Marine Molecular Biotechnology Subseries of Progress in Molecular and Subcellular Biology Series Editor: Werner E. G. Muller

Progress in Molecular and Subcellular Biology Series Editors: W.E.G. Muller (Managing Editor), Ph. Jeanteur, I. Kostovic, Y. Kuchino, A. Madeira-Coelho, R. E. Rhoads

Springer-Verlag Berlin Heidelberg GmbH

Werner E.G. Müller (Ed.)

Sponges (Porifera)

With 87 Figures, 48 in Color

Springer

Professor Dr. WERNER E.G. MÜLLER Institut für Physiologische Chemie Abt. Angewandte Molekularbiologie Johannes Gutenberg-Universität Duesbergweg 6 55099 Mainz Germany

ISSN 1611-6119 ISBN 978-3-642-62471-1 ISBN 978-3-642-55519-0 (eBook) DOI 10.1007/978-3-642-55519-0

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Preface to the Series

Recent developments in the applied field of natural products are impressive, and the speed of progress appears to be almost self-accelerating. The results emerging make it obvious that nature provides chemicals, secondary metabo­lites, of astonishing complexity. It is generally accepted that these natural pro­ducts offer new potential for human therapy and biopolymer science. The major disciplines which have contributed, and increasingly contribute, to pro­gress in the successful exploitation of this natural richness include molecular biology and cell biology, flanked by chemistry. The organisms of choice useful for such exploitation, live in the marine environment. They have the longest evolutionary history during which they could develop strategies to fight suc­cessfully against other, invading organisms and to form highly complex multicellular plants and animals in aqueous medium. The first multicellular organisms, the plants, appeared already 1000 million years ago (MYA), then the fungi emerged and, finally, animals developed (800 MYA).

Focusing on marine animals, the evolutionarily oldest phyla, the Porifera, the Cnidaria and the Bryozoa, as sessile filter feeders, are exposed not only to a huge variety of commensal, but also toxic microorganisms, bacteria and fungi. In order to overcome these threats, they developed a panel of defense systems, for example, an immune system, which is closely related to those exi­sting in higher metazoans, the Protostomia and Deuterostomia. In addition, due to this characteristic, they became outstandingly successful during evolu­tion: they developed a chemical defense system which enabled them to fight in a specific manner against invaders. These chemicals are of low molecular weight and of non-proteinaceous nature. Due to the chemical complexity and the presence of stereogenic centers in these compounds, a high diversity of compounds became theoretically possible. In a natural selective process, during evolution, only those compounds could persist which caused the most potent bioactivity and provided the most powerful protection for the host in which they were synthesized. This means that during evolution nature conti­nuously modified the basic structures and their derivatives for optimal func­tion. In principle, the approach used in combinatorial chemistry is the same, but turned out to be painful and only in few cases successful. In consequence, it is advisable to learn from nature for these strategies to select for bioactive drugs. Besides the mentioned metazoan phyla, other animal phyla, such as the higher-evolved animals, the mollusks or tunicates, or certain algal groups,

VI Preface

also produce compounds for their chemical defense which are of interest scientifically and for potential application.

There is, however, one drawback. Usually, the amount of starting material used as a source for the extraction of most bioactive compounds found in marine organisms is minute and, hence, not sufficient for their further appli­cation in biomedicine. Furthermore, the constraints of the conventions for the protection of nature limit the commercial exploitation of novel compounds, since only a small number of organisms can be collected from the biotope. Consequently, exploitation must be sustainable, i.e., it should not endanger the equilibrium of the biota in a given ecosystem. However, the protection of biodiversity in nature, in general, and those organisms living in the marine environment, in particular, holds an inherent opportunity if this activity is based on genetic approaches. From the research on molecular biodiversity, benefits for human society emerge which are of obvious commercial value; the transfer of basic scientific achievements to applicable products is the task and the subject of Marine Molecular Biotechnology. This discipline uses modern molecular and cell biological techniques for the sustainable produc­tion of bioactive compounds and for the improvement of fermentation tech­nologies in bioreactors.

