A synthetic biology approach for the fabrication of functional (fluorescent magnetic) bioorganic-inorganic hybrid materials in sponge primmorphs.

During evolution, sponges (Porifera) have honed the genetic toolbox and biosynthetic mechanisms for the fabrication of siliceous skeletal components (spicules). Spicules carry a protein scaffold embedded within biogenic silica (biosilica) and feature an amazing range of optical, structural, and mechanical properties. Thus, it is tempting to explore the low-energy synthetic pathways of spiculogenesis for the fabrication of innovative hybrid materials. In this synthetic biology approach, the uptake of multifunctional nonbiogenic nanoparticles (fluorescent, superparamagnetic) by spicule-forming cells of bioreactor-cultivated sponge primmorphs provides access to spiculogenesis. The ingested nanoparticles were detected within intracellular vesicles resembling silicasomes (silica-rich cellular compartments) and as cytosolic clusters where they lent primmorphs fluorescent/magnetic properties. During spiculogenesis, the nanoparticles initially formed an incomplete layer around juvenile, intracellular spicules. In the mature, extracellular spicules the nanoparticles were densely arranged as a surface layer that rendered the resulting composite fluorescent and magnetic. By branching off the conventional route of solid-state materials synthesis under harsh conditions, a new pathway has been opened to a versatile platform that allows adding functionalities to growing spicules as templates in living cells, using nonbiogenic nanoscale building blocks with multiple functionalities. The magnet-assisted alignment renders this composite with its fluorescent/magnetic properties potentially suitable for application in biooptoelectronics and microelectronics (e.g., microscale on-chip waveguides for applications of optical detection and sensing).

Self-assembly and photocatalytic activity of branched silicatein/silintaphin filaments decorated with silicatein-synthesized TiO2 nanoparticles.

The fundamental mechanisms of biomineralization and their translation into innovative synthetic approaches have yielded promising perspectives for the fabrication of biomimetic and bioinspired organic-inorganic hybrid materials. In siliceous sponges, the enzyme silicatein catalyzes the polycondensation of molecular precursors to nano-structured SiO2 that is deposited on self-assembled filaments consisting of the two silicatein isoforms (silicatein-α and -ß) and the scaffold protein silintaphin-1. Due to its broad substrate specificity silicatein is also able to convert in vitro various other precursors to non-biogenic materials (e.g., hydrolysis of titanium bis(ammonium lactato)-dihydroxide [TiBALDH] and subsequent polycondensation to titania [TiO2]). In the present approach, silicatein was bioengineered to carry a protein tag (Arg-tag) that confers binding affinity to TiO2. Then, by combining Arg-tagged silicatein-α with silicatein-ß and silintaphin-1, self-assembled branched hybrid protein microfilaments were fabricated. Upon subsequent incubation with TiBALDH the filaments were decorated with TiO2 and assayed for photocatalytic activity through photodegradation of the dye methylene blue. This is the first approach that considers concomitant application of two silicatein isoforms for the synthesis of bioinspired organic-inorganic hybrid materials. It is also the first time that the biocatalytic activity of the enzymes has been combined with both the structure-providing properties of silintaphin-1 and a TiO2 affinity protein tag to fabricate self-assembled branched protein filaments as template for a silicatein-synthesized TiO2 photocatalyst. The TiO2-decorated filaments might be explored as a practical alternative to approaches where biotemplates have to be laboriously isolated from their original biological source prior to TiO2 immobilization.

Acquisition of structure-guiding and structure-forming properties during maturation from the pro-silicatein to the silicatein form.

Silicateins are the key enzymes involved in the enzymatic polycondensation of the inorganic scaffold of the skeletal elements of the siliceous sponges, the spicules. The gene encoding pro-silicatein is inserted into the pCold TF vector, comprising the gene for the bacterial trigger factor. This hybrid gene is expressed in Escherichia coli and the synthesized fusion protein is purified. The fusion protein is split into the single proteins with thrombin by cleavage of the linker sequence present between the two proteins. At 23 °C, the 87 kDa trigger factor-pro-silicatein fusion protein is cleaved to the 51 kDa trigger factor and the 35 kDa pro-silicatein. The cleavage process proceeds and results in the release of the 23 kDa mature silicatein, a process which very likely proceeds by autocatalysis. Almost in parallel with its formation, the mature enzyme precipitates as pure 23 kDa protein. When the precipitate is dissolved in an urea buffer, the solubilized protein displays its full enzymatic activity which is enhanced multi-fold in the presence of the silicatein interactor silintaphin-1 or of poly(ethylene glycol) (PEG). The biosilica product formed increases its compactness if silicatein is supplemented with silintaphin-1 or PEG. The elastic modulus of the silicatein-mediated biosilica product increases in parallel with the addition of silintaphin-1 and/or PEG from 17 MPa (silicatein) via 61 MPa (silicatein:silintaphin-1) to 101 MPa (silicatein:silintaphin-1 and PEG). These data show that the maturation process from the pro-silicatein state to the mature form is the crucial step during which silicatein acquires its structure-guiding and structure-forming properties.

