPLIED AND ENVIRONMENTAL MICROBIOLOGY. These microorganisms are known to be a rich source of secondary metabolites (108). 122. panicea for the investigation of the metabolite pattern were collected in October 2009 at the Kiel Fjord. Demospongiae. possibly new metabolites than other strains. 103). Living specimens of H. low-GϩC-content Gram-positive bacteria. the antimicrobially active substances 2. samples of H. They gained great interest due to their association with a wide variety of microorganisms. Fax: 49-431-6004452. and panicea Pallas (Porifera. All Rights Reserved. Am Kiel-Kanal 44. and their chemical cytotoxic. and Johannes F. 51. Numerous studies concerning specific aspects of spongebacterium associations were accomplished using distinct methods for the evaluation of the microbial diversity (mostly molecular approaches) or the bioactivities (culture-dependent methods) or biosynthetic aspects (chemical analyses and molecular approaches) of secondary metabolites of the associated bacteria (19. Sponges are multicellular invertebrates and sessile filter feeders which are abundant in the oceans as well as in freshwater habitats (41).00780-10 Copyright © 2010. 76. 24106 Kiel. It was shown that strains in which either PKS or NRPS genes were identified produced a significantly higher number of metabolites and exhibited a larger number of unidentified. bioactivity determination. 30. E-mail: jimhoff@ifm-geomar. 54. All strains were phylogenetically identified by 16S rRNA gene sequence analyses and were found to belong to the genera Actinoalloteichus. Baltic Sea (Germany). and genetic exploration of biosynthetic genes. Halichondriida. Inga Kajahn. Phone: 49-431-6004450. the Deinococcus group. we attempt to close the gap of knowledge about Actinobacteria associated with H. Actinobacteria are the most promising bacterial group regarding secondary metabolite production. Halichondriidae) used in this study were collected several times from November 2002 to June 2004 at the Kiel Fjord. Demospongiae. Micromonospora. there is less comprehensive information about the integration of this knowledge into concepts for sponge-bacterium interactions based on small molecules. For the first time. Halichondriidae). Germany Received 30 March 2010/Accepted 1 April 2010 Representatives of Actinobacteria were isolated from the marine sponge Halichondria panicea collected from the Baltic Sea (Germany). Additionally. Jutta Wiese. June 2010. the presence of biosynthesis genes encoding polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in 30 strains was established. 100). No. Kerstin Nagel. Therefore. 3702–3714 0099-2240/10/$12. Betaproteobacteria. we present comprehensive insights into a great variety of produced natural products as well as their bioactivities.

Escherichia coli K-12 (DSM 498). An NRPS PCR product of the expected size was recovered from 24 strains (52%). The Actinobacteria strains isolated from H. Germany). and GQ863934 to GQ863945 and GU320045 (for PKS sequences). Chemical analyses of culture extracts. Reversed-phase HPLC experiments were performed by using a Phenomenex Onyx Monolithic C18 column (100 by 3. Chemical analyses of culture extracts and sponge extract. and subsequently resolved in methanol. Xanthomonas campestris (DSM 2405). These similarities give basic information on the presence of NRPS genes. 6 min. Pseudomonas syringae pv. PKS sequences with similarities to known PKS gene fragments were obtained from 13 (28%) of the Actinobacteria strains (see the supplemental material). To identify all strains. The supernatants were separated. Staphylococcus lentus (DSM 6672). All bacterial strains were screened for genes corresponding to the nonribosomal peptide synthetase (NRPS) adenylation domain and the polyketide synthase type II (PKS-II) KS␣ domain by the methods developed by Doekel and Marahiel and Metsa ¨-Ketela ¨ et al.02 to 0. comprising 39 strains. 100% solvent B. Identification of NRPS and PKS-II gene fragments. Similarities between the gene sequences of the investigated Actinobacteria and known sequences ranged from 47% (for a sequence from strain HB328 and the ketosynthase gene sequence from Streptomyces felleus BAF43343) to 100% (for a sequence from strain HB202 and a PKS sequence from Streptomyces sp.ebi. 73).000 rpm for 30 s. Assay mixtures were prepared by transferring 10-␮l aliquots of methanolic solutions of extracts or pure substances into two wells of a 96-well microtiter plate and evaporating the solvent in a vacuum centrifuge. Antimicrobial activities of culture extracts. with 40 of 46 strains producing detectable amounts of These crude extracts were used for the chemical analysis. 100 by 21. solvent B) gradient with 0. Pseudomonas fluorescens (NCIMB 10586). 16S rRNA gene sequence analyses were carried out according to the protocol of Thiel et al. panicea.31 ppm for protons and ␦C 49. The PCR products had expected sizes of 250 to 270 bp (for NRPS gene fragments) and 580 to 620 bp (for PKS gene fragments). (110). 122 different substances were identified from the secondary metabolite patterns of the analyzed Actinobacteria strains by using HPLCMS. indicating multiple NRPS gene clusters. Extraction of sponge specimens. Nucleotide sequence accession numbers. PCR products for both PKS-II and NRPS genes were detected. the transformation of resazurin to resorufin was assayed by measuring the fluorescence at 560 nm after excitation at 590 nm. A much higher level of . and 200-␮l aliquots of the resulting suspensions were added to the wells. For selected substances. 5 g/liter) were diluted to an optical density at 600 nm (OD600) of 0. and GU213495 to GU213502 (for 16S rRNA sequences). panicea turned out to be potent producers of secondary metabolites. bacteria were grown in 300-ml Erlenmeyer flasks each containing 100 ml of medium GYM4 (10 g glucose. 46 isolates were classified as Actinobacteria belonging to the genera Actinoalloteichus. 