Hence, marine molecular biotechnology is the discipline which strives to define and solve the problems regarding the sustainable exploitation of nature for human health and welfare, through the cooperation between scien­tists working in marine biology/molecular biology/microbiology and chemi­stry. Such collaboration is now going on successfully in several laboratories.

It is the aim of this new subset of thematically connected volumes within our series "Progress in Molecular and Subcellular Biology" to provide an actual forum for the exchange of ideas and expertise between colleagues wor­king in this exciting field of "Marine Molecular Biotechnology". It also aims to disseminate the results to those researchers who are interested in the recent achievements in this area or are just curious to learn how science can help to exploit nature in a sustainable manner for human prosperity.

WERNER E.G. MULLER

Foreword

I was excited and pleased when Professor W.E.G. Muller and his colleagues from the Center of Excellence "BIOTECmarin" invited me to contribute with this short greeting to the volume "Marine Molecular Biotechnology".

My interest in marine science was aroused by my colleagues in Marine Biol­ogy, who invited me to participate in their study of marine toxins, especially ciguatera toxins. It did not take long to discover that Japan was the leading country in marine chemistry research. I was surprised and disappointed that activity in the field in Europe was minimal. Italy was a notable exception, but even in maritime-oriented countries, notably Great Britain, activity was mar­ginal. In most countries, research was usually confined to a single investigator and institution. I had hoped that Germany with its long and proud academic history would spawn one or several centers for the study of marine organic chemistry.

VIII Foreword

At long last it has come about through the initiative and effort of Professor Muller and his colleagues from the Center of Excellence. And, not surpris­ingly, they are carrying the work to a new level of sophistication, that of mol­ecular biology, cell biology and biotechnology. My own background in terres­trial natural products required few adjustments beyond learning some marine biology and becoming a certified diver. I am also excited with the bold endeavor of Professor Muller and his colleagues from BIOTECmarin to carry marine research to a new frontier - molecular marine biology.

My best wishes for a successful and fruitful endeavor.

Department of Chemistry University of Hawai'i at Manao Honolulu, Hawai'i 96822, USA

PAUL J. SCHEUER

Foreword

The world's oceans harbor a huge number of organisms with an amazing variety of metabolic pathways. The exact number of marine species from bac­teria to multicellular animals/plants can still not be estimated. It is expected that the elucidation of their biochemical mechanisms can contribute to solu­tions of problems in the fields of biomedicine, chemistry and environmental sciences.

The financial support of R&D projects aimed at exploring the living resources of the oceans is one point -of-main -effort program within the frame "Marine Research" of the German Government. Since November 1997, a series of such R&D projects have been granted within the program "Research in Marine Natural Products". The aim of these research activities is the discov­ery, isolation and characterization ofbioactive compounds from marine ani­mals. It is hoped that the results will contribute to the development of new drugs applicable for the treatment of hitherto incurable diseases or lead to the production of new materials, e.g. in the field of nanotechnology. Furthermore, in addition, the development of novel processes allowing a sustainable uti­lization of natural resources for the production of bioactive products, follow­ing the guidelines of the International Convention on Biological Biodiversity, is the subject of financial support.

Financial support by the German Government should also contribute to a structural improvement of the cooperation between research teams working in marine science and in natural products with efforts proceeding in industry. The aim is to eliminate structural deficiencies, to improve and optimize already existing infrastructures, and to improve transfer of findings from public institutions to industrial enterprises. These challenging activities, which also imply efficient ways for protection of knowledge, should result within a middle- to long-term period in a self-supporting process that needs help also from industrial enterprises.

