ABSTRACT
Cellulose is the most abundant biopolymer on Earth, and certain organisms from bacteria to plants and animals synthesize cellulose as an extracellular polymer for various biological functions. Humans have used cellulose for millennia as a material and an energy source, and the advent of a lignocellulosic fuel industry will elevate it to the primary carbon source for the burgeoning renewable energy sector. Despite the biological and societal importance of cellulose, the molecular mechanism by which it is synthesized is now only beginning to emerge. On the basis of recent advances in structural and molecular biology on bacterial cellulose synthases, we review emerging concepts of how the enzymes polymerize glucose molecules, how the nascent polymer is transported across the plasma membrane, and how bacterial cellulose biosynthesis is regulated during biofilm formation. Additionally, we review evolutionary commonalities and differences between cellulose synthases that modulate the nature of the cellulose product formed.
Subject(s)
Cellulose/biosynthesis , Plants/metabolism , Catalytic Domain , Cell Wall/chemistry , Electron Transport , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/metabolism , Plants/enzymologyABSTRACT
Plant epidermal cell walls maintain the mechanical integrity of plants and restrict organ growth. Mechanical analyses can give insights into wall structure and are inputs for mechanobiology models of plant growth. To better understand the intrinsic mechanics of epidermal cell walls and how they may accommodate large deformations during growth, we analyzed a geometrically simple material, onion epidermal strips consisting of only the outer (periclinal) cell wall, ~7 µm thick. With uniaxial stretching by >40%, the wall showed complex three-phase stress-strain responses while cyclic stretching revealed reversible and irreversible deformations and elastic hysteresis. Stretching at varying strain rates and temperatures indicated the wall behaved more like a network of flexible cellulose fibers capable of sliding than a viscoelastic composite with pectin viscosity. We developed an analytic framework to quantify nonlinear wall mechanics in terms of stiffness, deformation, and energy dissipation, finding that the wall stretches by combined elastic and plastic deformation without compromising its stiffness. We also analyzed mechanical changes in slightly dehydrated walls. Their extension became stiffer and more irreversible, highlighting the influence of water on cellulose stiffness and sliding. This study offers insights into the structure and deformation modes of primary cell walls and presents a framework that is also applicable to tissues and whole organs.
Subject(s)
Cell Wall , Cellulose , Cellulose/chemistry , Cell Wall/chemistry , Cell Membrane , Pectins , Plant EpidermisABSTRACT
Nature is home to a variety of microorganisms that create materials under environmentally friendly conditions. While this offers an attractive approach for sustainable manufacturing, the production of materials by native microorganisms is usually slow and synthetic biology tools to engineer faster microorganisms are only available when prior knowledge of genotype-phenotype links is available. Here, we utilize a high-throughput directed evolution platform to enhance the fitness of whole microorganisms under selection pressure and identify genetic pathways to enhance the material production capabilities of native species. Using Komagataeibacter sucrofermentans as a model cellulose-producing microorganism, we show that our droplet-based microfluidic platform enables the directed evolution of these bacteria toward a small number of cellulose overproducers from an initial pool of 40,000 random mutants. Sequencing of the evolved strains reveals an unexpected link between the cellulose-forming ability of the bacteria and a gene encoding a protease complex responsible for protein turnover in the cell. The ability to enhance the fitness of microorganisms toward a specific phenotype and to unravel genotype-phenotype links makes this high-throughput directed evolution platform a promising tool for the development of new strains for the sustainable manufacturing of materials.
Subject(s)
Cellulose , Directed Molecular Evolution , Cellulose/metabolism , Cellulose/biosynthesis , Directed Molecular Evolution/methods , Acetobacteraceae/metabolism , Acetobacteraceae/genetics , Phenotype , MutationABSTRACT
Degrading cellulose is a key step in the processing of lignocellulosic biomass into bioethanol. Cellobiose, the disaccharide product of cellulose degradation, has been shown to inhibit cellulase activity, but the mechanisms underlying product inhibition are not clear. We combined single-molecule imaging and biochemical investigations with the goal of revealing the mechanism by which cellobiose inhibits the activity of Trichoderma reesei Cel7A, a well-characterized exo-cellulase. We find that cellobiose slows the processive velocity of Cel7A and shortens the distance moved per encounter; effects that can be explained by cellobiose binding to the product release site of the enzyme. Cellobiose also strongly inhibits the binding of Cel7A to immobilized cellulose, with a Ki of 2.1 mM. The isolated catalytic domain (CD) of Cel7A was also inhibited to a similar degree by cellobiose, and binding of an isolated carbohydrate-binding module to cellulose was not inhibited by cellobiose, suggesting that cellobiose acts on the CD alone. Finally, cellopentaose inhibited Cel7A binding at micromolar concentrations without affecting the enzyme's velocity of movement along cellulose. Together, these results suggest that cellobiose inhibits Cel7A activity both by binding to the "back door" product release site to slow activity and to the "front door" substrate-binding tunnel to inhibit interaction with cellulose. These findings point to strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy.