4 min. 76. The PCR products of four strains showed more than one sequence with similar lengths in the electropherogram. Several extracts were subjected to preparative HPLC with a Phenomenex Gemini-NX C18 column (5 ␮m. For storage. Sequences of these PCR products were compared with NRPS and PKS sequences in the EMBL nucleotide database available at the European Bioinformatics Institute website by using the Basic Local Alignment Search Tool (blastx) (2). For 7 strains (15%). Extraction of liquid cultures. deuterium-labeled methanol [MeOH-D4]).2 mg/ml phosphate-buffered saline) was added to each well and the plate was incubated at 28°C until the color of the negative control changed from blue to pink. In 16 strains (35%). and 20 of these products had sequences similar (40 to 96%) to known NRPS gene fragments. and the suspension was plated onto five different media according to the method of Wiese et al. Dereplication of substances was realized by comparison of mass spectrometry (MS) and UV data obtained by high-performance liquid chromatography (HPLC)-UV/MS analyses. Antimicrobial assays were performed with the following test organisms: B.. S. GQ863931 to GQ863933. Cultivation of liquid cultures. 60% solvent B. Incubation was at 22°C in the dark. 10 ␮l of a resazurin solution (0. Germany) at 13. Afterwards the homogenate was stirred overnight at room temperature.20 mm) and variable gradients of eluents H2O (solvent A) and MeCN (solvent B). subtilis (DSM 347). (124).05. pH 7. Micrococcus. Bruker Daltonics). panicea. with members of the last genus. 5% solvent B.2) for 5 days at 28°C with shaking (at 120 rpm) in the dark. Staufen. BAH68110). 110 Å. dried. aptata (DSM 50252). Micromonospora. Germany) at 13. being the most abundant (Table 1). Reinfeld. dried in vacuo. High-resolution mass spectra were acquired on a benchtop time-of-flight spectrometer (MicrOTOF. Ralstonia solanacearum (DSM 9544). (124) using Bacillus subtilis (DSM 347). The entire culture broth (100 ml) was extracted by homogenization with 50 ml ethyl acetate by using an Ultra-Turrax T25 basic disperser (IKA-Werke GmbH and Co. Nocardiopsis. In total. neither NRPS nor PKS gene fragments were detected. Each culture was inoculated separately with a 1-cm2 piece from a culture grown on a GYM4 agar plate for 4 weeks at 28°C in the dark. C. E.000 rpm. 1 liter water. 2010 COMPREHENSIVE INVESTIGATION OF MARINE ACTINOBACTERIA 3703 Isolation and identification of antimicrobially active Actinobacteria. For evaluation of the cell viability. Pure cultures were obtained by subsequent plating steps on tryptic soy broth (TSB) medium A (with 10 g/liter NaCl). lentus (DSM 6672). pure cultures were kept at Ϫ80°C using the Cryobank System according to the instructions of the manufacturer (Mast Diagnostica GmbH.0 ppm for carbons. 2 ml/min) on a VWR Hitachi Elite LaChrom system coupled to an electrospray ionization (ESI) ion trap detector (Esquire 4000. glabrata (DSM 6425). Comparison of the 16S rRNA gene sequences of the spongederived strains was performed using the Basic Local Alignment Search Tool (nucleotide blast) with the EMBL nucleotide database available at the European Bioinformatics Institute website (http://www. GQ863946 to GQ863965 (for NRPS sequences).VOL. Overnight cultures of the test organisms in TSB medium (tryptic soy broth [Difco]. These crude extracts were used for antimicrobial assays and chemical analyses. All actinobacterial strains were analyzed for the presence of PKS-II and NRPS genes (Table 1. 4 g yeast extract. NMR solvent. RESULTS Isolation and identification of Actinobacteria from H. 12 g/ (2) and the Ribosomal Database Project II (RDP-II) website (21). Antibiotic activities of the living cultures were determined by an overlay assay as described by Wiese et al. The nucleotide sequence data reported in the present study were deposited in the GenBank nucleotide sequence database under the accession numbers GQ863900 to GQ863929. Staufen. and Streptomyces. Antibiotically active strains were selected for the present study.00 mm) and applying an H2O (solvent A)-acetonitrile (MeCN. GU213489 to GU213493. (24. The resulting supernatant was separated from the cell residue. a 1H nuclear magnetic resonance (NMR) analysis was carried out. and subsequently resolved in 5 ml methanol. Approximately 1 g of sponge material was macerated in sterilized habitat water. Identification of NRPS and PKS-II gene fragments. and Erwinia amylovora (DSM 50901). The resulting chemical data were compared with data from the Antibase (63) and the Chapman & Hall/CRC Chemical Database Dictionary of Natural Products (15). see also the supplemental material). For the analyses of secondary metabolite profiles. flow. UV. NaCl. and NMR techniques (Tables 2 and 3). coli K-12 (DSM 498). but correlation to specific metabolites is limited due to the lengths of the PCR fragments. and Candida glabrata (DSM 6425) as test strains. 4 g malt extract. Axia packed. After incubation for 15 h at 600 rpm and 28°C. Bruker Daltonics) with positive ESI. Among a large number of bacteria isolated from the sponge H. Approximately 100 g wet sponge tissue was extracted by homogenization with 500 ml methanol by using an Ultra-Turrax T25 basic disperser (IKA-Werke GmbH and Co. NMR spectra were recorded on a Bruker AV 600 spectrometer (at 600 and 150 MHz) by using the signals of the residual solvent protons as internal references (chemical shift ␦H 3. The resulting values were compared to those for a positive control (100 ␮g/well chloramphenicol for bacteria and 200 ␮g/well cycloheximide for Candida) and a negative control (no compound) on the same plate.1% HCOOH added to both solvents (gradient program: 0 min.. The numbers and diversity of produced compounds indicated by the metabolite profiles were evaluated with respect to the presence of PKS and NRPS biosynthetic genes.

