The V-type ATPase enhances photosynthesis in marine phytoplankton and further links phagocytosis to symbiogenesis.

Diatoms, dinoflagellates, and coccolithophores are dominant groups of marine eukaryotic phytoplankton that are collectively responsible for the majority of primary production in the ocean. These phytoplankton contain additional intracellular membranes around their chloroplasts, which are derived from ancestral engulfment of red microalgae by unicellular heterotrophic eukaryotes that led to secondary and tertiary endosymbiosis. However, the selectable evolutionary advantage of these membranes and the physiological significance for extant phytoplankton remain poorly understood.

Development of IglC and GroEL recombinant vaccines for francisellosis in Nile tilapia, Oreochromis niloticus.

Warm-water piscine francisellosis is a granulomatous bacterial disease caused by Francisella orientalis (Fo). The disease has been detected in a wide range of fish species globally, causing mortalities as high as 90% and significant economic losses. Currently there are no commercially available vaccines and few treatment options exist.

Bionic 3D printed corals.

Corals have evolved as optimized photon augmentation systems, leading to space-efficient microalgal growth and outstanding photosynthetic quantum efficiencies. Light attenuation due to algal self-shading is a key limiting factor for the upscaling of microalgal cultivation. Coral-inspired light management systems could overcome this limitation and facilitate scalable bioenergy and bioproduct generation.

Dynamic subcellular translocation of V-type H -ATPase is essential for biomineralization of the diatom silica cell wall.

Diatom cell walls, called frustules, are main sources of biogenic silica in the ocean and their intricate morphology is an inspiration for nanoengineering. Here we show dynamic aspects of frustule biosynthesis involving acidification of the silica deposition vesicle (SDV) by V-type H  ATPase (VHA). Transgenic Thalassiosira pseudonana expressing the VHA B subunit tagged with enhanced green fluorescent protein (VHA -eGFP) enabled subcellular protein localization in live cells.

Characterization of a New Protein Family Associated With the Silica Deposition Vesicle Membrane Enables Genetic Manipulation of Diatom Silica.

Diatoms are known for their intricate, silicified cell walls (frustules). Silica polymerization occurs in a compartment called the silica deposition vesicle (SDV) and it was proposed that the cytoskeleton influences silica patterning through the SDV membrane (silicalemma) via interactions with transmembrane proteins. In this work we identify a family of proteins associated with the silicalemma, named SAPs for Silicalemma Associated Proteins.

Development of a silicon limitation inducible expression system for recombinant protein production in the centric diatoms Thalassiosira pseudonana and Cyclotella cryptica.

Background: An inducible promoter for recombinant protein expression provides substantial benefits because under induction conditions cellular energy and metabolic capability can be directed into protein synthesis. The most widely used inducible promoter for diatoms is for nitrate reductase, however, nitrogen metabolism is tied into diverse aspects of cellular function, and the induction response is not necessarily robust. Silicon limitation offers a means to eliminate energy and metabolic flux into cell division processes, with little other detrimental effect on cellular function, and a protein expression system that works under those conditions could be advantageous.

Expression of Histophilus somni IbpA DR2 protective antigen in the diatom Thalassiosira pseudonana.

Increasing demand for the low-cost production of valuable proteins has stimulated development of novel expression systems. Many challenges faced by existing technology may be overcome by using unicellular microalgae as an expression platform due to their ability to be cultivated rapidly, inexpensively, and in large scale. Diatoms are a particularly productive type of unicellular algae showing promise as production organisms.

Clarification of Photorespiratory Processes and the Role of Malic Enzyme in Diatoms.

Evidence suggests that diatom photorespiratory metabolism is distinct from other photosynthetic eukaryotes in that there may be at least two routes for the metabolism of the photorespiratory metabolite glycolate. One occurs primarily in the mitochondria and is similar to the C2 photorespiratory pathway, and the other processes glycolate through the peroxisomal glyoxylate cycle. Genomic analysis has identified the presence of key genes required for glycolate oxidation, the glyoxylate cycle, and malate metabolism, however, predictions of intracellular localization can be ambiguous and require verification.

