SILICON METABOLISM IN DIATOMS: IMPLICATIONS FOR GROWTH

Diatoms are the world's largest contributors to biosilicification and are one of the predominant contributors to global carbon fixation. Silicon is a major limiting nutrient for diatom growth and hence is a controlling factor in primary productivity. Because our understanding of the cellular metabolism of silicon is limited, we are not fully knowledgeable about intracellular factors that may affect diatom productivity in the oceans. The goal of this review is to present an overview of silicon metabolism in diatoms and to identify areas for future research. Numerous studies have characterized parameters of silicic acid uptake by diatoms, and molecular characterization of transport has begun with the isolation of genes encoding the transporter proteins. Multiple types of silicic acid transporter gene have been identified in a single diatom species, and multiple types appear to be present in all diatom species. The controlled expression and perhaps localization of the transporters in the cell may be factors in the overall regulation of silicic acid uptake. Transport can also be regulated by the rate of silica incorporation into the cell wall, suggesting that an intracellular sensing and control mechanism couples transport with incorporation. Sizable intracellular pools of soluble silicon have been identified in diatoms, at levels well above saturation for silica solubility, yet the mechanism for maintenance of supersaturated levels has not been determined. The mechanism of intracellular transport of silicon is also unknown, but this must be an important part of the silicification process because of the close coupling between silica incorporation and uptake. Although detailed ultrastructural analyses of silica deposition have been reported, we know little about the molecular details of this process. However, proteins occluded within silica that promote silicification in vitro have recently been characterized, and the application of molecular techniques holds the promise of great advances in this area. Cellular energy for silicification and transport comes from aerobic respiration without any direct involvement of photosynthetic energy. As such, diatom silicon metabolism differs from that of other major limiting nutrients such as nitrogen and phosphorous, which are closely linked to photosynthetic metabolism. Cell wall silicification and silicic acid transport are tightly coupled to the cell cycle, which results in a dependency in the extent of silicification on growth rate. Silica dissolution is an important part of diatom cellular silicon metabolism, because dissolution must be prevented in the living cell, and because much of the raw material for mineralization in natural assemblages is supplied by dissolution of dead cells. Perhaps part of the reason for the ecological success of diatoms is due to their use of a silicified cell wall, which has been calculated to impart a substantial energy savings to organisms that have them. However, the growth of diatoms and other siliceous organisms has depleted the oceans of silicon, such that silicon availability is now a major factor in the control of primary productivity. Much new progress in understanding silicon metabolism in diatoms is expected because of the application of molecular approaches and sophisticated analytical techniques. Such insight is likely to lead to a greater understanding of the role of silicon in controlling diatom growth, and hence primary productivity, and of the mechanisms involved in the formation of the intricate silicified structures of the diatom cell wall.     

The Probable Mechanism for Silicon Capture by Diatom Algae: Assimilation of Polycarbonic Acids with Diatoms—Is Endocytosis a Key Stage in Building of Siliceous Frustules?

Many organisms including unicellular (diatoms, radiolaria, and chrysophytes), higher plants (rice and horsetail) and animals (sponges) use silica as a main part of skeletons. The bioavailable form of silicon is silicic acid and the mechanism of silicic acid penetration into living cells is still an enigma. Macropinocytosis was assumed as a key stage of the silicon capture by diatoms but assimilation of monomeric silicic acid by this way requires enormous amounts of water to be passed through the cell. We hypothesized that silicon can be captured by diatoms via endocytosis in the form of partially condensed silicic acid (oligosilicates) whose formation on the diatom surface was supposed. Oligosilicates are negatively charged nanoparticles and similar to coils of poly(acrylic acid) (PAA). We have synthesized fluorescent tagged PAA as well as several neutral and positively charged polymers. Cultivation of the diatom Ulnaria ferefusiformis in the presence of these polymers showed that only PAA is able to penetrate into siliceous frustules. The presence of PAA in the frustules was confirmed with chromatography and PAA causes various aberrations of the valve morphology. Growth of U. ferefusiformis and two other diatoms in the presence of tri- and tetracarbonic fluorescent tagged acids points to the ability of diatoms to recognize substances that bear four acidic groups and to include them into siliceous frustules. Thus, partial condensation of silicic acid is a plausible first stage of silicon assimilation.     

