RESUMEN
Morphogenesis of the intricate patterns of diatom silica cell walls is a protein-guided process, yet to date only very few such silica biomineralization proteins have been identified. Therefore, it is currently unknown whether all diatoms share conserved proteins of a basal silica forming machinery, and whether unique proteins are responsible for the morphogenesis of species-specific silica patterns. To answer these questions, we extracted proteins from the silica of three diatom species (Thalassiosira pseudonana, Thalassiosira oceanica, and Cyclotella cryptica) by complete demineralization of the cell walls. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis of the extracts identified 92 proteins that we name 'soluble silicome proteins' (SSPs). Surprisingly, no SSPs are common to all three species, and most SSPs showed very low similarity to one another in sequence alignments. In-depth bioinformatics analyses revealed that SSPs could be grouped into distinct classes based on short unconventional sequence motifs whose functions are yet unknown. The results from the in vivo localization of selected SSPs indicates that proteins, which lack sequence homology but share unconventional sequence motifs may exert similar functions in the morphogenesis of the diatom silica cell wall.
Asunto(s)
Diatomeas , Biomineralización , Cromatografía Liquida , Diatomeas/metabolismo , Proteoma/metabolismo , Dióxido de Silicio/química , Dióxido de Silicio/metabolismo , Espectrometría de Masas en TándemRESUMEN
Biofilm-forming benthic diatoms are key primary producers in coastal habitats, where they frequently dominate sunlit intertidal substrata. The development of gliding motility in raphid diatoms was a key molecular adaptation that contributed to their evolutionary success. However, the structure-function correlation between diatom adhesives utilized for gliding and their relationship to the extracellular matrix that constitutes the diatom biofilm is unknown. Here, we have used proteomics, immunolocalization, comparative genomics, phylogenetics and structural homology analysis to investigate the evolutionary history and function of diatom adhesive proteins. Our study identified eight proteins from the adhesive trails of Craspedostauros australis, of which four form a new protein family called Trailins that contain an enigmatic Choice-of-Anchor A (CAA) domain, which was acquired through horizontal gene transfer from bacteria. Notably, the CAA-domain shares a striking structural similarity with one of the most widespread domains found in ice-binding proteins (IPR021884). Our work offers new insights into the molecular basis for diatom biofilm formation, shedding light on the function and evolution of diatom adhesive proteins. This discovery suggests that there is a transition in the composition of biomolecules required for initial surface colonization and those utilized for 3D biofilm matrix formation.
Asunto(s)
Diatomeas , Diatomeas/metabolismo , Adhesivos/metabolismo , Transferencia de Gen Horizontal , Biopelículas , BacteriasRESUMEN
In 2004, Thalassiosira pseudonana was the first eukaryotic marine alga to have its genome sequenced. Since then, this species has quickly emerged as a valuable model species for investigating the molecular underpinnings of essentially all aspects of diatom life, particularly bio-morphogenesis of the cell wall. An important prerequisite for the model status of T. pseudonana is the ongoing development of increasingly precise tools to study the function of gene networks and their encoded proteins in vivo. Here, we briefly review the current toolbox for genetic manipulation, highlight specific examples of its application in studying diatom metabolism, and provide a peek into the role of diatoms in the emerging field of silica biotechnology.
Asunto(s)
Diatomeas , Dióxido de Silicio , Dióxido de Silicio/metabolismo , Diatomeas/genética , Diatomeas/metabolismo , Genoma , BiologíaRESUMEN
Diatoms are single-celled microalgae with silica-based cell walls (frustules) that are abundantly present in aquatic habitats, and form the basis of the food chain in many ecosystems. Many benthic diatoms have the remarkable ability to glide on all natural or man-made underwater surfaces using a carbohydrate- and protein-based adhesive to generate traction. Previously, three glycoproteins, termed FACs (Frustule Associated Components), have been identified from the common fouling diatom Craspedostauros australis and were implicated in surface adhesion through inhibition studies with a glycan-specific antibody. The polypeptide sequences of FACs remained unknown, and it was unresolved whether the FAC glycoproteins are indeed involved in adhesion, or whether this is achieved by different components sharing the same glycan epitope with FACs. Here we have determined the polypeptide sequences of FACs using peptide mapping by LC-MS/MS. Unexpectedly, FACs share the same polypeptide backbone (termed CaFAP1), which has a domain structure of alternating Cys-rich and Pro-Thr/Ser-rich regions reminiscent of the gel-forming mucins. By developing a genetic transformation system for C. australis, we were able to directly investigate the function of CaFAP1-based glycoproteins in vivo. GFP-tagging of CaFAP1 revealed that it constitutes a coat around all parts of the frustule and is not an integral component of the adhesive. CaFAP1-GFP producing transformants exhibited the same properties as wild type cells regarding surface adhesion and motility speed. Our results demonstrate that FAC glycoproteins are not involved in adhesion and motility, but might rather act as a lubricant to prevent fouling of the diatom surface.
