Nils Kröger

Isolation and biochemical characterization of underwater adhesives from diatoms

Many aquatic organisms are able to colonize surfaces through the secretion of underwater adhesives. Diatoms are unicellular algae that have the capability to colonize any natural and man-made submerged surfaces. There is great technological interest in both mimicking and preventing diatom adhesion, yet the biomolecules responsible have so far remained unidentified. A new method for the isolation of diatom adhesive material is described and its amino acid and carbohydrate composition determined. The adhesive materials from two model diatoms show differences in their amino acid and carbohydrate compositions, but also share characteristic features including a high content of uronic acids, the predominance of hydrophilic amino acid residues, and the presence of 3,4-dihydroxyproline, an extremely rare amino acid. Proteins containing dihydroxyphenylalanine, which mediate underwater adhesion of mussels, are absent. The data on the composition of diatom adhesives are consistent with an adhesion mechanism based on complex coacervation of polyelectrolyte-like biomolecules.

Pentalysine clusters mediate silica targeting of silaffins in Thalassiosira pseudonana

Background: Morphogenesis of diatom biosilica depends on a silaffin-dependent organic matrix inside intracellular vesicles (SDVs). Results: Silaffin-derived 12–14-mer peptides containing five modified lysines and several phosphoserines (pentalysine clusters) are sufficient for silica targeting in vivo. Conclusion: Pentalysine clusters function as address tags for SDV targeting of silaffins. Significance: Elucidating the molecular mechanisms for biogenesis of mineral-forming vesicles is essential for understanding biomineral morphogenesis. The biological formation of inorganic materials (biomineralization) often occurs in specialized intracellular vesicles. Prominent examples are diatoms, a group of single-celled eukaryotic microalgae that produce their SiO2 (silica)-based cell walls within intracellular silica deposition vesicles (SDVs). SDVs contain protein-based organic matrices that control silica formation, resulting in species specifically nanopatterned biosilica, an organic-inorganic composite material. So far no information is available regarding the molecular mechanisms of SDV biogenesis. Here we have investigated by fluorescence microscopy and subcellular membrane fractionation the intracellular transport of silaffin Sil3. Silaffins are a group of phosphoproteins constituting the main components of the organic matrix of diatom biosilica. We demonstrate that the N-terminal signal peptide of Sil3 mediates import into a specific subregion of the endoplasmic reticulum. Additional segments from the mature part of Sil3 are required to reach post-endoplasmic reticulum compartments. Further transport of Sil3 and incorporation into the biosilica (silica targeting) require protein segments that contain a high density of modified lysine residues and phosphoserines. Silica targeting of Sil3 is not dependent on a particular peptide sequence, yet a lysine-rich 12–14-amino acid peptide motif (pentalysine cluster), which is conserved in all silaffins, strongly promotes silica targeting. The results of the present work provide the first insight into the molecular mechanisms for biogenesis of mineral-forming vesicles from an eukaryotic organism.

Biochemical Composition and Assembly of Biosilica-associated Insoluble Organic Matrices from the Diatom Thalassiosira pseudonana

The nano- and micropatterned biosilica cell walls of diatoms are remarkable examples of biological morphogenesis and possess highly interesting material properties. Only recently has it been demonstrated that biosilica-associated organic structures with specific nanopatterns (termed insoluble organic matrices) are general components of diatom biosilica. The model diatom Thalassiosira pseudonana contains three types of insoluble organic matrices: chitin meshworks, organic microrings, and organic microplates, the latter being described in the present study for the first time. To date, little is known about the molecular composition, intracellular assembly, and biological functions of organic matrices. Here we have performed structural and functional analyses of the organic microrings and organic microplates from T. pseudonana. Proteomics analysis yielded seven proteins of unknown function (termed SiMat proteins) together with five known silica biomineralization proteins (four cingulins and one silaffin). The location of SiMat1-GFP in the insoluble organic microrings and the similarity of tyrosine- and lysine-rich functional domains identifies this protein as a new member of the cingulin protein family. Mass spectrometric analysis indicates that most of the lysine residues of cingulins and the other insoluble organic matrix proteins are post-translationally modified by short polyamine groups, which are known to enhance the silica formation activity of proteins. Studies with recombinant cingulins (rCinY2 and rCinW2) demonstrate that acidic conditions (pH 5.5) trigger the assembly of mixed cingulin aggregates that have silica formation activity. Our results suggest an important role for cingulins in the biogenesis of organic microrings and support the hypothesis that this type of insoluble organic matrix functions in biosilica morphogenesis.