Biogenic inorganic polysilicates (biosilica): formation and biomedical applications.

The siliceous sponges, the demosponges and hexactinellid glass sponges, are unique in their ability to form biosilica structures with complex architectures through an enzyme-catalyzed mechanism. The biosilica skeleton of these sponges with its hierarchically structure and exceptional opto-mechanical properties has turned out to be an excellent model for the design of biomimetic nanomaterials with novel property combinations. In addition, biosilica shows morphogenetic activity that offers novel applications in the field of bone tissue engineering and repair. In recent years, much progress has been achieved towards the understanding of the principal enzymes, the silicateins that form the sponge skeletal elements, the spicules, and their self-assembling and structure-guiding properties. The discovery of the silicatein-interacting, scaffolding proteins provided new insights in the mechanism of spiculogenesis. The now available toolbox of enzymes and proteins that are involved in biosilica formation and the biosilica material synthesized by them are of great interest for a variety of applications from nanobiotechnology to nanomedicine.

Deep metazoan phylogeny: when different genes tell different stories.

Molecular phylogenetic analyses have produced a plethora of controversial hypotheses regarding the patterns of diversification of non-bilaterian animals. To unravel the causes for the patterns of extreme inconsistencies at the base of the metazoan tree of life, we constructed a novel supermatrix containing 122 genes, enriched with non-bilaterian taxa. Comparative analyses of this supermatrix and its two non-overlapping multi-gene partitions (including ribosomal and non-ribosomal genes) revealed conflicting phylogenetic signals. We show that the levels of saturation and long branch attraction artifacts in the two partitions correlate with gene sampling. The ribosomal gene partition exhibits significantly lower saturation levels than the non-ribosomal one. Additional systematic errors derive from significant variations in amino acid substitution patterns among the metazoan lineages that violate the stationarity assumption of evolutionary models frequently used to reconstruct phylogenies. By modifying gene sampling and the taxonomic composition of the outgroup, we were able to construct three different yet well-supported phylogenies. These results show that the accuracy of phylogenetic inference may be substantially improved by selecting genes that evolve slowly across the Metazoa and applying more realistic substitution models. Additional sequence-independent genomic markers are also necessary to assess the validity of the phylogenetic hypotheses.

Nocturnin in the demosponge Suberites domuncula: a potential circadian clock protein controlling glycogenin synthesis in sponges.

Sponges are filter feeders that consume a large amount of energy to allow a controlled filtration of water through their aquiferous canal systems. It has been shown that primmorphs, three-dimensional cell aggregates prepared from the demosponge Suberites domuncula and cultured in vitro, change their morphology depending on the light supply. Upon exposure to light, primmorphs show a faster and stronger increase in DNA, protein and glycogen content compared with primmorphs that remain in the dark. The sponge genome contains nocturnin, a light/dark-controlled clock gene, the protein of which shares a high sequence similarity with the related molecule of higher metazoans. The sponge nocturnin protein was found showing a poly(A)-specific 3'-exoribonuclease activity. In addition, the cDNA of the glycogenin gene was identified for subsequent expression studies. Antibodies against nocturnin were raised and used in parallel with the cDNA to determine the regional expression of nocturnin in intact sponge specimens; the highest expression of nocturnin was seen in the epithelial layer around the aquiferous canals. Quantitative PCR analyses revealed that primmorphs after transfer from light to dark show a 10-fold increased expression in the nocturnin gene. In contrast, the expression level of glycogenin decreases in the dark by 3-4-fold. Exposure of primmorphs to light causes a decrease in nocturnin transcripts and a concurrent increase in glycogenin transcripts. It was concluded that sponges are provided with the molecular circadian clock protein nocturnin that is highly expressed in the dark where it controls the stability of a key metabolic enzyme, glycogenin.