Genome and methylome of the oleaginous diatom reveal genetic flexibility toward a high lipid phenotype.

Background: Improvement in the performance of eukaryotic microalgae for biofuel and bioproduct production is largely dependent on characterization of metabolic mechanisms within the cell. The marine diatom which was originally identified in the Aquatic Species Program, is a promising strain of microalgae for large-scale production of biofuel and bioproducts, such as omega-3 fatty acids.

Results: We sequenced the nuclear genome and methylome of this oleaginous diatom to identify the genetic traits that enable substantial accumulation of triacylglycerol.

Applications of Imaging Flow Cytometry for Microalgae.

The ability to image large numbers of cells at high resolution enhances flow cytometric analysis of cells and cell populations. In particular, the ability to image intracellular features adds a unique aspect to analyses, and can enable correlation between molecular phenomena resulting in alterations in cellular phenotype. Unicellular microalgae are amenable to high-throughput analysis to capture the diversity of cell types in natural samples, or diverse cellular responses in clonal populations, especially using imaging cytometry.

Probing fatty acid metabolism in bacteria, cyanobacteria, green microalgae and diatoms with natural and unnatural fatty acids.

In both eukaryotes and prokaryotes, fatty acid synthases are responsible for the biosynthesis of fatty acids in an iterative process, extending the fatty acid by two carbon units every cycle. Thus, odd numbered fatty acids are rarely found in nature. We tested whether representatives of diverse microbial phyla have the ability to incorporate odd-chain fatty acids as substrates for their fatty acid synthases and their downstream enzymes.

Transcript level coordination of carbon pathways during silicon starvation-induced lipid accumulation in the diatom Thalassiosira pseudonana.

Diatoms are one of the most productive and successful photosynthetic taxa on Earth and possess attributes such as rapid growth rates and production of lipids, making them candidate sources of renewable fuels. Despite their significance, few details of the mechanisms used to regulate growth and carbon metabolism are currently known, hindering metabolic engineering approaches to enhance productivity. To characterize the transcript level component of metabolic regulation, genome-wide changes in transcript abundance were documented in the model diatom Thalassiosira pseudonana on a time-course of silicon starvation.

Diatom silica biomineralization: Parallel development of approaches and understanding.

Diatom silica cell walls present an intriguing application of biomineralization in a single celled organism. The ability of diatoms to make an enormous variety of silica structures on the nano- to micro-scale is unparalleled in nature. The process is a whole-cell endeavor, involving diverse cellular components that coordinate "bottom up" and "top down" structure formation processes to reproducibly convert genetic information into physical structure.

Evidence for a regulatory role of diatom silicon transporters in cellular silicon responses.

The utilization of silicon by diatoms has both global and small-scale implications, from oceanic primary productivity to nanotechnological applications of their silica cell walls. The sensing and transport of silicic acid are key aspects of understanding diatom silicon utilization. At low silicic acid concentrations (<30 μM), transport mainly occurs through silicic acid transport proteins (SITs), and at higher concentrations it occurs through diffusion.

Electronically transparent graphene replicas of diatoms: a new technique for the investigation of frustule morphology.

The morphogenesis of the silica cell walls (called frustules) of unicellular algae known as diatoms is one of the most intriguing mysteries of the diatoms. To study frustule morphogenesis, optical, electron and atomic force microscopy has been extensively used to reveal the frustule morphology. However, since silica frustules are opaque, past observations were limited to outer and fracture surfaces, restricting observations of interior structures.

Development of flow cytometric procedures for the efficient isolation of improved lipid accumulation mutants in a sp. microalga.

The successful development of microalgae-based biofuel production will rely on improvements in the amount and rate of fuel molecule precursor accumulation. Mutagenesis and selection is an attractive approach to improve fuel molecule productivity. Previous screening methods have been laborious, the numbers of mutants isolated have been small, and overall performance of mutants may have been compromised by the presence of deleterious secondary mutations generated by random mutagenesis that affect other cellular processes and growth.

Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth.