Asunto(s)
Diatomeas , Diatomeas/genética , Mucinas/metabolismo , Cromatografía Liquida , Ecosistema , Espectrometría de Masas en Tándem , Glicoproteínas/metabolismoRESUMEN
Coastal regions contribute an estimated 20% of annual gross primary production in the oceans, despite occupying only 0.03% of their surface area. Diatoms frequently dominate coastal sediments, where they experience large variations in light regime resulting from the interplay of diurnal and tidal cycles. Here, we report on an extensive diurnal transcript profiling experiment of the motile benthic diatom Seminavis robusta. Nearly 90% (23 328) of expressed protein-coding genes and 66.9% (1124) of expressed long intergenic non-coding RNAs showed significant expression oscillations and are predominantly phasing at night with a periodicity of 24 h. Phylostratigraphic analysis found that rhythmic genes are enriched in highly conserved genes, while diatom-specific genes are predominantly associated with midnight expression. Integration of genetic and physiological cell cycle markers with silica depletion data revealed potential new silica cell wall-associated gene families specific to diatoms. Additionally, we observed 1752 genes with a remarkable semidiurnal (12-h) periodicity, while the expansion of putative circadian transcription factors may reflect adaptations to cope with highly unpredictable external conditions. Taken together, our results provide new insights into the adaptations of diatoms to the benthic environment and serve as a valuable resource for the study of diurnal regulation in photosynthetic eukaryotes.
Asunto(s)
Adaptación Fisiológica , Ritmo Circadiano/genética , Diatomeas/citología , Diatomeas/fisiología , Expresión Génica , Ciclo Celular/genética , Pared Celular/genética , Pared Celular/metabolismo , Cloroplastos/genética , Enzimas/genética , Enzimas/metabolismo , Evolución Molecular , Mitocondrias/genética , Filogenia , Plancton/genética , Plancton/fisiología , ARN Largo no CodificanteRESUMEN
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.
Asunto(s)
Diatomeas/ultraestructura , Matriz Extracelular/metabolismo , Dióxido de Silicio/metabolismo , Pared Celular/química , Pared Celular/ultraestructura , Diatomeas/química , Matriz Extracelular/química , Dióxido de Silicio/químicaRESUMEN
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.
Asunto(s)
Pared Celular/metabolismo , Diatomeas/metabolismo , Oligopéptidos/metabolismo , Péptidos/metabolismo , Dióxido de Silicio/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Pared Celular/ultraestructura , Vesículas Citoplasmáticas/metabolismo , Diatomeas/genética , Diatomeas/ultraestructura , Retículo Endoplásmico/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Confocal , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Modelos Biológicos , Datos de Secuencia Molecular , Oligopéptidos/genética , Péptidos/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Señales de Clasificación de Proteína/genética , Transporte de ProteínasRESUMEN
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.
Asunto(s)
Adhesivos/aislamiento & purificación , Diatomeas/química , Adhesivos/análisis , Aminoácidos/análisis , Carbohidratos/análisis , Espectroscopía de Resonancia MagnéticaRESUMEN
Diatoms are eukaryotic microalgae that produce species-specifically structured cell walls made of SiO(2) (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.
Asunto(s)
Pared Celular/química , Diatomeas/ultraestructura , Morfogénesis , Proteínas/química , Dióxido de Silicio , Pared Celular/ultraestructura , Matriz Extracelular/química , Datos de Secuencia Molecular , Nanotecnología/métodos , Péptidos , Fosfoproteínas , PoliaminasRESUMEN
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.