Subject(s)
Cellobiose , Cellulase , Cellulose , Hypocreales , Cellobiose/metabolism , Cellulase/metabolism , Cellulase/antagonists & inhibitors , Cellulose/metabolism , Hypocreales/enzymology , Hypocreales/metabolism , Single Molecule Imaging/methods , Catalytic Domain , Fungal Proteins/metabolism , Fungal Proteins/antagonists & inhibitors , Fungal Proteins/chemistryABSTRACT
Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that oxidatively degrade various polysaccharides, such as cellulose. Despite extensive research on this class of enzymes, the role played by their C-terminal regions predicted to be intrinsically disordered (dCTR) has been overlooked. Here, we investigated the function of the dCTR of an LPMO, called CoAA9A, up-regulated during plant infection by Colletotrichum orbiculare, the causative agent of anthracnose. After recombinant production of the full-length protein, we found that the dCTR mediates CoAA9A dimerization in vitro, via a disulfide bridge, a hitherto-never-reported property that positively affects both binding and activity on cellulose. Using SAXS experiments, we show that the homodimer is in an extended conformation. In vivo, we demonstrate that gene deletion impairs formation of the infection-specialized cell called appressorium and delays penetration of the plant. Using immunochemistry, we show that the protein is a dimer not only in vitro but also in vivo when secreted by the appressorium. As these peculiar LPMOs are also found in other plant pathogens, our findings open up broad avenues for crop protection.
Subject(s)
Fungal Proteins , Polysaccharides , Protein Multimerization , Scattering, Small Angle , Fungal Proteins/genetics , Fungal Proteins/metabolism , X-Ray Diffraction , Polysaccharides/metabolism , Cellulose/metabolismABSTRACT
Biofilms are a widespread multicellular form of bacterial life. The spatial structure and emergent properties of these communities depend on a polymeric extracellular matrix architecture that is orders of magnitude larger than the cells that build it. Using as a model the wrinkly macrocolony biofilms of Escherichia coli, which contain amyloid curli fibers and phosphoethanolamine (pEtN)-modified cellulose as matrix components, we summarize here the structure, building, and function of this large-scale matrix architecture. Based on different sigma and other transcription factors as well as second messengers, the underlying regulatory network reflects the fundamental trade-off between growth and survival. It controls matrix production spatially in response to long-range chemical gradients, but it also generates distinct patterns of short-range matrix heterogeneity that are crucial for tissue-like elasticity and macroscopic morphogenesis. Overall, these biofilms confer protection and a potential for homeostasis, thereby reducing maintenance energy, which makes multicellularity an emergent property of life itself.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Bacteria , Biofilms , Biology , Escherichia coli/genetics , Extracellular Matrix/chemistryABSTRACT
Potassium vanadium fluorophosphate (KVPO4F) is regarded as a promising cathode candidate for potassium-ion batteries due to its high working voltage and satisfactory theoretical capacity. However, the usage of electrochemically inactive binders and redundant current collectors typically results in inferior electrochemical performance and low energy density, thus implying the important role of rational electrode structure design. Herein, we have reported a scalable and cost-effective synthesis of a cellulose-derived KVPO4F self-supporting electrode, which features a special surface hydroxyl chemistry, three-dimensional porous and conductive framework, as well as super flexible and stable architecture. The cellulose not only serves as a flexible substrate, a pore-forming agent, and a versatile binder for KVPO4F/conductive carbon but also enhances the K-ion migration ability. Benefiting from the special hydroxyl chemistry-induced storage mechanism and electrode structural stability, the flexible freestanding KVPO4F cathode exhibits high-rate performance (53.0% capacity retention with current densities increased 50-fold, from 0.2 C to 10 C) and impressive cycling stability (capacity retention up to 74.9% can be achieved over 1,000 cycles at a rate of 5 C). Such electrode design and surface engineering strategies, along with a deeper understanding of potassium storage mechanisms, provide invaluable guidance for better electrode design to boost the performance of potassium-ion energy storage systems.