Among the 16 strains which gave no hints of the presence of PKS-II or NRPS . pep Other. arom pk. no metabolite could be detected. Identified compounds are listed in Tables 2 and 3. a total of 108 different substances were identified. ENVIRON. pep Other Other Other. aromatic polyketide. Other arom pk arom pk pep pep X X X X X X X PKS fragment PKS and NRPS fragmentse NRPS fragment NRPS fragment NRPS fragment NRPS fragment NRPS fragment NRPS fragment NRPS fragment PKS and NRPS fragments PKS and NRPS fragments NRPS NRPS fragment NRPS fragmente e Other Other. pep Other. peptide. other.5%.3704 SCHNEEMANN ET AL. In the 30 Actinobacteria strains with positive PCR results for PKS genes (13 strains) and/or NRPS genes (24 strains). while 17 strains (57%) produced substances (a total of 88 compounds) that have not been identified so far and are probably new natural products (Table 1). pep Other. Other. Strains exhibiting no PKS-II or NRPS amplicons synthesized only a small spectrum of compounds. and chemical analyses Strain Most closely related type straina 16S rRNA gene sequence accession no. mainly antimycin derivatives. Pep. diversity of natural products was found in strains with proof of PKS or NRPS genes than in those with negative PCR results (23 different structural groups versus 7 different structural groups) (Fig. rufus LMG 20087 Streptomyces scabiei ATCC 49173 Streptomyces sampsonii ATCC 25495 Streptomyces platensis JCM 4662 Streptomyces fulvorobeus LMG 19901 Streptomyces griseus ATCC 25497 Streptomyces fulvorobeus LMG 19901 Streptomyces griseus ATCC 51928 Streptomyces mediolani LMG 20093 Streptomyces sampsonii ATCC 25495 Streptomyces fulvorobeus LMG 19901 Streptomyces sampsonii ATCC 25495 Streptomyces luridiscabiei S63 Streptomyces atroolivaceus LMG 19306 Streptomyces fulvorobeus LMG 19901 Streptomyces griseus ATCC 25497 Streptomyces intermedius DSM 40372 Streptomyces griseus JCM 4623 Streptomyces koyangensis VK-A60 Streptomyces sampsonii ATCC 25495 Streptomyces fulvorobeus LMG 19901 Streptomyces mediolani LMG 20093 Streptomyces pulveraceus LMG 20322 Streptomyces mediolani LMG 20093 Micromonospora matsumotoense IMSNU 22003 Micrococcus luteus ATCC 4698 Streptomyces mediolani LMG 20093 Micromonospora matsumotoense IMSNU 22003 Micromonospora matsumotoense IMSNU 22003 Actinoalloteichus cyanogriseus C7654 Streptomyces platensis JCM 4662 Streptomyces odorifer DSM 40347 Streptomyces fulvorobeus LMG 19901 Streptomyces coelescens ICSSB 1021 Streptomyces sanglieri A5843 Streptomyces fulvorobeus LMG 19901 Micromonospora aurantiaca DSM 43813 Streptomyces griseus ATCC 2549 Nocardiopsis alba DSM 43377 D63872 Z76686 D63871 Z76688 Z76688 D63872 AB045882 AJ781351 D63862 D63871 AB045882 AJ781331 D63872 AJ781331 AF112160 AJ781354 D63871 AJ781331 D63871 AF361784 AJ781320 AJ781331 D63872 Z76686 AB045872 AY079156 D63871 AJ781331 AJ781354 AJ781377 AJ781354 AF152109 AF542073 AJ781354 AF152109 AF152109 AY094367 AB045882 Z76682 AJ781331 AF503496 AY094363 AJ781331 X92604 D63872 X97883 PKS and NRPS fragments Other Other Other Other X X PKS and NRPS fragments NRPS fragment NRPS fragment NRPS fragment PKS and NRPS fragmentse Other Pep Other Other Other. APPL. e A PCR product(s) in the expected size range was obtained. MICROBIOL. arom pk Other. Other Other. Affiliation of the obtained sequence to known genes was not possible. In only one of these strains. pep X X X X X NRPS fragment NRPS fragment NRPS fragment PKS and NRPS fragments PKS fragment PKS fragment PKS fragment NRPS fragment PKS fragment PKS fragment Other. Actinobacteria from H. d X indicates the occurrence of a new compound. detection of PKS-II and NRPS gene fragments. Other Other Other. NCBI accession number. secondary metabolite(s) other than a peptide or aromatic polyketide. panicea: identification. TABLE 1. arom pk Other Other X X X X X X The analyzed sequences from each strain and the corresponding type strain had a level of similarity greater than or equal to 98. pep Other Other Other Pepf Other Other.b Gene fragment(s) identified Chemically identified product(s)c Detection of new compound(s)d HB062 HB084 HB094 HB095 HB096 HB099 HB100 HB101 HB102 HB105 HB107 HB108 HB110 HB113 HB114 HB115 HB116 HB117 HB118 HB122 HB132 HB138 HB140 HB142 HB149 HB156 HB157 HB181 HB184 HB200 HB202 HB238 HB241 HB243 HB253 HB254 HB272 HB274 HB288 HB291 HB298 HB320 HB328 HB375 HB381 HB383 a b c Streptomyces griseus ATCC 25497 Streptomyces intermedius DSM 40372 Streptomyces sampsonii ATCC 25495 Streptomyces rutgersensis DSM 40077 Streptomyces rutgersensis DSM 40077 Streptomyces griseus ATCC 25497 Streptomyces platensis JCM 4662 Streptomyces libani subsp. 1). Elucidation of the structures of these compounds is in progress. f Proven NRPS biosynthetic pathway.