Asunto(s)
Apicomplexa/genética , Plastidios/genética , Proteínas Protozoarias/genética , ARN Mensajero/genética , ARN Protozoario/genética , Apicomplexa/metabolismo , Apicomplexa/patogenicidad , Ciclo Celular/fisiología , Plastidios/metabolismo , Transporte de Proteínas/genética , Proteínas Protozoarias/metabolismo , ARN Mensajero/biosíntesis , ARN Protozoario/biosíntesisRESUMEN
The formation of SiO(2)-based cell walls by diatoms (a large group of unicellular microalgae) is a well established model system for the study of molecular mechanisms of biological mineral morphogenesis (biomineralization). Diatom biomineralization involves highly phosphorylated proteins (silaffins and silacidins), analogous to other biomineralization systems, which also depend on diverse sets of phosphoproteins (e.g. mammalian teeth and bone, mollusk shells, and sponge silica). The phosphate moieties on biomineralization proteins play an essential role in mineral formation, yet the kinases catalyzing the phosphorylation of these proteins have remained poorly characterized. Recent functional genomics studies on the diatom Thalassiosira pseudonana have revealed >100 proteins potentially involved in diatom silica formation. Here we have characterized the biochemical properties and biological function of one of these proteins, tpSTK1. Multiple tpSTK1-like proteins are encoded in diatom genomes, all of which exhibit low but significant sequence similarity to kinases from other organisms. We show that tpSTK1 has serine/threonine kinase activity capable of phosphorylating silaffins but not silacidins. Cell biological and biochemical analysis demonstrated that tpSTK1 is an abundant component of the lumen of the endoplasmic reticulum. The present study provides the first molecular structure of a kinase that appears to catalyze phosphorylation of biomineral forming proteins in vivo.
Asunto(s)
Proteínas Algáceas/metabolismo , Diatomeas/enzimología , Retículo Endoplásmico/enzimología , Proteínas Quinasas/metabolismo , Dióxido de Silicio/metabolismo , Proteínas Algáceas/genética , Diatomeas/genética , Retículo Endoplásmico/genética , Genoma/fisiología , Fosforilación/fisiología , Proteínas Quinasas/genéticaRESUMEN
Sexual reproduction is a fundamental phase in the life cycle of most diatoms. Despite its role as a source of genetic variation, it is rarely reported in natural circumstances and its molecular foundations remain largely unknown. Here, we integrate independent transcriptomic datasets to prioritize genes responding to sex inducing pheromones (SIPs) in the pennate diatom Seminavis robusta. We observe marked gene expression changes associated with SIP treatment in both mating types, including an inhibition of S phase progression, chloroplast division, mitosis, and cell wall formation. Meanwhile, meiotic genes are upregulated in response to SIP, including a sexually induced diatom specific cyclin. Our data further suggest an important role for reactive oxygen species, energy metabolism, and cGMP signaling during the early stages of sexual reproduction. In addition, we identify several genes with a mating type specific response to SIP, and link their expression pattern with physiological specialization, such as the production of the attraction pheromone diproline in mating type - (MT-) and mate-searching behavior in mating type + (MT+). Combined, our results provide a model for early sexual reproduction in pennate diatoms and significantly expand the suite of target genes to detect sexual reproduction events in natural diatom populations.
Asunto(s)
Diatomeas , Atractivos Sexuales , Diatomeas/genética , Feromonas , Reproducción , TranscriptomaRESUMEN
Benthic diatoms are the main primary producers in shallow freshwater and coastal environments, fulfilling important ecological functions such as nutrient cycling and sediment stabilization. However, little is known about their evolutionary adaptations to these highly structured but heterogeneous environments. Here, we report a reference genome for the marine biofilm-forming diatom Seminavis robusta, showing that gene family expansions are responsible for a quarter of all 36,254 protein-coding genes. Tandem duplications play a key role in extending the repertoire of specific gene functions, including light and oxygen sensing, which are probably central for its adaptation to benthic habitats. Genes differentially expressed during interactions with bacteria are strongly conserved in other benthic diatoms while many species-specific genes are strongly upregulated during sexual reproduction. Combined with re-sequencing data from 48 strains, our results offer insights into the genetic diversity and gene functions in benthic diatoms.