ABSTRACT
Finely controlled flow forces in extrusion-based additive manufacturing can be exploited to program the self-assembly of malleable nanostructures in soft materials by integrating bottom-up design into a top-down processing approach. Here, we leverage the processing parameters offered by direct ink-writing (DIW) to reconfigure the photonic chiral nematic liquid crystalline phase in hydroxypropyl cellulose (HPC) solutions prior to deposition on the writing substrate to direct structural evolution from a particular initial condition. Moreover, we incorporate polyethylene glycol (PEG) into iridescent HPC inks to form a physically cross-linked network capable of inducing kinetic arrest of the cholesteric/chiral pitch at length scales that selectively reflect light throughout the visible spectrum. Based on thorough rheological measurements, we have found that printing the chiral inks at a shear rate where HPC molecules adopt pseudonematic state results in uniform chiral recovery following flow cessation and enhanced optical properties in the solid state. Printing chiral inks at high shear rates, on the other hand, shifts the monochromatic appearance of the extruded filaments to a highly angle-dependent state, suggesting a preferred orientation of the chiral domains. The optical response of these filaments when exposed to mechanical deformation can be used in the development of optical sensors.
ABSTRACT
Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From â¼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.
Subject(s)
Cellulose 1,4-beta-Cellobiosidase , Enzyme Assays , Genome, Fungal , Mutation , Protein Engineering , Aspergillus oryzae/enzymology , Aspergillus oryzae/genetics , Cellulose 1,4-beta-Cellobiosidase/chemistry , Cellulose 1,4-beta-Cellobiosidase/classification , Cellulose 1,4-beta-Cellobiosidase/genetics , Cellulose 1,4-beta-Cellobiosidase/metabolism , Genome, Fungal/genetics , Protein Engineering/methods , Substrate Specificity , Talaromyces/enzymology , Talaromyces/genetics , Trichoderma/enzymology , Trichoderma/genetics , Trichoderma/metabolism , BiocatalysisABSTRACT
Lytic polysaccharide monooxygenases (LPMOs) oxidatively depolymerize recalcitrant polysaccharides, which is important for biomass conversion. The catalytic domains of many LPMOs are linked to carbohydrate-binding modules (CBMs) through flexible linkers, but the function of these CBMs in LPMO catalysis is not well understood. In this study, we utilized MtLPMO9L and MtLPMO9G derived from Myceliophthora thermophila to investigate the impact of CBMs on LPMO activity, with particular emphasis on their influence on H2O2 tolerance. Using truncated forms of MtLPMO9G generated by removing the CBM, we found reduced substrate binding affinity and enzymatic activity. Conversely, when the CBM was fused to the C terminus of the single-domain MtLPMO9L to create MtLPMO9L-CBM, we observed a substantial improvement in substrate binding affinity, enzymatic activity, and notably, H2O2 tolerance. Furthermore, molecular dynamics simulations confirmed that the CBM fusion enhances the proximity of the active site to the substrate, thereby promoting multilocal cleavage and impacting the exposure of the copper active site to H2O2. Importantly, the fusion of CBM resulted in more efficient consumption of H2O2 by LPMO, leading to improved enzymatic activity and reduced auto-oxidative damage of the copper active center.
Subject(s)
Catalytic Domain , Hydrogen Peroxide , Mixed Function Oxygenases , Polysaccharides , Sordariales , Copper/metabolism , Hydrogen Peroxide/adverse effects , Hydrogen Peroxide/metabolism , Mixed Function Oxygenases/metabolism , Polysaccharides/metabolism , Sordariales/enzymology , Sordariales/metabolism , Molecular Dynamics SimulationABSTRACT
Cassava, a pivotal tropical crop, exhibits rapid growth and possesses a substantial biomass. Its stem is rich in cellulose and serves as a crucial carbohydrate storage organ. The height and strength of stems restrict the mechanised operation and propagation of cassava. In this study, the triple helix transcription factor MeGT2.6 was identified through yeast one-hybrid assay using MeCesA1pro as bait, which is critical for cellulose synthesis. Over-expression and loss-of-function lines were generated, and results revealed that MeGT2.6 could promote a significant increase in the plant height, stem diameter, cell size and thickness of SCW of cassava plant. Specifically, MeGT2.6 upregulated the transcription activity of MeGA20ox1 and downregulated the expression level of MeGA2ox1, thereby enhancing the content of active GA3, resulting in a large cell size, high plant height and long stem diameter in cassava. Moreover, MeGT2.6 upregulated the transcription activity of MeCesA1, which promoted the synthesis of cellulose and hemicellulose and produced a thick secondary cell wall. Finally, MeGT2.6 could help supply additional substrates for the synthesis of cellulose and hemicellulose by upregulating the invertase genes (MeNINV1/6). Thus, MeGT2.6 was found to be a multiple regulator; it was involved in GA metabolism and sucrose decomposition and the synthesis of cellulose and hemicellulose.