Hardening of bio-silica in sponge spicules involves an aging process after its enzymatic polycondensation: evidence for an aquaporin-mediated water absorption.

BACKGROUND: Spicules, the siliceous skeletal elements of the siliceous sponges, are synthesized enzymatically via silicatein. The product formed, bio-silica, constitutes their inorganic matrix. It remained unexplored which reactions are involved in molding of the amorphous bio-silica and formation of a solid and rigid biomaterial. METHODS: Cell and molecular biological techniques have been applied to analyze processes resulting in the hardening of the enzymatically synthesized bio-silica. The demosponge Suberites domuncula has been used for the studies. RESULTS: Cell aggregates (primmorphs) from the sponge S. domuncula, grown in the presence of Mn-sulfate, form spicules that comprise, instead of a smooth, a rough and porous surface which is decorated with irregular bio-silica deposits. During this process, the expression of the aquaporin-8 gene becomes down-regulated. Further in vitro studies showed that aquaporin is required for dehydration, and hardening of bio-silica following its enzymatic formation. The data show that in cell aggregates grown in the presence of Mn-sulfate, aquaporin-8 is down-regulated. We conclude that in cell aggregates grown in the presence of Mn-sulfate, the removal of reaction water, produced during the bio-silica polycondensation reaction, is inhibited. GENERAL SIGNIFICANCE: This study highlights that besides the silicatein-driven polycondensation reaction, the spicule formation also requires a phase of syneresis that results in a hardening of the material.

Interaction of the retinoic acid signaling pathway with spicule formation in the marine sponge Suberites domuncula through activation of bone morphogenetic protein-1.

BACKGROUND: The formation of the spicules in siliceous sponges involves the formation of cylinder-like structures in the extraspicular space, composed of the enzyme silicatein and the calcium-dependent lectin. SCOPE OF REVIEW: Molecular cloning of the cDNAs (carotene dioxygenase, retinal dehydrogenase, and BMB-1 [bone morphogenic protein-1]) from the demosponge Suberites domuncula was performed. These tools were used to understand the retinoid metabolism in the animal by qRT-PCR, immunoblotting and TEM. MAJOR CONCLUSIONS: We demonstrate that silintaphin-2, a silicatein-interacting protein, is processed from a longer-sized 15-kDa precursor to a truncated, shorter-sized 13kDa calcium-binding protein via proteolytic cleavage at the dipeptide Ala↓Asp, mediated by BMP-1. The expression of this protease as well as the expression of two key enzymes of the carotinoid metabolism, the ß,ß-carotene-15,15'-dioxygenase and the retinal dehydrogenase/reductase, were found to be strongly up-regulated by retinoic acid. Hence retinoic acid turned out to be a key factor in skeletogenesis in the most ancient still existing metazoans, the sponges. GENERAL SIGNIFICANCE: It is shown that retinoic acid regulates the formation of the organic cylinder that surrounds the axis of the spicules and enables, as a scaffold, the radial apposition of new silica layers and hence the growth of the spicules.

Biosilica: Molecular Biology, Biochemistry and Function in Demosponges as well as its Applied Aspects for Tissue Engineering.

Biomineralization, biosilicification in particular (i.e. the formation of biogenic silica, SiO(2)), has become an exciting source of inspiration for the development of novel bionic approaches following 'nature as model'. Siliceous sponges are unique among silica-forming organisms in their ability to catalyze silica formation using a specific enzyme termed silicatein. In this study, we review the present state of knowledge on silicatein-mediated 'biosilica' formation in marine demosponges, the involvement of further molecules in silica metabolism and their potential applications in nano-biotechnology and bio-medicine. While most forms of multicellular life have developed a calcium-based skeleton, a few specialized organisms complement their body plan with silica. Only sponges (phylum Porifera) are able to polymerize silica enzymatically mediated in order to generate massive siliceous skeletal elements (spicules) during a unique reaction, at ambient temperature and pressure. During this biomineralization process (i.e. biosilicification), hydrated, amorphous silica is deposited within highly specialized sponge cells, ultimately resulting in structures that range in size from micrometres to metres. This peculiar phenomenon has been comprehensively studied in recent years, and in several approaches, the molecular background was explored to create tools that might be employed for novel bioinspired biotechnological and biomedical applications. Thus, it was discovered that spiculogenesis is mediated by the enzyme silicatein and starts intracellularly. The resulting silica nanoparticles fuse and subsequently form concentric lamellar layers around a central protein filament, consisting of silicatein and the scaffold protein silintaphin-1. Once the growing spicule is extruded into the extracellular space, it obtains final size and shape. Again, this process is mediated by silicatein and silintaphin-1/silintaphin-1, in combination with other molecules such as galectin and collagen. The molecular toolbox generated so far allows the fabrication of novel micro- and nano-structured composites, contributing to the economical and sustainable synthesis of biomaterials with unique characteristics. In this context, first bioinspired approaches implement recombinant silicatein and silintaphin-1 for applications in the field of biomedicine (biosilica-mediated regeneration of tooth and bone defects) with promising results.