Biologically derived fuels are viable alternatives to traditional fossil fuels, and microalgae are a particularly promising source, but improvements are required throughout the production process to increase productivity and reduce cost. Metabolic engineering to increase yields of biofuel-relevant lipids in these organisms without compromising growth is an important aspect of advancing economic feasibility. We report that the targeted knockdown of a multifunctional lipase/phospholipase/acyltransferase increased lipid yields without affecting growth in the diatom Thalassiosira pseudonana.

Characterization and localization of insoluble organic matrices associated with diatom cell walls: insight into their roles during cell wall formation.

Organic components associated with diatom cell wall silica are important for the formation, integrity, and function of the cell wall. Polysaccharides are associated with the silica, however their localization, structure, and function remain poorly understood. We used imaging and biochemical approaches to describe in detail characteristics of insoluble organic components associated with the cell wall in 5 different diatom species.

Metabolic and cellular organization in evolutionarily diverse microalgae as related to biofuels production.

Microalgae are among the most diverse organisms on the planet, and as a result of symbioses and evolutionary selection, the configuration of core metabolic networks is highly varied across distinct algal classes. The differences in photosynthesis, carbon fixation and processing, carbon storage, and the compartmentation of cellular and metabolic processes are substantial and likely to transcend into the efficiency of various steps involved in biofuel molecule production. By highlighting these differences, we hope to provide a framework for comparative analyses to determine the efficiency of the different arrangements or processes.

Whole transcriptome analysis of the silicon response of the diatom Thalassiosira pseudonana.

Background: Silicon plays important biological roles, but the mechanisms of cellular responses to silicon are poorly understood. We report the first analysis of cell cycle arrest and recovery from silicon starvation in the diatom Thalassiosira pseudonana using whole genome microarrays.

Results: Three known responses to silicon were examined, 1) silicified cell wall synthesis, 2) recovery from silicon starvation, and 3) co-regulation with silicon transporter (SIT) genes.

Expression, purification, and reconstitution of a diatom silicon transporter.

The synthesis and manipulation of silicon materials on the nanoscale are core themes in nanotechnology research. Inspiration is increasingly being taken from the natural world because the biological mineralization of silicon results in precisely controlled, complex silica structures with dimensions from the millimeter to the nanometer. One fascinating example of silicon biomineralization occurs in the diatoms, unicellular algae that sheath themselves in an ornate silica-based cell wall.

Characterization of the small RNA transcriptome of the diatom, Thalassiosira pseudonana.

This study presents the first characterization of endogenous small RNAs in a diatom, Thalassiosira pseudonana. Small RNAs act as transcriptional and translational regulators, controlling specific target genes involved in various cellular functions. Diatoms are unicellular photosynthetic organisms that play major roles in environmental processes, such as food webs and global carbon fixation.

Extensive and intimate association of the cytoskeleton with forming silica in diatoms: control over patterning on the meso- and micro-scale.

Background: The diatom cell wall, called the frustule, is predominantly made out of silica, in many cases with highly ordered nano- and micro-scale features. Frustules are built intracellularly inside a special compartment, the silica deposition vesicle, or SDV. Molecules such as proteins (silaffins and silacidins) and long chain polyamines have been isolated from the silica and shown to be involved in the control of the silica polymerization.

Dynamics of silica cell wall morphogenesis in the diatom Cyclotella cryptica: substructure formation and the role of microfilaments.

Diatoms are unicellular algae that make cell walls out of silica with highly ornate features on the nano- to microscale. The complexity and variety of diatom cell wall structures exceeds those possible with synthetic materials chemistry approaches. Understanding the design and assembly processes involved in diatom silicification should provide insight into patterning on the unicellular level, and information for biomimetic approaches for materials synthesis.

3D imaging of diatoms with ion-abrasion scanning electron microscopy.

Ion-abrasion scanning electron microscopy (IASEM) takes advantage of focused ion beams to abrade thin sections from the surface of bulk specimens, coupled with SEM to image the surface of each section, enabling 3D reconstructions of subcellular architecture at approximately 30nm resolution. Here, we report the first application of IASEM for imaging a biomineralizing organism, the marine diatom Thalassiosira pseudonana. Diatoms have highly patterned silica-based cell wall structures that are unique models for the study and application of directed nanomaterials synthesis by biological systems.