VOL. UV MS. panicea-associated actinobacterial strains positive for a PKS and/or NRPS gene fragment by PCR No. HB288 HB157 HB157 HB157. cytotoxic. Identified metabolites from H. cytotoxic Antibacterial Active against Gram-positive bacteria and brine shrimp Cytotoxic NDa Acaricidal Active against Gram-positive bacteria. MS. MS. HB181. UV. UV MS. NMR MS. NRPS-specific PCR UV UV UV UV UV MS. MS. cytotoxic Pepsin inhibitor ␤-Lactamase inhibitor ND Antifungal Antifungal Antibacterial. MS. MS. MS. inhibitor of ATP-citrate lyase Inhibitor of ATP-citrate lyase Dereplication analyses MS. 2010 COMPREHENSIVE INVESTIGATION OF MARINE ACTINOBACTERIA 3705 TABLE 2. UV. MS. MS. HB291 HB117. MS. fungi. antiviral. NRPS-specific PCR UV UV. UV UV UV UV UV UV UV UV UV UV UV. HB288 HB157. HB156 HB156 HB156 HB156. MS. MS. MS. UV MS. NMR MS. MS. antiprotozoan. NMR MS. 1 2 3 4 5 6 7 8 9 10 11 12 13 HB062 HB100 HB100 HB101 HB101 HB102 HB107 HB107 HB107 HB113. MS. UV MS. UV UV UV UV UV. HB181. HB288 Streptazone B1 (90) Venturicidin A (95) Irumamycin (83) Bundlin A (115) Neoenactin B1 (98) Ashimycin A (112) Validamycin D (42) N-Demethylstreptomycin (31) Detoxin E1 (84) Antibiotic X 14873A (67) Valistatin (58) Phencomycin ethyl ester (91) Pepstatin BU (5) N-Acetylglycylclavaminic acid (6. NRPS-specific PCR UV UV UV. MS.8isoquinolinedione (70) 5-(6Ј-Methyl-7Ј-oxo-octyl)-SH-furan-2-one (76) Polyene with 7 or 8 double bonds Fredericamycin A (117) Antimycin A4 (8) Antimycin A3 (8) Antimycin A2 (8) Alkaloid Macrolide Lactone Macrolide Hydroxamic acid Aminoglycoside Aminoglycoside Aminoglycoside Depsipeptide Polyether Tripeptide Phenazine Peptide ␤-Lactam Glycoside Polyene Macrolide Lactone Sesquiterpene ketone Quinone Lactone Polyene Aromatic polyketide Macrolide Macrolide Macrolide MS. NMR Continued on following page . 76. UV MS. UV MS. photosensitizer Pepsin inhibitor Pepsin inhibitor Antifungal Antibacterial. MS. MS. NMR MS. insecticide. UV. NRPS-specific PCR UV UV. neuromuscular blocking agent. UV MS. NRPS-specific PCR UV. 92) Isovaleryl-6-deoxy-␣-L-talopyranoside (11) Polyene with 8 or 9 double bonds Galbonolide A (28) 5-Hydroxy-3-(1-hydroxy-2-methylpropyl)4-methyl-2(5H)-furanone (14) Albaflavenone (36) 3. MS. MS. polyene Porphyrin Peptide Peptide ␤-Lactam antibiotic Nucleoside analog Dipeptide Lactone Peptide Macrolide Aminoglycoside Polyether Activity(ies) Antibacterial Phytotoxic Zinc chelator. HB140 HB113 HB113 Source(s) Compound (reference) 6ٞ-O-␣-D-Mannopyranosylmannosidostreptomycin (45) Oxazolomycin (53) Coproporphyrin (113) Pepstatin AC (5) Pepstatin CU (5) Clavamycin D (57) 4-Thiouracil (25) Bovinocidin (4) Trihomononactic acid (93) Antibiotic NA 22598A1 (62) 3-O-(␣-L-Mycarosyl)erythronolide B (71) Gualamycin (114) Antibiotic X 14931A (120) Chemical group Aminoglycoside ␤-Lactone. HB181. antifungal Insect chitinase inhibitor Insecticide Antifungal Antibacterial. MS. chitinase and phosphatase inhibitor Antibacterial Active against Gram-positive bacteria Cytotoxic Antifungal Cytotoxic generator of free radicals Inhibitor of ATP-citrate lyase Cytotoxic. MS. UV MS. UV MS. MS.4-Dimethyl-7-(methylamino)-5. antifungal. MS. UV MS. and protozoa ND Antibacterial. HB291 HB117. antiparasitic Cytotoxic Antifungal Antifungal Active against Gram-positive bacteria and fungi Antifungal Antibacterial Antifungal Antimicrobial Cytotoxic Active against Gram-positive bacteria Aminopeptidase M inhibitor Antibacterial. cytotoxic Antibacterial. MS. MS. NRPS-specific PCR UV UV UV 14 15 HB113 HB113 17-Methylenespiramycin I (68) Streptomycin (116) Macrolide Aminoglycoside MS. HB328 Antibiotic M-2846 (105) 1-Epimanzamine D (127) Antibiotic A 75943 (75) N-Didemethylallosamidin (81) Methylallosamidin (99) Allosamidin (99) Dihydromaltophilin (34) Senacarcin A (79) Pyrocoll (22) Macrolide Alkaloid ␦-Lactone Aminoglycoside Aminoglycoside Aminoglycoside Macrolactam Phenazine Alkaloid MS. MS. MS. UV. HB291 HB156 HB157 HB157. MS. HB291 HB132 HB132 HB138 HB138 HB138 HB138 HB138 HB138 HB138 HB138 HB140 HB140 HB140 HB142. UV 16 17 18 19 20 21 22 23 24 HB113 HB113 HB113 HB113 HB113 HB113 HB117. MS. HB272 HB113. MS. UV. UV MS. HB181 HB157. NMR 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 HB117. phospholipase D inhibitor Active against Gram-positive bacteria Antibacterial. cytotoxic Bone resorption inhibitor Chitinase inhibitor. UV.