Differential expression of the demosponge (Suberites domuncula) carotenoid oxygenases in response to light: protection mechanism against the self-produced toxic protein (Suberitine).

The demosponge Suberites domuncula has been described to contain high levels of a proteinaceous toxin, Suberitine, that displays haemolytic activityIn the present study this 7-8 kDa polypeptide has been isolated and was shown to exhibit also cytotoxic effects on cells of the same species. Addition of retinal, a recently identified metabolite of ß-carotene that is abundantly present in S. domuncula was found to reduce both the haemolytic and the cell toxic activity of Suberitine at a molar ratio of 1:1. Spectroscopic analyses revealed that the interaction between ß-carotene and Suberitine can be ascribed to a reversible energy transfer reaction. The enzyme that synthesises retinal in the sponge system is the ß,ß-carotene-15,15'-dioxygenase [carotene dioxygenase]. In order to clarify if this enzyme is the only ß-carotene-metabolizing enzyme a further oxygenase had been identified and cloned, the (related) carotenoid oxygenase. In contrast to the dioxygenase, the carotenoid oxygenase could not degrade ß-carotene or lycopene in Escherichia coli strains that produced these two carotenoids; therefore it had been termed related-carotenoid oxygenase. Exposure of primmorphs to light of different wavelengths from the visible spectrum resulted after 3 days in a strong upregulation of the dioxygenase in those 3D-cell aggregates that had been incubated with ß-carotene. The strongest effect is seen with blue light at a maximum around 490 nm. It is concluded that the toxin Suberitine is non-covalently modified by retinal, the cleavage product from ß-carotene via the enzyme carotene dioxygenase, a light inducible oxygenase. Hence, this study highlights that in S. domuncula the bioactive metabolite, retinal, has the property to detoxify its homologous toxin.

Isolation of the silicatein-α interactor silintaphin-2 by a novel solid-phase pull-down assay.

The skeleton of siliceous sponges consists of amorphous biogenous silica (biosilica). Biosilica formation is driven enzymatically by means of silicatein(s). During this unique process of enzymatic polycondensation, skeletal elements (spicules) that enfold a central proteinaceous structure (axial filament), mainly comprising silicatein, are formed. However, only the concerted action of silicatein and other proteins can explain the genetically controlled diversity of spicular morphotypes, from simple rods with pointed ends to intricate structures with up to six rays. With the scaffold protein silintaphin-1, a first silicatein interactor that facilitates the formation of the axial filament and, consequently, of the growing spicule was discovered. In this study, a new interactor has been identified by both a conventional yeast two-hybrid library screening and a newly established pull-down assay. For the latter approach, silicatein-α has been bioengineered to carry a Glu tag, which confers binding affinity to hydroxyapatite. After immobilization on a solid-phase matrix (hydroxyapatite), the Glu-tagged silicatein was used as bait for the identification of interactors. Both approaches revealed a 15 kDa polypeptide, and its identity was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Colocalization of silintaphin-2 and silicatein-α within the axial filament and on the spicule surface was shown by immunohistological analyses. Subsequent autoradiography demonstrated the Ca(2+) binding affinity of this silicatein interactor. These findings indicate that both proteins operate in concert during spiculogenesis. Besides binding of calcium, silintaphin-2 shares several structural features with certain acidic, secreted extracellular matrix proteins that facilitate tissue mineralization in Metazoa. Hence, silintaphin-2 might mediate signal transduction during spiculogenesis or may play a more direct role during biosilica formation, in concert with silicatein.

Molecular biomineralization: toward an understanding of the biogenic origin of polymetallic nodules, seamount crusts, and hydrothermal vents.