The Center of Excellence "BIOTECmarin", which is supported by the cen­tral government as well as by the government of the regional states, meets the criteria in a distinguished manner. In this virtual center, scientists working in different disciplines of natural sciences as well as engineers concentrate and potentiate their efforts; the results are channeled into a newly formed unit that should allow a rapid utilization of the research results gathered for applied and commercial exploitation. The target organisms of the research

x Foreword

efforts in "BIOTECmarin" are sponges and their associated microorganisms that are known to be prominent producers of natural compounds. They pro­duce highly active secondary metabolites with a wide range of chemical struc­tures. The biological activities of the compounds are directed against a broad range of metabolic processes; hence the starting organisms, sponges and their associated microorganisms, should be considered as valuable resources (both with respect to the scientific potentials and their biomedical application) which should be exploited only in a sustainable way.

We welcome that the members of the Center of Excellence "BIOTECmarin" present their extensive expertise as well as techniques and simultaneously outline the present state of knowledge in this field of research in the mono­graph "Marine Molecular Biotechnology".

Projekttrager Jiilich UDO SCHOTTLER

im Forschungszentrum Jiilich GmbH

Preface

The Center of Excellence "BIOTECmarin" was founded in 2001 with the sup­port of the Ministries of Research and Technology of the German Government as well as of the regional states. It is the aim of this center to combine and poten­tiate the expertise of the different groups involved in basic science in the fields of taxonomy/biology/microbiology/molecular biology/chemistry with the emphasis on the transfer of bundled knowledge to applied research.

Sponges (phylum Porifera) are the central focus of the activities in the cen­ter. They are known to be extremely rich sources of bioactive compounds, mainly of secondary metabolites. The main efforts will be devoted to cell cul­ture and mariculture of sponges to assure a sustainable exploitation of bioac­tive compounds from biological starting materials. These activities are flan­ked by improved technologies to cultivate the bacteria and fungi associated with the sponges. It is the hope that, by elucidating the strategies of the inter­actions between microorganisms and their hosts (sponges), by modern cell and molecular biological methods, a more comprehensive cultivation of the symbiotic organisms will be possible. The next step in the transfer of know­ledge to biotechnological applications is the isolation, characterization and structural determination of the bioactive compounds by sophisticated chemi­cal approaches. Furthermore, combined efforts will also result in new insights into processes of biomineralization that are characteristic for sponges, e.g. the formation of the siliceous skeleton. Finally, those secondary metabolites that prove to be of biomedical interest, and are produced either by the host or by the associated microorganisms, will be synthesized either by chemical or bio­chemical techniques, or by a combination of both. The success of these studies will also depend on the progress that is made in the elucidation of the genome structure and the repertoire of the sponge genes. The functional genome of one model sponge, Suberites domuncula, will be analyzed in detail. The results gathered will provide an insight into the metabolic capacities of the sponges.

The studies in this focused area performed by the groups at the center (see Photo), bioprospecting the biopotential existing in sponges and their micro­organisms, are fascinating, continually uncovering new surprises, ranging from unexpected findings in (1) molecular evolution contributing to the understanding of the transition of the ancestors of the Metazoa to the Pori­fera, as the oldest metazoan phylum, in (2) chemistry, discovery of novel com­pounds, to (3) the elucidation of the molecular basis underlying the symbio­tic relationship of sponges with their microorganisms.

XII Preface

Photo. Members of the center who participated with colleagues from other European countries during the first meeting on "Modern developments in marine biotechnology" in Rovinj (Croatia); 22-23 August 2002. Back row (left to right): R. Batel, F. Briimmer, W.E.G. MUller, U. Hentschel, G. Bringmann, J. Imhoff. Front row: G. Le Pennec (group of H.J. Breter), L. Schillak, C. Thoms (group of P. Proksch), H.C. Schroder

It needs hardly to be stressed that the findings disclosed in the center will be immediately checked for their potential in applied biotechnology, an approach which will result not only in scientific papers, but also in "applied" products, in patents.

We are grateful to several colleagues for their help in refereeing the chap­ters and for their additional advice.