Asunto(s)
Adaptación Fisiológica/genética , Diatomeas/genética , Ecosistema , Evolución Molecular , Genoma/genética , Diatomeas/clasificación , Diatomeas/metabolismo , Agua Dulce , Tamaño del Genoma , Genómica/métodos , Polimorfismo de Nucleótido Simple , Agua de Mar , Especificidad de la Especie , Transcriptoma/genéticaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Throughout all kingdoms of life, a large number of adhesive biomolecules have evolved to allow organisms to adhere to surfaces underwater. Proteins play an important role in the adhesion of numerous marine invertebrates (e.g. mussels, sea stars, sea urchins) whereas much less is known about the biological adhesives from marine plants, including the diatoms. Diatoms are unicellular microalgae that together with bacteria dominate marine biofilms in sunlit habitats. In this study we present the first proteomics analyses of the diatom adhesive material isolated from the tenacious fouling species Amphora coffeaeformis. We identified 21 proteins, of which 13 are diatom-specific. Ten of these proteins share a conserved C-terminal domain, termed GDPH domain, which is widespread yet not ubiquitously present in all diatom classes. Immunofluorescence localization of a GDPH domain bearing protein (Ac629) as well as two other proteins identified in this study (Ac1442, Ac9617) demonstrated that these are components of the adhesive trails that are secreted by cells that glide on surfaces. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'.
Asunto(s)
Diatomeas/fisiología , Proteoma/análisis , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Incrustaciones Biológicas , Adhesión Celular , Diatomeas/genética , Propiedades de SuperficieRESUMEN
Molluscan shells are a model system to understand the fundamental principles of mineral formation by living organisms. The diversity of unconventional mineral morphologies and 3D mineral-organic architectures that comprise these tissues, in combination with their exceptional mechanical efficiency, offers a unique platform to study the formation-structure-function relationship in a biomineralized system. However, so far, morphogenesis of these ultrastructures is poorly understood. Here, a comprehensive physical model, based on the concept of directional solidification, is developed to describe molluscan shell biomineralization. The capacity of the model to define the forces and thermodynamic constraints that guide the morphogenesis of the entire shell construct-the prismatic and nacreous ultrastructures and their transitions-and govern the evolution of the constituent mineralized assemblies on the ultrastructural and nanostructural levels is demonstrated using the shell of the bivalve Unio pictorum. Thereby, explicit tools for novel bioinspired and biomimetic bottom-up materials design are provided.
RESUMEN
The intricate, genetically controlled biosilica nano- and micropatterns produced by diatoms are a testimony for biology's ability to control mineral formation (biomineralization) at the nanoscale and regarded as paradigm for nanotechnology. Previously, several protein families involved in diatom biosilica formation have been identified, and many of them remain tightly associated with the final biosilica structure. Determining the locations of biosilica-associated proteins with high precision is, therefore expected to provide clues to their roles in biosilica morphogenesis. To achieve this, we introduce here single-molecule localization microscopy to diatoms based on photo-activated light microscopy (PALM) to overcome the diffraction limit. We identified six photo-convertible fluorescent proteins (FPs) that can be utilized for PALM in the cytoplasm of model diatom Thalassiosira pseudonana. However, only three FPs were also functional when embedded in diatom biosilica. These were employed for PALM-based localization of the diatom biosilica-associated protein Silaffin-3 (tpSil3) with a mean precision of 25 nm. This allowed for the identification of distinct accumulation areas of Sil3 in the biosilica, which cannot be resolved by confocal fluorescence microscopy. The enhanced microscopy technique introduced here for diatoms will aid in elucidating the molecular mechanism of silica biomineralization as well as other aspects of diatom cell biology.
Asunto(s)
Diatomeas/metabolismo , Proteínas Luminiscentes/metabolismo , Microscopía Electrónica de Rastreo , Imagen Individual de MoléculaRESUMEN
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.
Asunto(s)
Diatomeas/enzimología , Diatomeas/genética , Regulación Enzimológica de la Expresión Génica , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Regiones Terminadoras Genéticas , Secuencia de Aminoácidos , Clonación Molecular , Regulación de la Expresión Génica de las Plantas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Datos de Secuencia Molecular , Nitrato-Reductasa , Nitrato Reductasas/genética , Nitrato Reductasas/metabolismo , Nitratos/farmacología , Plantas Modificadas Genéticamente , Biosíntesis de Proteínas , Compuestos de Amonio Cuaternario/farmacología , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/análisis , Homología de Secuencia de Aminoácido , Transcripción Genética , Transformación GenéticaRESUMEN
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.