Polymetallic nodules and crusts, hydrothermal vents from the Deep Sea are economically interesting, since they contain alloying components, e.g., manganese or cobalt, that are used in the production of special steels; in addition, they contain rare metals applied for plasma screens, for magnets in hard disks, or in hybrid car motors. While hydrothermal vents can regenerate in weeks, polymetallic nodules and seamount crusts grow slowly. Even though the geochemical basis for the growth of the nodules and crusts has been well studied, the contribution of microorganisms to the formation of these minerals remained obscure. Recent HR-SEM (high-resolution scanning electron microscopy) analyses of nodules and crusts support their biogenic origin. Within the nodules, bacteria with surface S-layers are arranged on biofilm-like structures, around which Mn deposition starts. In crusts, coccoliths represent the dominant biologically formed structures that act as bio-seeds for an initial Mn deposition. In contrast, hydrothermal vents have apparently an abiogenic origin; however, their minerals are biogenically transformed by bacteria. In turn, strategies can now be developed for biotechnological enrichment as well as selective dissolution of metals from such concretions. We are convinced that the recent discoveries will considerably contribute to our understanding of the participation of organic matrices in the enrichment of those metals and will provide the basis for feasibility studies for biotechnological applications.

Biosilica-based strategies for treatment of osteoporosis and other bone diseases.

Osteoporosis is a common disease in later life, which has become a growing public health problem. This degenerative bone disease primarily affects postmenopausal women, but also men may suffer from reduced bone mineral density. The development of prophylactic treatments and medications of osteoporosis has become an urgent issue due to the increasing proportion of the elderly in the population. Apart from medical/hormonal treatments, current strategies for prophylaxis of osteoporosis are primarily based on calcium supplementation as a main constituent of bone hydroxyapatite mineral. Despite previous reports suggesting an essential role in skeletal growth and development, the significance of the trace element silicon in human bone formation has attracted major scientific interest only rather recently. The interest in silicon has been further increased by the latest discoveries in the field of biosilicification, the formation of the inorganic silica skeleton of the oldest still extant animals on Earth, the sponges, which revealed new insights in the biological function of this element. Sponges make use of silicon to build up their inorganic skeleton which consists of biogenously formed polymeric silica (biosilica). The formation of biosilica is mediated by specific enzymes, silicateins, which have been isolated, characterized, and expressed in a recombinant way. Epidemiological studies revealed that dietary silicon reduces the risk of osteoporosis and other bone diseases. Recent results allowed for the first time to understand the molecular mechanism underlying the protective effect of silicic acid/biosilica against osteoporosis. Biosilica was shown to modulate the ratio of expression of two cytokines involved in bone formation-RANKL and osteoprotegerin. Hence, biosilica has been proposed to have a potential in prophylaxis and therapy of osteoporosis and related bone diseases.

The unique invention of the siliceous sponges: their enzymatically made bio-silica skeleton.

Sponges are sessile filter feeders that, among the metazoans, evolved first on Earth. In the two classes of the siliceous sponges (the Demospongiae and the Hexactinellida), the complex filigreed body is stabilized by an inorganic skeleton composed of amorphous silica providing them a distinct body shape and plan. It is proposed that the key innovation that allowed the earliest metazoans to form larger specimens was the enzyme silicatein. This enzyme is crucial for the formation of the siliceous skeleton. The first sponge fossils with body preservation were dated back prior to the "Precambrian-Cambrian" boundary [Vendian (610-545 Ma)/Ediacaran (542-580 Ma)]. A further molecule required for the formation of a hard skeleton was collagen, fibrous organic filaments that need oxygen for their formation. Silicatein forming the spicules and collagen shaping their morphology are the two organic components that control the appositional growth of these skeletal elements. This process starts in both demosponges and hexactinellids intracellularly and is completed extracellularly where the spicules may reach sizes of up to 3 m. While the basic strategy of their formation is identical in both sponge classes, it differs on a substructural level. In Hexactinellida, the initial silica layers remain separated, those layers bio-fuse (bio-sinter) together in demosponges. In some sponge taxa, e.g., the freshwater sponges from the Lake Baikal, the individual spicules are embedded in an organic matrix that is composed of the DUF protein. This protein comprises clustered stretches of amino acid sequences composed of pronounced hydrophobic segments, each spanning around 35 aa. We concluded with the remark of Thompson (1942) highlighting that "the sponge-spicule is a typical illustration of the theory of 'bio-crystallisation' to form 'biocrystals' ein Mittelding between an inorganic crystal and an organic secretion." Moreover, the understanding of the enzymatic formation of the spicules conferred sponge biosilica a considerable economical actuality as a prime raw material of this millennium.

