Nicole Poulsen

Diatoms—From Cell Wall Biogenesis to Nanotechnology

Diatoms are single-celled algae that produce intricately structured cell walls made of nanopatterned silica (SiO(2)). The cell wall structure is a species-specific characteristic demonstrating that diatom silica morphogenesis is genetically encoded. Understanding the molecular mechanisms by which a single cell executes the morphogenetic program for the formation of an inorganic material (biomineralization) is not only a fascinating biological problem, but also of great interest for nanomaterials science and technology. Recently, analysis of the organic components associated with diatom silica, the development of techniques for molecular genetic manipulation of diatoms, and two diatom genome sequencing projects are providing insight into the composition and mechanism of the remarkable biosilica-forming machinery.

Biosilica formation in diatoms: characterization of native silaffin-2 and its role in silica morphogenesis.

The biological formation of inorganic materials with complex form (biominerals) is a widespread phenomenon in nature, yet the molecular mechanisms underlying biomineral morphogenesis are not well understood. Among the most fascinating examples of biomineral structures are the intricately patterned, silicified cell walls of diatoms, which contain tightly associated organic macromolecules. From diatom biosilica a highly polyanionic phosphoprotein, termed native silaffin-2 (natSil-2), was isolated that carries unconventional amino acid modifications. natSil-2 lacked intrinsic silica formation activity but was able to regulate the activities of the previously characterized silica-forming biomolecules natSil-1A and long-chain polyamines. Combining natSil-2 and natSil-1A (or long-chain polyamines) generated an organic matrix that mediated precipitation of porous silica within minutes after the addition of silicic acid. Remarkably, the precipitate displayed pore sizes in the range 100–1000 nm, which is characteristic for diatom biosilica nanopatterns.

A new molecular tool for transgenic diatoms

Research in diatom biology has entered the postgenomic era since the recent completion of the Thalassiosira pseudonana genome project. However, the molecular tools available for genetic manipulation of diatoms are still sparse, impeding the functional analysis of diatom genes in vivo. Here we describe the first method for inducible gene expression in transgenic diatoms. This method uses a DNA cassette containing both promoter (Pnr) and terminator (Tnr) elements derived from the nitrate reductase gene of the diatom Cylindrotheca fusiformis. By using green fluorescent protein (gfp) cDNA as a reporter gene, it is demonstrated that gene expression under the control of the Pnr/Tnr cassette is switched off when cells are grown in the presence of ammonium ions and becomes switched on within 4 h when cells are transferred to medium containing nitrate. Incubating cells in nitrogen‐free medium switches on transcription of the gfp gene, yet gfp mRNA does not become translated into protein. This block on translation is released by the addition of nitrate, resulting in rapid onset of GFP production with a drastically reduced delay time of only 1 h. Altogether we have demonstrated that the Pnr/Tnr cassette enables inducible gene expression and control of both the level and timing of mRNA and protein expression in transgenic diatoms.

Molecular genetic manipulation of the diatom Thalassiosira pseudonana (bacillariophyceae)

Here, we describe the first system for genetic transformation of Thalassiosira pseudonana (Hustedt) Hasle et Heimdal, the only diatom for which a complete genome sequence is presently available. This method is based on microparticle bombardment followed by selection of transformants using the antibiotic nourseothricin. It exhibits the highest transformation efficiency compared with transformation systems for other diatom species. To achieve the high transformation efficiency, it is important to allow recovery of the bombarded T. pseudonana cells in non‐selective suspension culture before spreading on nourseothricin containing agar plates. It is demonstrated that T. pseudonana is readily susceptible to co‐transformation allowing for the simultaneous introduction of a non‐selective gene together with the selection marker gene. Both introduced genes are stably inherited even in the absence of the antibiotic selection pressure. We have developed two T. pseudonana‐specific expression vectors that can drive constitutive expression (vector pTpfcp) and inducible expression (vector pTpNR) of introduced genes. In combination with the available genome data the T. pseudonana transformation system is expected to provide a powerful tool for functional genomics in diatoms.