W.E.G. MULLER

Coordinator of the Center of Excellence "BIOTECmarin"

Contents

Analysis of the Sponge [Porifera] Gene Repertoire: Implications for the Evolution of the Metazoan Body Plan . . . . . . . . . . . W.E.G. Muller, I.M. Muller

1 2 3 3.1 3.1.1 3.1.2 3.1.3 3.1.4 3.1.5 3.2 3.2.1 3.2.2 3.2.3 4 4.1 4.2 4.3 5 6 7 8 8.1

9

9.1 9.2 10

Introduction . . . . . . . . . . . . . . . . . Sponges. . . . . . . . . . . . . . ..... . Adhesion Between Cells . . . . . . . Cell-Cell Adhesion in Sponges ...... . Galectin ....................... . The 36-kDa Putative AF . . . . . . . . . . . . . . . . The 86-kDa AF-Associated Polypeptide ..... The Core Structure of the AF . . .. . . . . . The Putative AR . . . . . . . . . . . . Cell-Matrix Adhesion in Sponges .. . ... . Collagen ................. . Fibronectin . . . . . . . . . . . . . . . . . . . . . . . Integrin ........................ . Growth and Differentiation . . . . . . . Primmorphs . . . Canal Formation . . . . . . . . . . . Proliferation ................. . Migration of Cells - Contraction in Cell Layers Elements of a Neuronal Network . . . . . . . . . Secretion of Skeletal Elements ... Morphogens Proteins ............... . ... . Apoptosis .............. . ... . Induction of Apoptosis in Sponges ........... . Induction of Expression of Apoptotic Genes in Sponges . Conclusion: Contribution to the Origin of the Metazoan Body Plan References ................. .

1

2 3 5 6 7

8 9 9 9

10 10 12 13 14 15 15 18 20 21 21 22 22 25 25 25

26 28

XIV

Sponge-Associated Bacteria: General Overview and Special Aspects of Bacteria Associated with Halichondria panicea J.E Imhoff, R. Stohr

1 2 2.1 2.2 2.3 2.4 2.5

3 3.1 3.2

3.3 4

Introduction General Considerations . Microscope Observations Cultivation of Sponge-Associated Bacteria Molecular Genetic Analysis of Sponge-Associated Bacteria Symbiosis Between Bacteria and Sponges . . . . . . . . . . Biologically Active Substances from Sponge-Associated Bacteria .... Bacteria Associated with Halichondria panicea . Eubacteria Isolated from Halichondria panicea Molecular Diversity of Eubacteria Within Halichondria panicea . . . . . . . . . . . Diversity of the CytophagalFlavobacteria Group Conclusions

Contents

35

35 37 37 38 40 42

43 46 47

49 52 53

References ..................... 54

Microbial Diversity of Marine Sponges ................ 59 U. Hentschel, L. Fieseler, M. Wehrl, C. Gernert, M. Steinert, J. Hacker

1 2 2.1 2.2 2.3 3 3.1 3.2 3.2.1 3.2.2 3.2.3 4

4.1 4.1.1 4.1.2 4.l.3 4.2 4.3 4.4

Introduction . . . . . . . . . . . . . . . . . . . . Sponge-Microbe Associations ......... . Sponges as Ancient Niches for Microorganisms Bacterial Localization . . . . . . . . . Bacterial Morphotypes ....... . Tools of Molecular Microbial Ecology Cultivation -Dependent Techniques Cultivation-Independent Techniques Fluorescence In Situ Hybridization Denaturing Gradient Gel Electrophoresis 16S rDNA Library Construction ..... A Uniform Microbial Community in Sponges from Different Oceans Sponge Model Systems Aplysina aerophoba Rhopaloides odorabile . Theonella swinhoei .. The Microbial Signature of Sponges Sponge-Microbe Interaction Model The Paradigm of Sponge-Microbial Symbiosis

59 60 60 62 64 6S 66 67 69 70 70

71 71 71 73 74 74 77 78

Contents

5

6

Biotechnological Potential of Sponge-Associated Microorganisms . . . . . . . . . . . Conclusions and Future Directions References ............. .