Demosponge EST sequencing reveals a complex genetic toolkit of the simplest metazoans.

Sponges (Porifera) are among the simplest living and the earliest branching metazoans. They hold a pivotal role for studying genome evolution of the entire metazoan branch, both as an outgroup to Eumetazoa and as the closest branching phylum to the common ancestor of all multicellular animals (Urmetazoa). In order to assess the transcription inventory of sponges, we sequenced expressed sequence tag libraries of two demosponge species, Suberites domuncula and Lubomirskia baicalensis, and systematically analyzed the assembled sponge transcripts against their homologs from complete proteomes of six well-characterized metazoans--Nematostella vectensis, Caenorhabditis elegans, Drosophila melanogaster, Strongylocentrotus purpuratus, Ciona intestinalis, and Homo sapiens. We show that even the earliest metazoan species already have strikingly complex genomes in terms of gene content and functional repertoire and that the rich gene repertoire existed even before the emergence of true tissues, therefore further emphasizing the importance of gene loss and spatio-temporal changes in regulation of gene expression in shaping the metazoan genomes. Our findings further indicate that sponge and human genes generally show similarity levels higher than expected from their respective positions in metazoan phylogeny, providing direct evidence for slow rate of evolution in both "basal" and "apical" metazoan genome lineages. We propose that the ancestor of all metazoans had already had an unusually complex genome, thereby shifting the origins of genome complexity from Urbilateria to Urmetazoa.

Sponge biosilica formation involves syneresis following polycondensation in vivo.

Syneresis is a process observed during the maturation/aging of silica gels obtained by sol-gel synthesis that results in shrinkage and expulsion of water due to a rearrangement and increase in the number of bridging siloxane bonds. Here we describe how the process of biosilica deposition during spicule ("biosilica" skeleton of the siliceous sponges) formation involves a phase of syneresis that occurs after the enzyme-mediated polycondensation reaction. Primmorphs from the demosponge Suberites domuncula were used to study syneresis and the inhibition of this mechanism. We showed by scanning electron microscopy that spicules added to primmorphs that have been incubated with manganese sulfate fuse together through the deposition of silica spheres and bridges. Energy-dispersive X-ray mapping of the newly formed deposits showed high silicon and oxygen content. These biosilica deposits contain a comparably higher percentage of water than mature/aged spicules. Quantitative real-time polymerase chain reaction analyses revealed that the addition of silicate to primmorph cultures resulted in a marked upregulation of the expression of the aquaporin gene and of the genes encoding the silica anabolic enzyme silicatein-α and the silica catabolic enzyme silicase. On the other hand, addition of manganese sulfate, either alone or together with silicate, caused a strong reduction in the level of aquaporin transcripts, although this metal ion did not essentially affect the silicate-induced increase in silicatein-α and silicase gene expression. We conclude that the secondary silica deposits formed on spicules under physiological conditions in the presence of silicate fuse together and subsequently undergo syneresis, which is facilitated by the removal of water through aquaporin channels. In growing spicules, these processes of biosilica formation and syneresis in the lamellar monolithic structures precede the final step of "biosintering" during which the massive biosilica rods of the spicules are formed.

Chemical mimicry: hierarchical 1D TiO2@ZrO2 core-shell structures reminiscent of sponge spicules by the synergistic effect of silicatein-α and silintaphin-1.

In nature, mineralization of hard tissues occurs due to the synergistic effect of components present in the organic matrix of these tissues, with templating and catalytic effects. In Suberites domuncula, a well-studied example of the class of demosponges, silica formation is mediated and templated by an axial proteinaceous filament with silicatein-α, one of the main components. But so far, the effect of other organic constituents from the proteinaceous filament on the catalytic effect of silicatein-α has not been studied in detail. Here we describe the synthesis of core-shell TiO(2)@SiO(2) and TiO(2)@ZrO(2) nanofibers via grafting of silicatein-α onto a TiO(2) nanowire backbone followed by a coassembly of silintaphin-1 through its specifically interacting domains. We show for the first time a linker-free, one-step funtionalization of metal oxides with silicatein-α using glutamate tag. In the presence of silintaphin-1 silicatein-α facilitates the formation of a dense layer of SiO(2) or ZrO(2) on the TiO(2)@protein backbone template. The immobilization of silicatein-α onto TiO(2) probes was characterized by atomic force microscopy (AFM), optical light microscopy, and high-resolution transmission electron microscopy (HRTEM). The coassembly of silicatein-α and silintaphin-1 may contribute to biomimetic approaches that pursue a controlled formation of patterned biosilica-based biomaterials.