Complex-shaped microbial biominerals for nanotechnology.

Single-celled microorganisms such as diatoms and coccolithophores produce inorganic microparticles with genetically controlled hierarchical nanopatterns. Besides serving as paradigms to inspire new routes for materials synthesis, biominerals themselves, particularly diatom biosilica, are increasingly utilized as templates for the synthesis of novel functional materials. Over the past decade, a large variety of methods have been established that allow not only for the attachment or coating of desired materials onto diatom biosilica but also for complete chemical conversion without altering the characteristic micro- and nanoscale morphology. Examples include the synthesis of materials for photonics (surface-enhanced Raman spectroscopy, SERS, extraordinary optical transmission, EOT), ultraresponsive and sensitive gas sensors, gas storage materials, and highly active catalysts. More recently, emerging insight into the cellular mechanisms of biosilica formation has enabled the in vivo functionalization of diatom biosilica through advanced cultivation techniques and genetic engineering. As a naturally renewable material, biominerals hold the promise of serving as an inexpensive and easily available resource for a future nanotechnology-based industry.

A Tyrosine-Rich Cell Surface Protein in the Diatom Amphora coffeaeformis Identified through Transcriptome Analysis and Genetic Transformation

Diatoms are single-celled eukaryotic microalgae that are ubiquitously found in almost all aquatic ecosystems, and are characterized by their intricately structured SiO2 (silica)-based cell walls. Diatoms with a benthic life style are capable of attaching to any natural or man-made submerged surface, thus contributing substantially to both microbial biofilm communities and economic losses through biofouling. Surface attachment of diatoms is mediated by a carbohydrate- and protein- based glue, yet no protein involved in diatom underwater adhesion has been identified so far. In the present work, we have generated a normalized transcriptome database from the model adhesion diatom Amphora coffeaeformis. Using an unconventional bioinformatics analysis we have identified five proteins that exhibit unique amino acid sequences resembling the amino acid composition of the tyrosine-rich adhesion proteins from mussel footpads. Establishing the first method for the molecular genetic transformation of A. coffeaeformis has enabled investigations into the function of one of these proteins, AC3362, through expression as YFP fusion protein. Biochemical analysis and imaging by fluorescence microscopy revealed that AC3362 is not involved in adhesion, but rather plays a role in biosynthesis and/or structural stability of the cell wall. The methods established in the present study have paved the way for further molecular studies on the mechanisms of underwater adhesion and biological silica formation in the diatom A. coffeaeformis.

Compartmentalisation of enzymes for cascade reactions through biomimetic layer-by-layer mineralization

Cellular metabolic pathways are paradigms for the rapid and waste-free conversion of molecules into useful products through multiple enzyme-catalyzed steps (cascade reactions). Attempts to establish efficient cascade reactions for technological applications have focused on mimicking natures high degree of organization by controlling the positioning of enzymes through immobilization in tailor-made compartments. The present work utilized peptide-mediated layer-by-layer mineralization as a facile and generic method for the compartmentalisation of multi-enzyme systems in nanoscale silica layers. It is demonstrated that, in a multilayer system, the overall rate of the reaction cascade was primarily affected by the placement of the enzyme catalyzing the first step, with the placement of the enzyme possessing the lowest catalytic efficiency also being an important factor. As the rate-limiting enzymes were positioned closer to the external silica surface, the overall rate of cascade reactions increased. Furthermore, distributing the enzymes into different adjacent silica compartments yielded higher overall cascade reaction rates compared to placement of the enzymes into the same silica layer. The synthetic methods and kinetic analyses presented here provide guidance for improving the performance of immobilized multi-enzyme systems for a wide range of technological applications.