Full Absolute Stereo structures of Natural Products Directly from Crude Extracts: the HPLC-MS/MS-NMR-CD 'Triad' G. Bringmann, G. Lang

xv

79 82 83

89

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 2 Exemplarily for Naphthylisoquinoline Alkaloids:

Constitutions and Relative Configurations by LC-MS/MS-NMR . . . . . . . . . . . . . . . . 90

3 Complemented by the LC-CD Option for the Online Assignment of Absolute Configurations: the Triad Is Complete! . . . . . . . . . . . . . . . . . 93

4 Application of the Triad to the Online Structural Elucidation of New Naphthylisoquinoline Alkaloids and Related Compounds .................... 94

5 An Application to Natural Phenylanthraquinones -Including Quantum Chemical CD Calculations and Total Synthesis .................. 97

6 Stereochemistry of Axially Chiral Biscarbazoles in Plant Extracts, by LC-CD Coupling and CD Calculations 103

7 Without (True) Stereo genic Axes or Centers, but Chiral: a Bis-Bibenzyl Macrocycle ............ 104

8 First Time in Marine Natural Products Analysis: the Analytical Triad HPLC-MS/MS-NMR-CD 105

9 Conclusions 109 References .................. .

Bioactive Natural Products from Marine Invertebrates and Associated Fungi ............... . P. Proksch, R. Ebel, R.A. Edrada, V. Wray, K. Steube

1

2 2.1

2.2 3

Introduction: Some Current Issues of Marine Natural Products Research Ecological Functions of Sponge Alkaloids Defensive Pyridoacridine Alkaloids from the Tropical Sponge Oceanapia sp. ....... . Chemical Defense of Mediterranean Aplysina Sponges Pharmacologically Active Constituents from Marine Invertebrates ............... .

111

117

117 121

122 124

127

XVI

3.1

3.2

3.3

4

4.1

4.2

5

Swinhoeiamide A: a New Calyculin Derivative from the Sponge Theonella swinhoei Bromopyrrole Alkaloids from the Sponge Stylissa carteri (syn. Axinella carteri) ......... . Staurosporine Derivatives from the Tunicate Eudistoma toealensis and the Flatworm Pseudoceros sp. Sponge-Associated Fungi as a New Source for Bioactive Metabolites .. . . . . . . . . . . . . . . . New Natural Products Isolated from Fungi Associated with the Marine Sponge Aplysina aerophoba . . . . . . New Natural Products Isolated from Fungi Associated with the Marine Sponge Xestospongia exigua Conclusions References .................. .

Sustainable Use of Marine Resources: Cultivation of Sponges F. Brummer, M. Nickel

1 2 3 4 5 6

Introduction . . . . . . . . . . . . . In Situ Cultivation of Bath Sponges Sponge Farming . . . . . . . . . . . Ex Situ Maintenance of Sponges in Aquaria . In Vitro Cultivation of Sponges Conclusions and Future Directions References ............. .

Sustainable Production of Bioactive Compounds from Sponges: Primmorphs as Bioreactors . . . . H.C. Schroder, F. Brummer, E. Fattorusso, A. Aiello, M. Menna, S. de Rosa, R. Batel, W.E.G. Muller

1 Introduction ............. • • • • I •••••••

2 Origin of Biologically Active Compounds from Sponges 3 Biologically Active Compounds from S. domuncula 3.1 Neurotoxic Compound 3.2 Quinolinic Acid ..... 4 The Primmorph System . 4.1 Characteristics . 4.2 Medium Design ..... 4.3 Other Factors ...... 4.4 Immunohistological Analysis of Primm or phs 5 Production of Bioactive Compounds

in the Primmorph System 5.1 Avarol ......... 5.2 (2'-5')Oligoadenylates ...

Contents

127

128

129

132

133

135 137 138

143

144 146 147 149 154 157 158

163

164 168 170 170 172 175 176 177 179 180

180 181 183

Contents

6 6.1 6.2 7

Future Directions ..................... . Immortalization/Cell Lines . . . . . . . . . . . . . . . . . Transfection Conclusions References .