Silicon nanotechnologies of pigmented heterokonts

Many pigmented heterokonts are able to synthesize elements of their cell walls (the frustules) of dense biogenic silica. These include diatom algae, which occupy a significant place in the biosphere. The siliceous frustules of diatoms have species-specific patterns of surface structures between 10 and a few hundred nanometers. The present review considers possible mechanisms of uptake of silicic acid from the aquatic environment, its transport across the plasmalemma, and intracellular transport and deposition of silica inside the specialized Silica Deposition Vesicle (SDV) where elements of the new frustule are formed. It is proposed that a complex of silicic acid with positively charged proteins silaffins and polypropylamines remains a homogeneous solution during the intracellular transport to SDV, where biogenic silica precipitates. The high density of the deposited biogenic silica may be due to removal of water from the SDV by aquaporins followed by syneresis--a process during which pore water is expelled from the network of the contracting gel. The pattern of aquaporins in the silicalemma, the membrane embracing the SDV, can determine the pattern of species-specific siliceous nanostructures.     

Computational modelling of diatom silicic acid transporters predicts a conserved fold with implications for their function and evolution

Diatoms are an important group of algae that can produce intricate silicified cell walls (frustules). The complex process of silicification involves a set of enigmatic integral membrane proteins that are thought to actively transport the soluble precursor of biosilica, dissolved silicic acid. Full-length silicic acid transporters are found widely across the diatoms while homologous shorter proteins have now been identified in a range of other organisms. It has been suggested that modern silicic acid transporters arose from the union of such partial sequences. Here, we present a computational study of the silicic acid transporters and related transporter-like sequences to help understand the structure, function and evolution of this class of membrane protein. The AlphaFold software predicts that all of the protein sequences studied here share a common fold in the membrane domain which is entirely different from the predicted folds of non-homologous silicic acid transporters from plants. Substrate docking reveals how conserved polar residues could interact with silicic acid at a central solvent-accessible binding site, consistent with an alternating access mechanism of transport. The structural conservation between these proteins supports a model where modern silicon transporters evolved from smaller ancestral proteins by gene fusion.     

Conservative motif CMLD in silicic acid transport proteins of diatom algae

Sequencing of fragments of genes coding for silicic acid transport (SIT) proteins of diatoms of evolutionary distant classes (centric Chaetoceros muelleri Lemmermann, pennate araphid Synedra acus Kützing, pennate raphid Phaeodactylum tricornutum Bohlin, and pennate with keeled raphe system Cylindrotheca fusiformis Reimann et Lewin), revealed the presence in these proteins of a conservative amino acid motif CMLD. Hydropathy profiles suggest that CMLD occupies a position between two transmembrane strands which do not contain lysine and arginine residues. The two strands are good candidates for the role of the channel along which transport of silicic acid occurs. CMLD is a rare motif. Diatoms are known to need Zn2+ for the incorporation of silica. Presumably, CMLD is the site of Zn2+ binding of SITs. We found that the growth of diatoms is inhibited by a negatively charged alkylating reagent 5-(2-iodoacetamidoethyl)aminonaphtalene-1-sulfonic acid which cannot penetrate through the cell membrane. Cysteine of CMLD can be a target of this reagent. Synthetic peptide NCMLDY forms a complex with Zn2+, as revealed by the fact that the ion considerably reduces the rate of alkylation of the peptide.     

Silica-containing inclusions in the cytoplasm of diatom <Emphasis Type="Italic">Synedra acus</Emphasis>

Cells of the araphid pennate diatom Synedra acus subsp. radians contain large inclusion (1–15 μm) storing silica, as revealed by transmission electron microscopy and EDX-analysis. The size of the inclusions increases with the time of cultivation of diatoms. Approximate concentration of SiO₂ in the inclusions is 1–4%. It is assumed that silica in the inclusions is present as gelatinized silica-gel. These results explain the possible mechanism of transport of silicic acid into the cell against the concentration gradient.     