Silicanin-1 is a conserved diatom membrane protein involved in silica biomineralization

BackgroundBiological mineral formation (biomineralization) proceeds in specialized compartments often bounded by a lipid bilayer membrane. Currently, the role of membranes in biomineralization is hardly understood.ResultsInvestigating biomineralization of SiO2 (silica) in diatoms we identified Silicanin-1 (Sin1) as a conserved diatom membrane protein present in silica deposition vesicles (SDVs) of Thalassiosira pseudonana. Fluorescence microscopy of GFP-tagged Sin1 enabled, for the first time, to follow the intracellular locations of a biomineralization protein during silica biogenesis in vivo. The analysis revealed incorporation of the N-terminal domain of Sin1 into the biosilica via association with the organic matrix inside the SDVs. In vitro experiments showed that the recombinant N-terminal domain of Sin1 undergoes pH-triggered assembly into large clusters, and promotes silica formation by synergistic interaction with long-chain polyamines.ConclusionsSin1 is the first identified SDV transmembrane protein, and is highly conserved throughout the diatom realm, which suggests a fundamental role in the biomineralization of diatom silica. Through interaction with long-chain polyamines, Sin1 could serve as a molecular link by which the SDV membrane exerts control on the assembly of biosilica-forming organic matrices in the SDV lumen.

PSCD Domains of Pleuralin-1 from the Diatom Cylindrotheca fusiformis: NMR Structures and Interactions with Other Biosilica-Associated Proteins.

Diatoms are eukaryotic unicellular algae characterized by silica cell walls and associated with three unique protein families, the pleuralins, frustulins, and silaffins. The NMR structure of the PSCD4 domain of pleuralin-1 from Cylindrotheca fusiformis contains only three short helical elements and is stabilized by five unique disulfide bridges. PSCD4 contains two binding sites for Ca(2+) ions with millimolar affinity. NMR-based interaction studies show an interaction of the domain with native silaffin-1A as well as with α-frustulins. The interaction sites of the two proteins mapped on the PSCD4 structure are contiguous and show only a small overlap. A plausible functional role of pleuralin could be to bind simultaneously silaffin-1A located inside the cell wall and α-frustulin coating the cell wall, thus connecting the interfaces between hypotheca and epitheca at the girdle bands. Restrained molecular dynamics calculations suggest a bead-chain-like structure of the central part of pleuralin-1.

Reconstituting the formation of hierarchically porous silica patterns using diatom biomolecules

The genetically-controlled formation of complex-shaped inorganic materials by living organisms is an intriguing phenomenon. It illustrates our incomplete understanding of biological morphogenesis and demonstrates the feasibility of ecologically benign routes for materials technology. Amorphous SiO2 (silica) is taxonomically the most widespread biomineral, with diatoms, a large group of single-celled microalgae, being the most prolific producers. Silica is the main component of diatom cell walls, which exhibit species-specific patterns of pores that are hierarchically arranged and endow the material with advantageous properties. Despite recent advances in characterizing diatom biomolecules involved in biosilica morphogenesis, the mechanism of this process has remained controversial. Here we describe the in vitro synthesis of diatom-like, porous silica patterns using organic components that were isolated from biosilica of the diatom Cyclotella cryptica. The synthesis relies on the synergism of soluble biomolecules (long-chain polyamines and proteins) with an insoluble nanopatterned organic matrix. Biochemical dissection of the process revealed that the long-chain polyamines rather than the proteins are essential for efficient in vitro synthesis of the hierarchically porous silica patterns. Our results support the organic matrix hypothesis for morphogenesis of diatom biosilica and introduce organic matrices from diatoms as a new tool for the synthesis of meso- to microporous inorganic materials.

Morphogenese der Silica-Zellwände von Diatomeen

Diatoms are unicellular microalgae that form SiO2(silica)-based cell walls with complex shapes and nano- to microscale patterns. Such biologically produced inorganic structures are paradigms for materials synthesis in nanotechnology. Morphogenesis of diatom silica is controlled by unusual biomolecules including intrinsically disordered proteins. Recently, new insight has been gained into the mechanism by which such ”unorderly” biomolecules are able to control formation of highly regular silica patterns.