UV. UV.6-dimethyl-8octyl-4. UV. UV MS.8R)-8-Ethyl-7-hydroxy2. UV MS. NMR MS. UV. NMR MS.5-dioxonan-3yl͔-3-formamido-2-hydroxybenzamide (37) Blastamycin (37) Kitamycin A (39) 5-{3-͓(3-Formamido-2hydroxybenzoyl)amino͔-2. MS. NMR MS. NMR UV. MS. NMR 52 53 54 55 56 57 58 59 HB157 HB157 HB181 HB181 HB181. UV MS. HB243 HB181 HB181 HB181 Antimycin A0 (8) Antimycin A5 (8) Histidinomycin (48) Nebramycin (111) Antibiotic X 14873G (67) Mycospocidin (78) 5-Hydroxy-4-oxonorvaline (52) 3-Formamido-2-hydroxy-N͓(2R. TABLE 2—Continued APPL. MICROBIOL. UV MS.6. antiviral. NMR MS. UV MS. ichthyotoxin. MS. immunosuppressant ND MS.6S. MS. insecticide Cytotoxic. antifungal. MS. No. NMR MS.7R. UV 61 62 63 HB181 HB181 HB181 Macrolide (antimycin) Macrolide (antimycin) Macrolide (antimycin) Antifungal. ENVIRON. NRPS-specific PCR UV MS. NRPS-specific PCR UV UV MS. UV Continued on following page . UV. algicide Cytotoxic Cytotoxic Antifungal.9-dioxo-1. topoisomerase II inhibitor ND Active against Gram-positive bacteria Active against Gram-positive bacteria Active against Gram-positive bacteria Active against Gram-positive bacteria Active against Gram-positive bacteria Active against Gram-positive bacteria Active against Gram-positive bacteria Active against Gram-positive bacteria Plant growth regulator ND ND ND Antibacterial Antibacterial Inhibitor of dipeptidyl peptidase IV Antifungal Antibacterial ND Active against Gram-positive bacteria. UV. MS. UV 86 87 88 89 HB272 HB272 HB272 HB274 Daryamide C (7) Antibiotic A 58365B (80) Antibiotic SW 163B (106) Dihydro-4-(hydroxymethyl)-3-(3methylbutanonyl)-2(3H)-furanone (15) Polyketide Nucleoside Octadepsipeptide Furanone MS.5-dioxonan-7-yl} pentanoic acid (37) Bafilomycin D (60) Antibiotic BE 10988 (82) N-Methyltyrosyl-Nmethyltyrosylleucylalanin (3) Streptophenazin A (74) Streptophenazin B (74) Streptophenazin C (74) Streptophenazin D (74) Streptophenazin E (74) Streptophenazin F (74) Streptophenazin G (74) Streptophenazin H (74) Rotihibin B (32) Heptakis-(6-deoxy)-cyclomaltoheptose (15) Antibiotic GAI 3 (15) Antibiotic GAI 2 (15) Fortimicin KR1 (107) Massetolide K (26) Sulfostin (1) Fosfazinomycin A (35) Tobramycin (59) N-Acetylvalylleucylargininal (69) Hemipyocyanine (102) Macrolide Macrolide Peptide Aminoglycoside Polyether Nucleoside Amino acid Macrolide (antimycin) MS.8R)-7-hydroxy-2. MS. UV 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 HB181 HB200 HB200 HB202 HB202 HB202 HB202 HB202 HB202 HB202 HB202 HB238 HB238 HB238 HB238 HB238 HB238 HB243 HB243 HB243 HB243 HB272 Macrolide Thiazolylindole Peptide Phenazine Phenazine Phenazine Phenazine Phenazine Phenazine Phenazine Phenazine Peptide Glycoside Glycoside Glycoside Aminocyclitol Lipopeptide Alkaloid Aminophosphonic acid Aminoglycoside Peptide Phenazine Inhibitor of membrane ATPases. UV. MS.3706 SCHNEEMANN ET AL.6-dimethyl-4. UV. UV MS.7R. UV MS. UV MS. UV UV UV UV. UV MS. NRPS-specific PCRb MS. HB288 Compound (reference) Antimycin A1 (8) Chemical group Macrolide Activity(ies) Antiviral inhibitor of ATPcitrate lyase. UV MS. NRPS-specific PCR MS. NRPS-specific PCR MS.9-dioxo-1. UV MS.3S. UV 60 HB181 Macrolide (antimycin) ND MS. 51 Source(s) HB157.6S. UV MS. HB181. UV.5-dioxonan-3yl͔benzamide (37) N-͓(2R. NMR MS.8trimethyl-4. inhibitor of ubiquinolcytochrome C oxidoreductase Antifungal ND Antifungal Antimicrobial Active against Gram-positive bacteria Active against Gram-positive bacteria Antifungal ND Dereplication analyses MS.9-dioxo-1. NMR MS. inhibitor of ATP-citrate lyase Plant growth inhibitor ND MS. UV. UV. UV. NMR MS. UV. NMR UV.

In total. Seven (47%) yielded neither antimicrobial activity nor a gene fragment. X. NMR 95 96 97 98 99 100 101 102 103 104 105 106 107 108 a b HB298 HB298 HB320 HB320 HB320 HB320 HB328 HB328 HB328 HB375 HB375 HB375 HB375 HB375 Inthomycin A (118) Inthomycin B or C (40) Blanchaquinone (20) Antibiotic MA 1189 (102) Antibiotic K 502A (125) Homo-nonactyl-nonactoate (94) Futalosine (43) Antibiotic X 14889A (66) Mutalomycin (29) Diazepinomicin (18) Fortimicin AH (27) Rakicidin A (72) Rakicidin B (72) Polyene with ca. Just four glycosides were identified. HB118. antiviral. 2010 COMPREHENSIVE INVESTIGATION OF MARINE ACTINOBACTERIA TABLE 2—Continued 3707 No. 76. Of 20 Actinobacteria strains containing NRPS gene fragments. antibiotic K 502A and blanchaquinone. These two substances were secondary metabolites identified in strain HB243. Bioactivities of identified compounds known from literature. All strains were selected according to their antibiotic activities by an overlay assay. activities against E. More rarely. UV UV. UV. 1). activities against Gram-positive bacteria and fungi as well as cytotoxic activities were described for a number of these metabolites. Eleven (73%) of the 15 extracts with no antimicrobial activity did not show any NRPS or PKS gene fragment. antifungal. MS. MS. the other 19 structural groups were represented by only one or a few metabolites (Fig. because only small gene fragments were amplified. an attempt was made to correlate the structural properties of produced secondary metabolites with the presence of PKS-II and NRPS genes. 90 91 92 93 94 HB274 HB288 HB288 HB291 HB298 Source(s) Compound (reference) 4-Hydroxythreonine (121) Albopeptin A (49) PD 125375 (96) Lankamycin (33) Germicidin (86) Chemical group Amino acid Peptide Pyrrol. The identified substances belong to 23 different structural groups. Antimicrobial activities of culture extracts. coli (9%). UV ND. A PCR product of the estimated size was obtained. UV. MS. fluorescens (7%). Sequence similarity could not be detected. MS. we were not able to find any microbial activity. and aminoglycosides (13 compounds). MS. immunosuppressive. cytotoxic. no corresponding PKS products were found under the applied culture conditions. antibacterial. phenazines (13 compounds). MS. Only 3 of those 12 metabolite-producing bacteria were capable of the biosynthesis of natural products (a total of 11 compounds) not listed in any database used (Table 1). UV UV. one of these was heptakis-(6-deoxy)-cyclomaltoheptose. 