Silicon uptake in diatoms revisited: a model for saturable and nonsaturable uptake kinetics and the role of silicon transporters

The silicic acid uptake kinetics of diatoms were studied to provide a mechanistic explanation for previous work demonstrating both nonsaturable and Michaelis-Menten-type saturable uptake. Using (68)Ge(OH)(4) as a radiotracer for Si(OH)(4), we showed a time-dependent transition from nonsaturable to saturable uptake kinetics in multiple diatom species. In cells grown under silicon (Si)-replete conditions, Si(OH)(4) uptake was initially nonsaturable but became saturable over time. Cells prestarved for Si for 24 h exhibited immediate saturable kinetics. Data suggest nonsaturability was due to surge uptake when intracellular Si pool capacity was high, and saturability occurred when equilibrium was achieved between pool capacity and cell wall silica incorporation. In Thalassiosira pseudonana at low Si(OH)(4) concentrations, uptake followed sigmoidal kinetics, indicating regulation by an allosteric mechanism. Competition of Si(OH)(4) uptake with Ge(OH)(4) suggested uptake at low Si(OH)(4) concentrations was mediated by Si transporters. At high Si(OH)(4), competition experiments and nonsaturability indicated uptake was not carrier mediated and occurred by diffusion. Zinc did not appear to be directly involved in Si(OH)(4) uptake, in contrast to a previous suggestion. A model for Si(OH)(4) uptake in diatoms is presented that proposes two control mechanisms: active transport by Si transporters at low Si(OH)(4) and diffusional transport controlled by the capacity of intracellular pools in relation to cell wall silica incorporation at high Si(OH)(4). The model integrates kinetic and equilibrium components of diatom Si(OH)(4) uptake and consistently explains results in this and previous investigations.     

When to say when: can excessive drinking explain silicon uptake in diatoms?

Diatoms are the single most important drivers of the oceanic silicon biogeochemical cycle. Due to their considerable promise in nanotechnology, there is tremendous interest in understanding the mechanism by which they produce their intricately and ornately decorated silica‐based cell wall. Although specific proteins have been implicated in some of the key steps of silicification, the exact mechanisms are poorly understood. Silicon transporters, identified in both diatoms and silicoflagellates, are hypothesized to mediate silicon uptake. Recently, macropinocytosis, the non‐specific engulfment of extracellular fluid, was proposed as a more energetically favorable uptake mechanism, which can also explain the long‐observed effect of salinity on frustule morphology. We explore the bioenergetic, membrane recycling, and vacuolar volume requirements that must be satisfied for pinocytosis‐mediated silicon uptake. These calculated requirements contrast starkly with existing data on diatom physiology, uptake kinetics, growth, and ultrastructure, leading us to conclude that pinocytosis cannot be the primary mechanism of silicon uptake.     

Characterization of a silicon transporter gene family in Cylindrotheca fusiformis: sequences, expression analysis, and identification of homologs in other diatoms

The transport of silicon is an integral part of the synthesis of the silicified cell wall of diatoms, yet knowledge of the number, features, and regulation of silicon transporters is lacking. We report the isolation and sequence determination of five silicon transporter (SIT) genes from Cylindrotheca fusiformis, and examine their expression patterns during cell wall synthesis. The encoded SIT amino acid sequences are highly conserved in their putative transmembrane domains. Nine conserved cysteines in this domain may account for the sensitivity of silicon uptake to sulfhydryl blocking agents. A less conserved C-terminal domain is predicted to form coiled-coil structures, suggesting that the SITs interact with other proteins. We show that SIT gene expression is induced just prior to, and during, cell wall synthesis. The genes are expressed at very different levels, and SIT1 is expressed in a different pattern from SIT 2–5. Hybridization experiments show that multiple SIT gene copies are present in all diatom species tested. From the data we infer that individual transporters play specific roles in silicon uptake, and propose that the cell regulates uptake by controlling the amount or location of each. The identification of all SIT genes in C. fusiformis will enhance our understanding of the mechanism and control of silicon transport in diatoms.     

Inspired by nature: an exploration of biocatalyzed siloxane bond formation and cleavage

The intricate siliceous architectures of diatom species have inspired our exploration of biosilicification. In vitro studies of natural systems within the area of silica biosynthesis are complicated. Previous studies, which included biomimetic approaches, often failed to recognize the chemistry of silicic acid and its analogues. To better understand the role of various proteins in the biosilicification process, recent studies have been conducted to test the ability of enzymes to catalyze the formation and cleavage of siloxane bonds. Notably, biocatalysis at silicon was observed. Further understanding of the biotransformation strategy in the design and synthesis of structurally complex materials would be beneficial.     

