Marine molecular biology: an emerging field of biological sciences.

An appreciation of the potential applications of molecular biology is of growing importance in many areas of life sciences, including marine biology. During the past two decades, the development of sophisticated molecular technologies and instruments for biomedical research has resulted in significant advances in the biological sciences. However, the value of molecular techniques for addressing problems in marine biology has only recently begun to be cherished. It has been proven that the exploitation of molecular biological techniques will allow difficult research questions about marine organisms and ocean processes to be addressed. Marine molecular biology is a discipline, which strives to define and solve the problems regarding the sustainable exploration of marine life for human health and welfare, through the cooperation between scientists working in marine biology, molecular biology, microbiology and chemistry disciplines. Several success stories of the applications of molecular techniques in the field of marine biology are guiding further research in this area. In this review different molecular techniques are discussed, which have application in marine microbiology, marine invertebrate biology, marine ecology, marine natural products, material sciences, fisheries, conservation and bio-invasion etc. In summary, if marine biologists and molecular biologists continue to work towards strong partnership during the next decade and recognize intellectual and technological advantages and benefits of such partnership, an exciting new frontier of marine molecular biology will emerge in the future.

Isolation and characterization of Wnt pathway-related genes from Porifera.

The Wnt signal acts by binding to Frizzled receptors, with the subsequent activation of two different signal transduction cascades, the canonical and the non-canonical Wnt pathways, involved in cell growth, differentiation, migration and fate. The canonical pathway functions through the translocation of beta-catenin to the nucleus and the activation of TCF/LEF transcription factors; it plays an important role in developmental patterning and cell fate decisions during embryogenesis. The non-canonical Wnt pathway is responsible for the planar cell polarity process in invertebrates, and for the convergent-extension movements during vertebrate gastrulation. The final effect of the non-canonical Wnt pathway is the rearrangement of the cell cytoskeleton, through the activation of the subfamily of Ras-like small GTPases. In a recent report we described for the first time the isolation of a Wnt-related gene, Sd-Frizzled, from the most basal animal phylum, the Porifera. In the present study we report the isolation and phylogenetic characterization of several Wnt pathway-related genes from the sponge Suberites domuncula: Sd-TCF/LEF, Sd-GSK3, a recently discovered molecule with a putative function as a Wnt regulator (Sd-LZIC), the small Rho GTPases Sd-RhoA, Sd-Cdc42, and their effector Sd-mrlc. Also the isolation of a secreted frizzled related protein sFRP from another sponge species (Lubomirskia baicalensis) is reported.

Antiangiogenic, antimicrobial, and cytotoxic potential of sponge-associated bacteria.

The bacteria associated with marine invertebrates are a rich source of bioactive metabolites. In the present study bacteria associated with the sponge Suberites domuncula and its primmorphs (3-dimensional aggregates containing proliferating cells) were isolated and cultured. These bacteria were extracted, and the extracts were assayed for antiangiogenic, hemolytic, antimicrobial, and cytotoxic activities. Our studies revealed that extract obtained from the bacterium (PB2) isolated from sponge primmorphs is a potent angiogenesis inhibitor. In the chick chorio-allantoic membrane (CAM) assay, it showed 50% activity at 5 microg ml(-1) and 100% activity at 10 and 20 microg ml(-1) concentrations. Extracts obtained from 5 bacterial strains isolated from sponge and its primmorphs showed hemolytic activity. The sponge-associated bacteria belonging to the alpha subdivision of Proteobacteria and the primmorph-associated bacterium identified as a possible novel Pseudomonas sp. displayed remarkable antimicrobial activity. It is important to note that these bacterial extracts were strongly active against multidrug-resistant clinical strains such as Staphylococcus aureus and Staphylococcus epidermidis, isolated from hospital patients. The bacterial extracts having antimicrobial activity also showed cytotoxicity against HeLa and PC12 cells. In summary, this investigation explores the importance of sponge-associated bacteria as a valuable resource for the discovery of novel bioactive molecules.

Matrix-mediated canal formation in primmorphs from the sponge Suberites domuncula involves the expression of a CD36 receptor-ligand system.

Sponges (Porifera), represent the phylogenetically oldest metazoan phylum still extant today. Recently, molecular biological studies provided compelling evidence that these animals share basic receptor/ligand systems, especially those involved in bodyplan formation and in immune recognition, with the higher metazoan phyla. An in vitro cell/organ-like culture system, the primmorphs, has been established that consists of proliferating and differentiating cells, but no canals of the aquiferous system. We show that after the transfer of primmorphs from the demosponge Suberites domuncula to a homologous matrix (galectin), canal-like structures are formed in these 3D-cell aggregates. In parallel with the formation of these structures a gene is expressed whose deduced protein falls into the CD36/LIMPII receptor family. The receptor was cloned and found to be strongly expressed after adhesion to the galectin matrix. This process was suppressed if primmorphs were co-incubated with a homologous polypeptide containing the CSVTCG domain, as found in thrombospondin-1 (and related) molecules of vertebrates. In situ hybridization studies revealed that the S. domuncula CD36/LIMPII receptor is localized in the pinacocytes that surround the canals of the sponge. Furthermore, a secondary metabolite from a sponge-associated bacterium was isolated and characterized, the 2-methylthio-1,4-naphthoquinone (MTN). MTN causes inhibition of cell proliferation of vertebrate tumor cells at concentrations of >80 ng/ml. However, doses of only 2 ng are required to potently inhibit angiogenesis in the chick chorio-allantoic membrane assay. At concentrations of 10 ng/ml this compound was also found to suppress the expression of the S. domuncula CD36/LIMPII; this result is a first indication that this secondary metabolite has a conserved functional activity: the suppression of the formation of the circulation system, from sponges to vertebrates.

Oxygen-controlled bacterial growth in the sponge Suberites domuncula: toward a molecular understanding of the symbiotic relationships between sponge and bacteria.

Sponges (phylum Porifera), known to be the richest producers among the metazoans of bioactive secondary metabolites, are assumed to live in a symbiotic relationship with microorganisms, especially bacteria. Until now, the molecular basis of the mutual symbiosis, the exchange of metabolites for the benefit of the other partner, has not been understood. We show with the demosponge Suberites domuncula as a model that the sponge expresses under optimal aeration conditions the enzyme tyrosinase, which synthesizes diphenols from monophenolic compounds. The cDNA isolated was used as a probe to determine the steady-state level of gene expression. The gene expression level parallels the level of specific activity in sponge tissue, indicating that without aeration the tyrosinase level drops drastically; this effect is reversible. The SB2 bacterium isolated from the sponge surface grew well in M9 minimal salt medium supplemented with the dihydroxylated aromatic compound protocatechuate; this carbon source supported growth more than did glucose. From the SB2 bacterium the protocatechuate gene cluster was cloned and sequenced. This cluster comprises all genes coding for enzymes involved in the conversion of protocatechuate to acetyl coenzyme A. Expression is strongly induced if the bacteria are cultivated on M9-protocatechuate medium; the genes pcaQ (encoding the putative transcriptional activator of the pca operon) and pcaDC were used for quantitative PCR analyses. We conclude that metabolites, in this case diphenols, which might be produced by the sponge S. domuncula are utilized by the sponge surface-associated bacterium for energy generation. This rationale will help to further uncover the symbiotic pathways between sponges and their associated "nonculturable" microorganisms; our approach is flanked by the establishment of an EST (expressed sequence tags) database in our laboratory.