11 were shown to produce one or more peptides. produced by strain HB238. not determined. 4 strains (25%) did not produce any detectable metabolite. peptides (16 compounds). P. MS. and algicidic). Frequent activities against B. UV UV UV UV UV UV UV NMR NMR PKS PKS-specific PCR MS. Even though we expected aromatic polyketides in 13 strains (Table 1) with a matching sequence for a PKS-II gene. UV MS. and E. MS. and HB157 produced the aromatic polyketide fredericamycin A and strain HB184 produced enterocin. 77 substances belonged to these 4 most prominent structural groups. UV.VOL. and C. Most frequent were polyketides (35 compounds). amylovora (9%) were observed. Strains HB062 and HB202 produced possibly new aromatic polyketides. The extracts revealed 18 different activity patterns ranging from no activity at all to activities against even seven of nine test organisms. genes. The results are summarized in Tables 2 and 3. inhibitor of porcine Naϩ/Kϩ-activated ATPase Antimicrobial. in some extracts. Subsequently. Strain HB320 produced two aromatic polyketides. R. a known lipopeptide which has been reported to be NRPS derived. On the other hand. 12 double bonds Polyketide Polyketide Anthraquinone Benzenediol Anthracycline Dilactone Nucleoside Polyether Polyether Alkaloid Aminocyclitol Lipopeptide Lipopeptide Polyene MS. Eight (53%) of these extracts additionally did not show any substance in the chromatogram (Tables 1 and 4).. In 10 of the strains yielding PCR products with the PKS-specific primers. MS. MS. amide Macrolide Pyrone Activity(ies) Antibacterial Antifungal Cytotoxic Antibacterial Retards germination of cress. We were able to detect the substances fosfazinomycin and tobramycin in the methanolic extract from H. herbicidal ND Antibacterial. For example. In 15 crude extracts.g. while in the remaining 12 strains. aromatic polyketides were identified in only 3 strains. For example. Chemical analysis of sponge extract. metabolites with exceptional activities. MS. as illustrated in Table 4. subtilis (44% extracts). lentus (33%). campestris (15%). glabrata (26%) were detected. cytotoxic 5-Lipoxygenase inhibitor ND ND Cytotoxic Active against Gram-positive bacteria Ionophore Antimicrobial Antibacterial Hypoxia-selective cytotoxic agent Cytotoxic Antifungal Dereplication analyses MS. Although specific prediction of products of NRPS and PKS genes according to the obtained sequences is problematic. a total of 27 compounds were identified. However. S. In particular. MS. only strain HB298 produced a single pyrone. only 31 (67%) of 46 culture extracts showed activity against at least one of the test strains (Table 4). MS. MS. syringae (9%). P. solanacearum (24%). panicea. Data on the biological activities of most of the secondary metabolites isolated from Actinobacteria and identified via HPLC-UV/MS analyses in this study were compiled by a profound literature search concerning reported biological activities (e. NRPS-specific PCR UV UV UV. the strains HB116. strain HB238 produced massetolide K (Table 2). like rotihibin B from . although the presence of PKS-II genes in these strains could not be confirmed. MS.

panicea-associated actinobacterial strains negative for a PKS and/or NRPS gene fragment by PCR No. UV MS. inhibitor of ubiquinolcytochrome C oxidoreductase Antifungal Antibacterial Antibacterial Inhibitor of ATP-citrate lyase Antifungal Antifungal Cytotoxic generator of free radicals Antibacterial. UV MS. UV MS. inhibitor of ATPcitrate lyase Inhibitor of ATP-citrate lyase Antiviral. NMR MS. UV MS.5-dioxonan-7-yl} pentanoic acid (37) Indole derivative Alkaloid Phenazine Macrolactam Phenazine Aromatic polyketide Aromatic carboxylic acid Macrolide (antimycin) MS. Alkaloid ND MS.3708 SCHNEEMANN ET AL.7R. HB118. HB096. not determined.5-dioxoan-7yl}fomate (15) Blastamycin (37) Urauchimycin A or B (46) Dehexylantimycin A1 (15) Antimycin A3 (8) Antimycin A2 (8) Antimycin A1 (8) Lactone Macrolide (antimycin) Cytotoxic ND a MS. UV. HB096. NMR MS. HB184 5-(6Ј-Methyl-7Ј-oxo-octyl)SH-furan-2-one (76) 3-Formamido-2-hydroxyN-͓(2R. UV . HB116. UV. UV 27 a HB241 ND. HB116. HB096. UV 25 26 HB184 HB184 Macrolide (antimycin) Macrolide (antimycin) ND ND MS. HB116. HB184 4 5 6 7 8 9 HB084. UV MS. HB116. antifungal. UV UV MS. HB118.6S. HB116. UV MS. MICROBIOL.9dioxo-1.9-dioxo-1. HB118. HB118 HB084.3S. HB118 HB095. UV MS. UV MS. HB116. cytotoxic. Source(s) Compound (reference) Chemical group Activity(ies) Dereplication analyses 1 2 HB084. HB184 HB084.9-dioxo1. UV MS. HB118. HB095. HB095. ENVIRON. HB096 HB095. HB184 3 HB084. APPL. HB116. NMR 18 19 20 21 22 23 24 HB122 HB122 HB122 HB122 HB184 HB184 HB184 Streptazone B1 or B2 (90) Saphenyl ester D (65) Xanthobaccin A (80) Aestivophoenin C (61) Enterocin (88) trans-Cinnamic acid (15) {8-Benzyl-3-͓(3formamido-2hydroxybenzoyl)amino͔2. HB096. HB095. HB184 HB084. UV MS. TABLE 3. antiparasitic Cytotoxic Antibacterial Antifungal Neuronal cell-protecting agent. NMR MS.6-dimethyl-4. HB184 HB084.8-trimethyl4. UV MS. UV. Identified metabolites from H. HB096. HB118. HB184 HB096 HB105 HB116.6-dimethyl-8-octyl-4. UV. HB095. HB118. UV MS. UV MS. ichthyotoxin.6.6-dimethyl-4. antiviral. HB096 HB095. UV Macrolide (antimycin) ND MS. antiprotozoan. antioxidant ND ND ND MS. HB118. HB116. UV. UV. NMR MS. HB116.5-dioxonan-7-yl} 3methylbutanoate (15) Antimycin (8) 5-{3-͓(3-Formamido-2hydroxybenzoyl)amino͔2.5-dioxoan3yl͔benzamide (37) {3͓(3-Formamido-2hydroxy-benzoyl) amido͔-2. NMR MS. HB118. HB118 HB122 Antimycin A0 (8) Gentamicin C1 (12) Gentamicin A2 (12) Antimycin A4 (8) Dihydromaltophilin (34) Polyene Fredericamycin A (117) Pyrocoll (22) Macrolide Aminoglycoside Aminoglycoside Macrolide Macrolactam Polyketide Aromatic polyketide Alkaloid Antifungal inhibitor of ATP-citrate lyase Antifungal ND Cytotoxic.8R)-7hydroxy-2. inhibitor of ATP-citrate lyase. HB184 HB084. UV. HB116. HB184 HB084. HB095.9dioxo-1. UV Macrolide (antimycin) Macrolide (antimycin) Macrolide (antimycin) Macrolide Macrolide Macrolide 10 11 12 13 14 15 16 17 HB084. NMR MS.