Approaches for a Sustainable Use of the Bioactive Potential in Sponges: Analysis of Gene Clusters, Differential Display of mRNA and DNA Chips . . . . . . . . . . . . . . . . . H.J. Breter, V.A. Grebenjuk, A. Skorokhod, W.E.G. Muller

1 2 2.1 2.2 2.2.1 2.2.2 2.3 2.4

3 4 5

5.1 5.2 5.3 5.3.1 5.3.2 5.3.3 5.3.4 5.3.5 6

Introduction . . . . . . . . . . . . . . . . . . . Genome of Porifera . . . . . . . . . . . . . . . Genome Size . . . . . . . . . . . . . . . . . . . . . . Gene Density . . . . . . . . . . . . . . . . . . . . . . . . . . . Receptor Tyrosine Kinase Cluster: G. cydonium ...... . Allograft Inflammatory Factor [AIF] Cluster: S. domuncula Intron Lengths . . . . . . . . . . . . . . . . . . . . . . Gene Number ................... . Nonrandom Distribution of Dinucleotide Repeats Burst of Gene Duplication ........... . Approaches to Identify Genes Involved in the Synthesis of Bioactive Compounds . . . . Analysis of the Expressed Genome of Sponges . . . . . . . . Differential Display of mRNA, DNA Chips .. . DNA-Array Assay ....................... . DNA Arrays .......................... . RNA Extraction and Preparation of Probes Hybridization .... . . . . . . . . . . . . . . . . Detection ......................... . Example ......................... . Conclusions ....... ........... . References ..................... .

Sorbicillactone A: a Structurally Unprecedented Bioactive

XVII

186 188 189 191 192

199

200 201 201 202 202 204 205 214 216 218

219 220 221 223 223 224 224 226 226 227 228

Novel-Type Alkaloid from a Sponge-Derived Fungus .... 231 G. Bringmann, G. Lang, J. Muhlbacher, K. Schaumann, S. Steffens, P.G. Rytik, U. Hentschel, J. Morschhauser, W.E.G. Muller

1 Introduction . . . . . . . . . . . . . . . . . 232 2 Isolation and Cultivation of the Fungus . . 233 3 Online Analysis of the Extract by the Triad

LC-MS/MS-NMR-CD: Hints at a Novel Structural Type ... 235 4 Isolation of the New Compound and Completion

of the Structural Elucidation . . . . . . . . . . . . . . . . .. 236

XVIII Contents

4.1 Assignment of the Constitution and the Relative Configuration ................... 236

4.2 Establishing the Absolute Configuration: by Quantum Chemical CD Calculations . . . . . . . . . . .. 238

5 Sorbicillactone A: a Unique, Novel-Type Structure and Its Presumable Biosynthetic Origin . . . . . 239

6 Sorbicillactone A: a Natural Product with Strong - and Selective - Bioactivities ... 241

6.1 Antifungal Activity .. . . . . . 242 6.2 Antibacterial Activity . . . . . . . . .. . . . 242 6.3 Antiprotozoic Activity . . . .. . . . . . . . . 242 6.4 Cytostatic Activity . . . . . . . . .. . . . . . . . . 243 6.5 Mode of Action of Sorbicillactone A on L5178y Cells 244 6.5.1 Determination of Apoptosis ..... 244 6.5.2 Reversibility of the Inhibitory Effect

of Sorbicillactone A on L5178y Cells . 245 6.5.3 Effect of Sorbicillactone A on the Synthesis

of Macromolecules In Vitro ......... 246 6.6 Antiviral (HIV-l) Effect Caused by Sorbicillactone A 247 6.6.1 Protection by Sorbicillactone A

Against Cytopathic Effects of HIV-1 . . . . . 247 6.6.2 Inhibition of Expression of HIV-1 Proteins . 248 7 Summary and Future Perspectives 249

References .. 250

Subject Index . . . . . . 255