Subject(s)
Cellulose , Gene Expression Regulation, Plant , Gibberellins , Manihot , Plant Proteins , Manihot/genetics , Manihot/metabolism , Cellulose/metabolism , Cellulose/biosynthesis , Plant Proteins/metabolism , Plant Proteins/genetics , Gibberellins/metabolism , Cell Wall/metabolism , Cell Enlargement , Transcription Factors/metabolism , Transcription Factors/genetics , Plant Stems/genetics , Plant Stems/metabolism , Plant Stems/growth & development , Polysaccharides/metabolismABSTRACT
Plant stems constitute the most abundant renewable resource on earth. The function of lysine (K)-2-hydroxyisobutyrylation (Khib), a novel post-translational modification (PTM), has not yet been elucidated in plant stem development. Here, by assessing typical pepper genotypes with straight stem (SS) and prostrate stem (PS), we report the first large-scale proteomics analysis for protein Khib to date. Khib-modifications influenced central metabolic processes involved in stem development, such as glycolysis/gluconeogenesis and protein translation. The high Khib level regulated gene expression and protein accumulation associated with cell wall formation in the pepper stem. Specially, we found that CaMYB61 knockdown lines that exhibited prostrate stem phenotypes had high Khib levels. Most histone deacetylases (HDACs, e.g., switch-independent 3 associated polypeptide function related 1, AFR1) potentially function as the "erasing enzymes" involved in reversing Khib level. CaMYB61 positively regulated CaAFR1 expression to erase Khib and promote cellulose and hemicellulose accumulation in the stem. Therefore, we propose a bidirectional regulation hypothesis of "Khib modifications" and "Khib erasing" in stem development, and reveal a novel epigenetic regulatory network in which the CaMYB61-CaAFR1 molecular module participating in the regulation of Khib levels and biosynthesis of cellulose and hemicellulose for the first time.
Subject(s)
Capsicum , Gene Expression Regulation, Plant , Lysine , Plant Proteins , Plant Stems , Proteomics , Plant Stems/genetics , Plant Stems/metabolism , Plant Stems/growth & development , Plant Proteins/metabolism , Plant Proteins/genetics , Capsicum/genetics , Capsicum/growth & development , Capsicum/metabolism , Lysine/metabolism , Protein Processing, Post-Translational , Cell Wall/metabolism , Cell Wall/genetics , Transcription Factors/metabolism , Transcription Factors/geneticsABSTRACT
Plant cell walls are essential for defining plant growth and development, providing structural support to the main body and responding to abiotic and biotic cues. Cellulose, the main structural polymer of plant cell walls, is synthesized at the plasma membrane by cellulose synthase complexes (CSCs). The construction and transport of CSCs to and from the plasma membrane is poorly understood but is known to rely on the coordinated activity of cellulose synthase-interactive protein 1 (CSI1), a key regulator of CSC trafficking. In this study, we found that Trs85, a TRAPPIII complex subunit, interacted with CSI1 in vitro. Using functional genetics and live-cell imaging, we have shown that trs85-1 mutants have reduced cellulose content, stimulated CSC delivery, an increased population of static CSCs and deficient clathrin-mediated endocytosis in the primary cell wall. Overall, our findings suggest that Trs85 has a dual role in the trafficking of CSCs, by negatively regulating the exocytosis and clathrin-mediated endocytosis of CSCs.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Cell Wall , Cellulose , Endocytosis , Glucosyltransferases , Protein Transport , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis/enzymology , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Glucosyltransferases/metabolism , Glucosyltransferases/genetics , Cell Wall/metabolism , Endocytosis/physiology , Cellulose/metabolism , Clathrin/metabolism , Cell Membrane/metabolism , Exocytosis/physiology , Mutation , Carrier ProteinsABSTRACT
Stomata are pores at the leaf surface that enable gas exchange and transpiration. The signaling pathways that regulate the differentiation of stomatal guard cells and the mechanisms of stomatal pore formation have been characterized in Arabidopsis thaliana. However, the process by which stomatal complexes develop after pore formation into fully mature complexes is poorly understood. We tracked the morphogenesis of young stomatal complexes over time to establish characteristic geometric milestones along the path of stomatal maturation. Using 3D-nanoindentation coupled with finite element modeling of young and mature stomata, we found that despite having thicker cell walls than young guard cells, mature guard cells are more energy efficient with respect to stomatal opening, potentially attributable to the increased mechanical anisotropy of their cell walls and smaller changes in turgor pressure between the closed and open states. Comparing geometric changes in young and mature guard cells of wild-type and cellulose-deficient plants revealed that although cellulose is required for normal stomatal maturation, mechanical anisotropy appears to be achieved by the collective influence of cellulose and additional wall components. Together, these data elucidate the dynamic geometric and biomechanical mechanisms underlying the development process of stomatal maturation.
