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Microalgae are a renewable and promising biomass for large-scale biofuel, food and nutrient production. However, their efficient exploitation depends on our knowledge of the cell wall composition and organization as it can limit access to high-value molecules. Here we provide an atomic-level model of the non-crystalline and insoluble glycoprotein-rich cell wall of Chlamydomonas reinhardtii. Using in situ solid-state and sensitivity-enhanced nuclear magnetic resonance, we reveal unprecedented details on the protein and carbohydrate composition and their nanoscale heterogeneity, as well as the presence of spatially segregated protein- and glycan-rich regions with different dynamics and hydration levels. We show that mannose-rich lower-molecular-weight proteins contribute to the cell wall cohesion by crosslinking high-molecular weight protein components, and that water provides plasticity to the cell-wall architecture. The methodology used here is transposable to study other microorganisms and plant materials – including those rich in cellulose – and their responses to contaminants and stresses.

Velvet worms capture prey and defend themselves by ejecting an adhesive slime which has been established as a model system for recyclable complex liquids. Triggered by mechanical agitation, the sticky fluid rapidly transitions into solid fibers. The assembly of slime proteins into stiff polymers is fully reversible and recyclable enabling the recovery of the soluble precursors. In order to understand the rapid and reversible mechanoresponsive behavior of this material, here, we study the nanostructural organization of slime components using small-angle scattering with neutrons and x-rays under physiological native conditions, after drying and re-hydration, and mechanical agitation. The scattering intensities are successfully described with a three-component model accounting for proteins of two dominant molecular weight fractions and for protein-based nanoglobules with a radius of ~40-45 nm, which is in line with the literature. However, in contrast to the previous assumption that high molecular weight (HMW) proteins -- the presumed building blocks of the fiber core -- are contained in the nanoglobules, we find that the majority of slime proteins exist as free proteins in solution, including the HMW fiber core precursors. Only less than 10 % of the slime proteins are contained in the nanoglobules, necessitating a reassessment of the previously proposed function of nanoglobules in fiber formation. Exploiting distinct differences in the x-ray and neutron scattering contrast of slime re-hydrated with light and heavy water (D2O) indicates that the majority of lipids available in the slime are contained in the nanoglobules, where they are homogeneously distributed. Surprisingly, mechanical agitation of slime in a completely filled container causes gelification; however, this neither leads to fiber formation nor alters the bulk structure of the slime significantly, suggesting that interfacial phenomena and directional shearing are required for the formation of stiff fibers in velvet worm slime.

Extracellular matrixes (ECMs), such as the cell walls and biofilms, are important for supporting cell integrity and function and regulating intercellular communication. These biomaterials are also of significant interest to the production of biofuels and the development of antimicrobial treatment. Solid-state nuclear magnetic resonance (ssNMR) and magic-angle spinning-dynamic nuclear polarization (MAS-DNP) are uniquely powerful for understanding the conformational structure, dynamical characteristics, and supramolecular assemblies of carbohydrates and other biomolecules in ECMs. This review highlights the recent high-resolution investigations of intact ECMs and native cells in many organisms spanning across plants, bacteria, fungi, and algae. We spotlight the structural principles identified in ECMs, discuss the current technical limitation and underexplored biochemical topics, and point out the promising opportunities enabled by the recent advances of the rapidly evolving ssNMR technology.

International audience; Microalgae are photosynthetic organisms widely distributed in nature and serve as a sustainable source of bioproducts. Their carbohydrate components are also promising candidates for bioenergy production and bioremediation, but the structural characterization of these heterogeneous polymers in cells remains a formidable problem. Here we present a widely applicable protocol for identifying and quantifying the glycan content using magic-angle-spinning (MAS) solid-state NMR (ssNMR) spectroscopy, with validation from glycosyl linkage and composition analysis deduced from mass-spectrometry (MS). Two-dimensional 13 C− 13 C correlation ssNMR spectra of a uniformly 13 C-labeled green microalga Parachlorella beijerinckii reveal that starch is the most abundant polysaccharide in a naturally cellulose-deficient strain, and this polymer adopts a well-organized and highly rigid structure in the cell. Some xyloses are present in both the mobile and rigid domains of the cell wall, with their chemical shifts partially aligned with the flat-ribbon 2-fold xylan identified in plants. Surprisingly, most other carbohydrates are largely mobile, regardless of their distribution in glycolipids or cell walls. These structural insights correlate with the high digestibility of this cellulose-deficient strain, and the in-cell ssNMR methods will facilitate the investigations of other economically important algae species.