Possible role of ubiquitin in silica biomineralization in diatoms: identification of a homologue with high silica affinity

In diatom silicon biomineralization peptides are believed to play a role in silica precipitation and the consequent structure direction of the cell wall. Characterization of such peptides should reveal the nature of this organic-inorganic interaction, knowledge that may eventually well be used to expand the existing range of artificial silicas ("biomimicking"). Biochemical studies on Navicula pelliculosa revealed a set of proteins, which have a high affinity for a solid silica matrix; some were only eluted from the matrix when SDS-denaturation was applied. One of the proteins with an affinity for silica, about 8.5 kDa, is shown to be a homologue of ubiquitin on the basis of its N-terminal amino acid sequence; ubiquitin itself is a highly conserved 8.6 kDa protein that is involved in protein degradation. This finding is in line with a model of silica biomineralization in diatoms that implies the removal of templating polypeptides when pores in the growing cell wall develop. Western blotting with specific anti-ubiquitin antibodies confirmed cross-reactivity. Immunocytochemical localization of ubiquitin indicates that it is present along the diatom cell wall and inside pores during different stages of valve formation.     

Characterization of a silicon transporter gene family in Cylindrotheca fusiformis : sequences, expression analysis, and identification of homologs in other diatoms

The transport of silicon is an integral part of the synthesis of the silicified cell wall of diatoms, yet knowledge of the number, features, and regulation of silicon transporters is lacking. We report the isolation and sequence determination of five silicon transporter (SIT) genes from Cylindrotheca fusiformis, and examine their expression patterns during cell wall synthesis. The encoded SIT amino acid sequences are highly conserved in their putative transmembrane domains. Nine conserved cysteines in this domain may account for the sensitivity of silicon uptake to sulfhydryl blocking agents. A less conserved C-terminal domain is predicted to form coiled-coil structures, suggesting that the SITs interact with other proteins. We show that SIT gene expression is induced just prior to, and during, cell wall synthesis. The genes are expressed at very different levels, and SIT1 is expressed in a different pattern from SIT 2–5. Hybridization experiments show that multiple SIT gene copies are present in all diatom species tested. From the data we infer that individual transporters play specific roles in silicon uptake, and propose that the cell regulates uptake by controlling the amount or location of each. The identification of all SIT genes in C. fusiformis will enhance our understanding of the mechanism and control of silicon transport in diatoms. Received: 17 June 1998 / Accepted: 22 September 1998     

BIOCHEMISTRY OF SILICA BIOMINERALIZATION IN DIATOMS

Diatoms are well known for the intricate patterns of their silica-based cell walls. The complex structures of diatom cell walls are species specific and become precisely reproduced during each cell division cycle, indicating a genetic control of silica biomineralization. Therefore, the formation of the diatom cell wall has been regarded as a paradigm for controlled production of nanostructured silica. However, the mechanisms allowing biosilicification to proceed at ambient temperature at high rates have remained enigmatic. Recently, we have shown that a set of highly cationic peptides (called silaffins) isolated from Cylindrotheca fusiformis shells are able to generate networks of silica nanospheres within seconds when added to a solution of silicic acid. Different silaffin species produce different morphologies of the precipitated silica. Silaffins contain covalently modified Lys-Lys elements. One of these lysine residues bears a novel type of protein modification, a polyamine consisting of 6–11 repeats of the N-methyl-propylamine unit. In addition to the silaffins, additional polyamine-containing substances have been isolated from a number of diatom species that may be involved in the control of biosilica morphology. Scanning electron microscopic analysis of diatom shells isolated in statu nascendi provide insights into the processes of pattern formation in biosilica. A model will be discussed that explains production of nanostructured biosilica in diatoms on the basis of these experimental results.     

Dissection of the frustules of the diatom Synedra acus under the action of picosecond impulses of submillimeter laser irradiation

Diatom algae realize highly intriguing processes of biosynthesis of siliceous structures in living cells under moderate conditions. Investigation of diatom physiology is complicated by frustule (siliceous exoskeleton). Frustules consist of valves and girdle bands which are adhered to each other by means of organic substances. Removal of the frustule from the lipid membrane of diatom cells would open new possibilities for study of silicon metabolism in diatoms. We found that submillimeter laser irradiation produced by a free-electron laser causes splitting of diatom frustules without destruction of cell content. This finding opens the way to direct study of diatom cell membrane and to isolation of cell organelles, including silica deposition vesicles. We suppose that the dissection action of the submillimeter irradiation results from unusual ultrasonic waves produced by the short (30–100 ps) but high-power (1 MW) terahertz laser impulses at 5.6 MHz frequency.     