which seems to be a plant growth regulator. . antimicrobial activity was measurable although substances with described antibacterial or cytotoxic activities were not identified. the extract from HB100 showed activity against R. However. which was attributed to the oxazolomycin isolated from this strain. It may be assumed that a genome with a higher number of biosynthetic gene clusters is more likely to result in a positive hit in an NRPS/PKS gene screening approach. strain HB238. Within this study. strain HB132 showed activity against different bacteria.VOL. The multiple antimicrobial activities of some extracts may be explained by the presence of antibiotics with broad spectra of activities. as reflected in actinobacterial genomes (10. 44). Hence. venturicidin A and irumamycin. 76. and HB157 produced the polyketide fredericamycin A. For example. but only two antifungal substances. is not working in the case of aromatic polyketides with unusual molecular constructions. the chosen primer system. by comparing data on the antimicrobial activities of the extracts (Table 4) with the data for identified metabolites and their known bioactivities from the literature (Tables 2 and 3). and HB288 were selected for isolation of metabolites. The biosynthetic gene cluster for this substance showed an unusual KS␣ domain which is not amplified by the primer system used. These findings may indicate the presence of new antibacterial compounds. The majority of Actinobacteria-derived compounds are shown to be complex polyketides and nonribosomal peptides. 2010 COMPREHENSIVE INVESTIGATION OF MARINE ACTINOBACTERIA 3709 FIG. we were able to relate the antimicrobial activities of the extracts to the corresponding identified antimicrobial metabolites in 31 culture extracts (Table 4). showing the same activity patterns (Table 4). Extracts from HB157 and HB288 inhibited the growth of C. all bacteria—except a single isolate—that tested positive in the genetic prescreening approach were not only able to produce a higher number of secondary metabolites but also provided greater diversity of structural types and more antimicrobial activity than those strains with a negative result for PCR amplification of PKS-II and NRPS genes. For this reason. The activities of the pure substances against our test organisms were examined. for six strains. strains HB100. other metabolites and other biosynthetic pathways do exist. HB118. Sequence comparison of the applied PKS-II primers and the fredericamycin A gene cluster did not reveal any homology. were detected. 1. positive results in a PCR-based screening for NRPS and PKS-II genes not only provide evidence of the production of corresponding metabolites but also may indicate the existence of further metabolic pathways of secondary metabolite synthesis. Antimicrobial activities of identified metabolites compared to crude extract activities. nevertheless. an idea which is supported by the detection of a so far unidentified substance in the extract from strain HB132. glabrata. Therefore. in addition. Overall. For example. Furthermore. the strains HB116. Comparison of the numbers of secondary metabolite-producing strains (pie diagrams) and the numbers of resulting compounds (bars) among strains with a negative NRPS/PKS PCR result and those with a positive PCR result. For example. the lack of detectable NRPS or PKS-II gene fragments does not definitely prove the absence of the respective biosynthetic gene clusters. molecular screening for PKS and NRPS genes is a valuable tool for preselection of strains for secondary metabolite production. DISCUSSION Correlation between NRPS and PKS-II biosynthetic gene fragment occurrence and secondary metabolite pattern. It is already known from literature that this substance shows such activity. However. For example. the number of strains that did not produce any compound under the applied culture conditions was also significantly higher within the group with a negative result. were identified (Tables 2 and 4). The antimicrobial activities of the culture extracts were congruent with those of the purified substances. Another explanation may be the occurrence of many different substances with complementary activities. two antibacterial metabolites and. HB157. To prove the correlation between antimicrobial activities of the crude extracts and the presence of secondary metabolites. solanacearum. the cytotoxic compound 4-thiouracil were identified in strain HB107 (Table 2). albeit favorable for the great majority of known PKS-II genes. which is consistent with the presence of antimycins A0 to A4.

56. subtilis E. glabrata P. amylovora R.. of extracts with activity % of extracts with activity a b X X X X Xb X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X Xb X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X 15 33 b X X X X X X Xb X X X X X X X X X X X X 20 44 X X X X X X X X X X X X X 4 9 3 7 12 26 4 9 7 15 4 9 11 24 31 67 29 63 X indicates positivity for antimicrobial activity. Antimicrobial activity established by: Testing of extracts Description of identified compounds in the literature HB062 HB084 HB094 HB095 HB096 HB099 HB100 HB101 HB102 HB105 HB107 HB108 HB110 HB113 HB114 HB115 HB116 HB117 HB118 HB122 HB132 HB138 HB140 HB142 HB149 HB156 HB157 HB181 HB184 HB200 HB202 HB238 HB241 HB243 HB253 HB254 HB272 HB274 HB288 HB291 HB298 HB320 HB328 HB375 HB381 HB383 No. Actinobacteria display enormous potential to produce secondary metabolites within bacterium-invertebrate communities (16. e. syringae X. 38. the activities of the compounds identified in the extracts from sponge-associated Actinobacteria will be discussed from an ecological point of view. the ecological roles of secondary metabolites produced by these microorganisms are not well understood. campestris E. solanacearum APPL. coli P. Antimicrobial activities of extracts from actinobacterial strainsa Active against: Strain S. Though the abundance of microorganisms in marine sponges is well examined. in defense against antagonists. . fluorescens C.3710 SCHNEEMANN ET AL. It was hypothesized that they may be relevant in biological interactions. 109).g. Actinobacteria represent a large proportion of the microbial community compared to the marine bacterioplankton (41. lentus B. 47. Ecological roles of compounds: beneficial effects. Correlation between the biological assay results for crude extracts and those for purified substances was possible. In the following. MICROBIOL. In the close bacterium-sponge associations. ENVIRON. TABLE 4.