Circumferential spicule growth by pericellular silica deposition in the hexactinellid sponge Monorhaphis chuni.

The giant basal spicule of the hexactinellid sponge Monorhaphis chuni represents the longest natural siliceous structure on Earth. This spicule is composed of concentrically arranged lamellae that are approximately 10 µm thick. In the present study, we investigated the formation of outer lamellae on a cellular level using microscopic and spectroscopic techniques. It is shown that the formation of an outermost lamella begins with the association of cell clusters with the surface of the thickening and/or growing spicule. The cells release silica for controlled formation of a lamella. The pericellular (silica) material fuses to a delimited and textured layer of silica with depressions approximately 20-30 µm in diameter. The newly formed layer initially displays 40 µm wide, well-structured banded ribbons and only attains its plain surface in a final step. The chemical composition in the depressions was studied using energy dispersive X-ray spectroscopy and by staining with Texas Red. The data suggest that those depressions are the nests for the silica-forming cells and that silica formation starts with a direct association of silica-forming cells with the outer surface of the spicule, where they remain and initiate the development of the next lamellae.

Inducible ASABF-type antimicrobial peptide from the sponge Suberites domuncula: microbicidal and hemolytic activity in vitro and toxic effect on molluscs in vivo.

Since sponges, as typical filter-feeders, are exposed to a high load of attacking prokaryotic and eukaryotic organisms, they are armed with a wide arsenal of antimicrobial/cytostatic low-molecular-weight, non-proteinaceous bioactive compounds. Here we present the first sponge agent belonging to the group of ASABF-type antimicrobial peptides. The ASABF gene was identified and cloned from the demosponge Suberites domuncula. The mature peptide, with a length of 64 aa residues has a predicted pI of 9.24, and comprises the characteristic CSα ß structural motif. Consequently, the S. domuncula ASABF shares high similarity with the nematode ASABFs; it is distantly related to the defensins. The recombinant peptide was found to display besides microbicidal activity, anti-fungal activity. In addition, the peptide lyses human erythrocytes. The expression of ASABF is upregulated after exposure to the apoptosis-inducing agent 2,2'-dipyridyl. During the process of apoptosis of surface tissue of S. domuncula, grazing gastropods (Bittium sp.) are attracted by quinolinic acid which is synthesized through the kynurenine pathway by the enzyme 3-hydroxyanthranilate 3,4-dioxygenase (HAD). Finally, the gastropods are repelled from the sponge tissue by the ASABF. It is shown that the effector peptide ASABF is sequentially expressed after the induction of the HAD gene and a caspase, as a central enzyme executing apoptosis.

Flashing light signaling circuit in sponges: endogenous light generation after tissue ablation in Suberites domuncula.

The skeleton of siliceous sponges (phylum Porifera: classes Demospongiae and Hexactinellida), composed of tightly interacting spicules that assemble to a genetically fixed scaffold, is formed of bio-silica. This inorganic framework with the quality of quartz glass has been shown to operate as light waveguide in vitro and very likely has a similar function in vivo. Furthermore, the molecular toolkit for endogenous light generation (luciferase) and light/photon harvesting (cryptochrome) has been identified in the demosponge Suberites domuncula. These three components of a light signaling system, spicules-luciferase-cryptochrome, are concentrated in the surface layers (cortex) of the poriferan body. Specimens from which this cortex has been removed/ablated do not emit light. However, with regeneration and reconstitution of the cortex the animals re-gain the capacity to flash light. This newly discovered characteristic of sponges to generate light prompted us to investigate the genetic basis for the endogenous light signaling system. As a potential transcription factor involved in the expression of luciferase and cryptochrome, a SOX-related protein has been identified. In dark-adapted animals or in tissue from below the cortex region, the medulla, no gene or protein expression of SOX-related protein, luciferase, and cryptochrome could be detected. However, during the regeneration of the cortex, a stage-specific expression pattern was recorded: SOX-related protein > luciferase > cryptochrome. We conclude that a flashing light signaling circuit exists, which might control the retinoic acid-induced differentiation of stem cells into pulsating and contracting sponge cells, that is, pinacocytes and myocytes.