Passivation of carbon dots via heteroatom doping has been shown to enhance their optical properties and tune their fluorescence signature. Additionally, the incorporation of polymeric precursors in carbon dot synthesis has gained considerable interest with benefits to biological applications namely bioimaging, drug delivery and sensing, among others. In order to combine the desirable attributes of both, fluorescence enhancement and increased biocompatibility, polymers composed of high aromaticity and nitrogen content can be used as efficient carbon dot passivating agents. Here, the synthesis of fluorescent polymer-passivated carbon dots was developed through a microwave-assisted pyrolysis reaction of galactose, citric acid and polydopamine. Passivation of the dots with polydopamine induces a 90 nm red-shift in the fluorescence maxima from 420 to 510 nm. Moreover, passivation results in excitation-independent fluorescence and a 3.5-fold increase in fluorescence quantum yield, which increases from 1.3 to 4.6%. The application of the carbon dots as imaging probes was investigated in in vitro and in vivo model systems. Cytotoxicity studies in J774 and CHO-K1 cell lines revealed reduced cell toxicity for the polydopamine-passivated carbon dots in comparison to their unpassivated counterpart. In BALB/c mice, biodistribution studies demonstrated that regardless of surface passivation, the dots predominantly remained in the circulatory system 90 minutes post inoculation suggesting their potential use for cardiovascular therapies.

International audience; Microalgae are ubiquitous primary organisms that play key roles in the aquatic environment where they are the basis of the food web. These microorganisms also produce a variety of molecules with high added value, which find applications in human and animal nutrition, as therapeutic agents and as biofuels. Lipid membranes abound in microalgae where they protect the cell and are essential building blocks of organelles such as the chloroplast. To better understand the architecture and dynamics of microalgal cell membranes and their constituents, it is important that they are investigated in their natural environment. Solid-state nuclear magnetic resonance (SS-NMR) is emerging as a powerful tool to tackle these complex assemblies in situ on cell extracts and also in whole cells. Several nuclei can be studied to probe algal membranes and, moreover, microalgae can be fully 13 C-or 15 N-labeled in a cost-effective manner. In the case of full organism labelling, the challenge of SS-NMR is to develop and identify experiments to filter specific constituent signals. In this chapter, we show how SS-NMR can provide a wealth of information on the structure, dynamics and composition of microalgal membranes. We also summarize the main SS-NMR approaches so far applied on lipid extracts or whole cells under near to native conditions and suggest potential avenues to improve our understanding of these systems.

Antimicrobial peptides (AMPs) may act by targeting the lipid membranes and disrupting the bilayer structure. In this study, three AMPs from the skin of Australian tree frogs, aurein 1.2, maculatin 1.1 and caerin 1.1, were investigated against Gram-negative Escherichia coli, Gram-positive Staphylococcus aureus, and vesicles that mimic their lipid compositions. Furthermore, equimolar mixtures of the peptides were tested to identify any synergistic interactions in antimicrobial activity. Minimum inhibition concentration and minimum bactericidal concentration assays showed significant activity against S. aureus but not against E. coli. Aurein was the least active while maculatin was the most active peptide and some synergistic effects were observed against S. aureus. Circular dichroism experiments showed that, in the presence of phospholipid vesicles, the peptides transitioned from an unstructured to a predominantly helical conformation (>50%), with greater helicity for POPG/TOCL compared to POPE/POPG vesicles. The helical content, however, was less in the presence of live E. coli and S. aureus, 25 and 5%, respectively. Equimolar concentrations of the peptides did not appear to form greater supramolecular structures. Dye release assays showed that aurein required greater concentration than caerin and maculatin to disrupt the lipid bilayers, and mixtures of the peptides did not cooperate to enhance their lytic activity. Overall, aurein, maculatin, and caerin showed moderate synergy in antimicrobial activity against S. aureus without becoming more structured or enhancement of their membrane-disrupting activity in phospholipid vesicles.

Starch is the most abundant energy storage molecule in plants and is an essential part of the human diet. This glucose polymer is composed of amorphous and crystalline domains in different forms (A and B types) with specific physicochemical properties that determine its bioavailability for an organism, as well as its value in the food industry. Using two-dimensional (2D) high resolution solid-state nuclear magnetic resonance (SS-NMR) on 13C-labelled starches that were obtained from Chlamydomonas reinhardtii microalgae, we established a complete and unambiguous assignment for starch and its constituents (amylopectin and amylose) in the two crystalline forms and in the amorphous state. We also assigned so far unreported non-reducing end groups and assessed starch chain length, crystallinity and amylose content. Starch was then characterized in situ, i.e., by 13C solid-state NMR of intact microalgal cells. Our in-cell methodology also enabled the identification of the effect of nitrogen starvation on starch metabolism. This work shows how solid-state NMR can enable the identification of starch structure, chemical modifications and biosynthesis in situ in intact microorganisms, eliminating time consuming and potentially altering purification steps.