and HB288) and bafilomycin (60) (produced by strain HB181). Erhard for bioactivity assays. produced by a Micromonospora species (97). some of the substances identified in sponge-associated Actinobacteria are definitely produced within the sponge in sufficient amounts to display their biological activities. Depending on the environmental conditions. So ¨nnichsen for running and processing NMR experiments. This inevitably raises the question of whether sponge-associated Actinobacteria provide a number of metabolites whose production has been attributed to sponges before. the peptide thiocoraline. Actinobacteria defend themselves against fungi in their environment. 74. For example. K. and coproporphyrin. 2010 COMPREHENSIVE INVESTIGATION OF MARINE ACTINOBACTERIA 3711 108) and thus may contribute to the survival of the organisms involved. it is of special interest that the first chemical analysis of crude extract from H. we expect that most of the other compounds described in this work. They also may serve as protective agents against digestion of the bacteria by the sponge itself. and HB291). Up to now. 87. Economic Affairs and Transport of the state of Schleswig-Holstein (Germany) in the framework of the Future Program for Economy. panicea rather have beneficial effects. a novel alphaproteobacterium was identified as the primary pathogen (119). and HB138) may defend Actinobacteria and their host against other bacteria (29. An example of such a substance is oxazolomycin (produced by strain HB100). detrimental organisms range from pathogenic bacteria and fungi to macroorganisms which colonize the surface or prey on the sponge. Known potential virulence factors in sponges are produced by fungi. It remains unknown whether all of the substances produced by Actinobacteria in laboratory cultures are also synthesized in the sponge habitat. panicea against infectious agents or predators may be mediated by the substances which were identified in this study. like antimycin A1 (8) (produced by strains HB157. Ecological roles of compounds: deleterious effects. This study was supported by the Ministry of Science. One possible function of these compounds is the chemical defense of the host (41).g. including antimicrobial and cytotoxic effects. . because they showed broad-spectrum activities. strain HB243. the substances produced by Actinobacteria possibly could participate in maintaining the balance of microbial biofilms within and on the sponge by preventing growth of deleterious microorganisms. One example is a modulation of colony morphology by phenazines of Pseudomonas aeruginosa (23). and HB328). and whether the compounds will be produced in sufficient amounts to interact with the environment. These substances may be protective against different eukaryotic antagonists like protists or vertebrates. and daryamide C (7) (produced by strain HB272). and HB328) and other antiprotozoan agents such as antibiotic X 14931A (produced by strain HB113) may protect the bacteria against digestion by protozoans (22). which inhibits ATPase (60). Analyses of the strains HB117. panicea are cytotoxic: streptazone B1 (90) (produced by strains HB117. HB122. 116. In summary. produced by strain HB100 (113). and HB157). Fungi are competitors for nutrients and also produce antibacterial substances. Insecticides and other agents against vertebrates and invertebrates. what factors are essential for production. were described previously as products of sponges (coproporphyrin was also identified as a product of bacteria). Ohlendorf for fruitful discussions. So far. cyanobacteria. Phytotoxic compounds and algicides may prevent the sponge from periphyton algae (53. identified as Streptomyces fulvorobeus (127). HB291. HB122. All these activities may affect not only potential predators but also the sponge. In the case of a sponge.. Nearly all compounds from Actinobacteria strains isolated from H. fredericamycin A (117) (produced by strains HB116. HB122. Many of the secondary metabolites identified in the Actinobacteria associated with H. Through the production of antifungal agents like pyrocoll (produced by strains HB117. Biological sources of the substances. panicea. viruses. Here.VOL. Beside beneficial interactions. e. and bacteria of the genera Bacillus and Pseudomonas (119). Thus. 104). 76. 89). antioxidant agents may also play a role in the regulation of growth and the composition of biofilms in H. HB181. as well as streptomycin (116) (produced by strain HB113). only a small selected fraction of the secondary metabolites of Actinobacteria will be expressed at a given time. which was originally isolated from a Palaun sponge and was produced by strain HB113. The protection of H. HB118. Schumann for cultivation experiments. some bacterium-derived substances have been isolated from sponge tissue (55. An increasing number of compounds originally thought to be biosynthesized by the sponges are actually produced by sponge-associated bacteria (89). it is not expected that these compounds are produced at inhibitory levels. Within this context. Hence. Kohlmeyer-Yilmaz as well as F. In one case. it is important that microbial secondary metabolites have been proposed to act as signaling molecules at subinhibitory concentrations. Actinobacteria have not been described as sponge pathogens. which may be taken as evidence that their concentrations are sufficient to be biologically active in situ. and G. Therefore. HB122. many redox-active secondary metabolites may have additional and relevant properties independent from their antioxidant/radicalscavenging activities. HB181. panicea revealed the occurrence of antimicrobially active substances identified in the sponge-associated Micromonospora sp. HB113. if not all. Pyrocoll (produced by strains HB117. some of the produced substances may exhibit deleterious effects on the sponge. A specific cocktail of chemical substances that is strongly dependent on environmental factors is expected to occur. may defend the sponge from predators. In conclusion. strain HB181 synthesized bafilomycin D. 118). panicea that we have identified within our study were known as bacterial products. and HB328 revealed the presence of the cytotoxic compounds senacarcin A (79) and pyrocoll (22). but antibiotic activities and cell-cell-signaling functions have been reported as well. In only two cases are the reported results ambiguous: 1-epimanzamine D. ACKNOWLEDGMENTS We gratefully thank B. may have their roles in the complex sponge system. A.” However. which is cofinanced by the European Union (EFRE). Antioxidative agents like aestivophoenin C (produced by strain HB122) may shield the sponge against the oxidation of sensitive molecules (61) and protect the cell nucleus and the cell membrane from these “scavengers. and we assume that the secondary metabolites produced by Actinobacteria associated with H. Antibacterial agents like the streptophenazines (produced by strain HB202) and different streptomycins (produced by strains HB062.

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