Silica Morphogenesis by Alternative Processing of Silaffins in the Diatom Thalassiosira pseudonana

For almost 200 years scientists have been fascinated by the ornate cell walls of the diatoms. These structures are made of amorphous silica, exhibiting species-spe- cific, mostly porous patterns in the nano- to micrometer range. Recently, from the diatom Cylindrotheca fusifor- mis unusual phosphoproteins (termed silaffins) and long chain polyamines have been identified and impli- cated in biosilica formation. However, analysis of the role of silaffins in morphogenesis of species-specific sil- ica structures has so far been hampered by the difficulty of obtaining structural data from these extremely com- plex proteins. In the present study, the five major silaf- fins from the diatom Thalassiosira pseudonana (tpSil1H, -1L, -2H, -2L, and -3) have been isolated, func- tionally analyzed, and structurally characterized, mak- ing use of the recently available genome data from this organism. Surprisingly, the silaffins of T. pseudonana and C. fusiformis share no sequence homology but are similar regarding amino acid composition and post- translational modifications. Silaffins tpSil1H and -2H are higher molecular mass isoforms of tpSil1L and -2L, respectively, generated in vivo by alternative processing of the same precursor polypeptides. Interestingly, only tpSil1H and -2H but not tpSil1L and -2L induce the for- mation of porous silica patterns in vitro, suggesting that the alternative processing event is an important step in morphogenesis of T. pseudonana biosilica.

Nanopatterned protein microrings from a diatom that direct silica morphogenesis

Diatoms are eukaryotic microalgae that produce species-specifically structured cell walls made of SiO2 (silica). Formation of the intricate silica structures of diatoms is regarded as a paradigm for biomolecule-controlled self-assembly of three-dimensional, nano- to microscale-patterned inorganic materials. Silica formation involves long-chain polyamines and phosphoproteins (silaffins and silacidins), which are readily soluble in water, and spontaneously form dynamic supramolecular assemblies that accelerate silica deposition and influence silica morphogenesis in vitro. However, synthesis of diatom-like silica structure in vitro has not yet been accomplished, indicating that additional components are required. Here we describe the discovery and intracellular location of six novel proteins (cingulins) that are integral components of a silica-forming organic matrix (microrings) in the diatom Thalassiosira pseudonana. The cingulin-containing microrings are specifically associated with girdle bands, which constitute a substantial part of diatom biosilica. Remarkably, the microrings exhibit protein-based nanopatterns that closely resemble characteristic features of the girdle band silica nanopatterns. Upon the addition of silicic acid the microrings become rapidly mineralized in vitro generating nanopatterned silica replicas of the microring structures. A silica-forming organic matrix with characteristic nanopatterns was also discovered in the diatom Coscinodiscus wailesii, which suggests that preassembled protein-based templates might be general components of the cellular machinery for silica morphogenesis in diatoms. These data provide fundamentally new insight into the molecular mechanisms of biological silica morphogenesis, and may lead to the development of self-assembled 3D mineral forming protein scaffolds with designed nanopatterns for a host of applications in nanotechnology.

A Systematic Screen to Discover and Analyze Apicoplast Proteins Identifies a Conserved and Essential Protein Import Factor

Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes – a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle.

Live Diatom Silica Immobilization of Multimeric and Redox-Active Enzymes

ABSTRACT Living organisms are adept in forming inorganic materials (biominerals) with unique structures and properties that exceed the capabilities of engineered materials. Biomimetic materials syntheses are being developed that aim at replicating the advantageous properties of biominerals in vitro and endow them with additional functionalities. Recently, proof-of-concept was provided for an alternative approach that allows for the production of biomineral-based functional materials in vivo. In this approach, the cellular machinery for the biosynthesis of nano-/micropatterned SiO2 (silica) structures in diatoms was genetically engineered to incorporate a monomeric, cofactor-independent (“simple”) enzyme, HabB, into diatom silica. In the present work, it is demonstrated that this approach is also applicable for enzymes with “complex” activity requirements, including oligomerization, metal ions, organic redox cofactors, and posttranslational modifications. Functional expression of the enzymes β-glucuronidase, glucose oxidase, galactose oxidase, and horseradish peroxidase in the diatom Thalassiosira pseudonana was accomplished, and 66 to 78% of the expressed enzymes were stably incorporated into the biosilica. The in vivo incorporated enzymes represent approximately 0.1% (wt/wt) of the diatom biosilica and are stabilized against denaturation and proteolytic degradation. Furthermore, it is demonstrated that the gene construct for in vivo immobilization of glucose oxidase can be utilized as the first negative selection marker for diatom genetic engineering.

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.