SYNCHRONIZED GROWTH OF THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE) PROVIDES NOVEL INSIGHTS INTO CELL‐WALL SYNTHESIS PROCESSES IN RELATION TO THE CELL CYCLE1

Cell‐cycle effects in phytoplankton have both general and specific influences over a variety of cellular processes. Understanding these effects requires that the majority of cells in a culture are progressing through the same cell‐cycle stage, which requires synchronous growth. We report the development of a silicon starvation–recovery synchrony for the first diatom with a sequenced genome, Thalassiosira pseudonana Hasle et Heimdale, which provides several novel insights into the process of cell‐wall formation. After 24 h of silicate starvation, flow cytometry measurements indicated that 80% of the cells were arrested in the early G1 phase of the cell cycle and then upon silicate replenishment progressed synchronously through the cycle. An early G1‐arrest point was not previously documented in diatoms. After silicate replenishment, girdle‐band synthesis was confined to a particular period in G1, and cells did not lengthen in accordance with each girdle band added, which has implications related to cell growth and separation processes in diatoms. Measurements of silicic acid uptake, intracellular pools, and silica incorporation into the cell wall, coupled with fluorescence visualization of newly synthesized cell‐wall structures, provide the first direct measurements of silica amounts in individual girdle bands and valves in a diatom. Fluorescence imaging indicated why valves in T. pseudonana do not have to reduce in size with each generation and enabled visualization of intermediates in structure formation. The development of a synchrony procedure for T. pseudonana enables correlation of cellular events with the cell cycle, which should facilitate the use of genomic information.     

Multiparametric Analyses Reveal the pH-Dependence of Silicon Biomineralization in Diatoms

Diatoms, the major contributors of the global biogenic silica cycle in modern oceans, account for about 40% of global marine primary productivity. They are an important component of the biological pump in the ocean, and their assemblage can be used as useful climate proxies; it is therefore critical to better understand the changes induced by environmental pH on their physiology, silicification capability and morphology. Here, we show that external pH influences cell growth of the ubiquitous diatom Thalassiosira weissflogii, and modifies intracellular silicic acid and biogenic silica contents per cell. Measurements at the single-cell level reveal that extracellular pH modifications lead to intracellular acidosis. To further understand how variations of the acid-base balance affect silicon metabolism and theca formation, we developed novel imaging techniques to measure the dynamics of valve formation. We demonstrate that the kinetics of valve morphogenesis, at least in the early stages, depends on pH. Analytical modeling results suggest that acidic conditions alter the dynamics of the expansion of the vesicles within which silica polymerization occurs, and probably its internal pH. Morphological analysis of valve patterns reveals that acidification also reduces the dimension of the nanometric pores present on the valves, and concurrently overall valve porosity. Variations in the valve silica network seem to be more correlated to the dynamics and the regulation of the morphogenesis process than the silicon incorporation rate. These multiparametric analyses from single-cell to cell-population levels demonstrate that several higher-level processes are sensitive to the acid-base balance in diatoms, and its regulation is a key factor for the control of pattern formation and silicon metabolism.     

A new calcium binding glycoprotein family constitutes a major diatom cell wall component.

Diatoms possess silica-based cell walls with species-specific structures and ornamentations. Silica deposition in diatoms offers a model to study the processes involved in biomineralization. A new wall is produced in a specialized vesicle (silica deposition vesicle, SDV) and secreted. Thus proteins involved in wall biogenesis may remain associated with the mature cell wall. Here it is demonstrated that EDTA treatment removes most of the proteins present in mature cell walls of the marine diatom Cylindrotheca fusiformis. A main fraction consists of four related glycoproteins with a molecular mass of approximately 75 kDa. These glycoproteins were purified to homogeneity. They consist of repeats of Ca2+ binding domains separated by polypeptide stretches containing hydroxyproline. The proteins in the EDTA extract aggregate and precipitate in the presence of Ca2+. Immunological studies detected related proteins in the cell wall of the freshwater diatom Navicula pelliculosa, indicating that these proteins represent a new family of proteins that are involved in the biogenesis of diatom cell walls.