Subject(s)
Arabidopsis , Cell Wall , Plant Stomata , Arabidopsis/physiology , Arabidopsis/growth & development , Arabidopsis/genetics , Plant Stomata/physiology , Plant Stomata/growth & development , Plant Stomata/cytology , Anisotropy , Cell Wall/metabolism , Cell Wall/physiology , Cellulose/metabolism , Finite Element Analysis , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/geneticsABSTRACT
Confocal microscopy has greatly aided our understanding of the major cellular processes and trafficking pathways responsible for plant growth and development. However, a drawback of these studies is that they often rely on the manual analysis of a vast number of images, which is time-consuming, error-prone, and subject to bias. To overcome these limitations, we developed Dot Scanner, a Python program for analyzing the densities, lifetimes, and displacements of fluorescently tagged particles in an unbiased, automated, and efficient manner. Dot Scanner was validated by performing side-by-side analysis in Fiji-ImageJ of particles involved in cellulose biosynthesis. We found that the particle densities and lifetimes were comparable in both Dot Scanner and Fiji-ImageJ, verifying the accuracy of Dot Scanner. Dot Scanner largely outperforms Fiji-ImageJ, since it suffers far less selection bias when calculating particle lifetimes and is much more efficient at distinguishing between weak signals and background signal caused by bleaching. Not only does Dot Scanner obtain much more robust results, but it is a highly efficient program, since it automates much of the analyses, shortening workflow durations from weeks to minutes. This free and accessible program will be a highly advantageous tool for analyzing live-cell imaging in plants.
Subject(s)
Image Processing, Computer-Assisted , Microscopy, Confocal , Software , Image Processing, Computer-Assisted/methods , Microscopy, Confocal/methods , Plant CellsABSTRACT
Cellulose is a critical component of secondary cell walls and woody tissues of plants. Cellulose synthase (CESA) complexes (CSCs) produce cellulose as they move within the plasma membrane, extruding glucan chains into the cell wall that coalesce and crystallize into cellulose fibrils. Here we examine COBRA-LIKE4 (COBL4), a GPI-anchored protein on the outer leaflet of the plasma membrane that is required for normal cellulose deposition in secondary cell walls. Characterization of the Arabidopsis (Arabidopsis thaliana) cobl4 mutant alleles called irregular xylem6, irx6-2 and irx6-3, showed reduced âº-cellulose content and lower crystallinity, supporting a role for COBL4 in maintaining cellulose quantity and quality. In live-cell imaging, mNeon Green-tagged CESA7 moved in the plasma membrane at higher speeds in the irx6-2 background compared to wild type. To test conservation of COBL4 function between herbaceous and woody plants, poplar (Populus trichocarpa) COBL4 homologs PtCOBL4a and PtCOBL4b were transformed into, and rescued, the Arabidopsis irx6 mutants. Using the Arabidopsis secondary cell wall-inducible VND7-GR system to study poplar COBL4 dynamics, YFP-tagged PtCOBL4a localized to the plasma membrane in regions of high cellulose deposition in secondary cell wall bands. As predicted for a lipid-linked protein, COBL4 was more mobile in the plane of the plasma membrane than CESA7 or a control plasma membrane marker. Following programmed cell death, COBL4 anchored to the secondary cell wall bands. These data support a role for COBL4 as a modulator of cellulose organization in the secondary cell wall, influencing cellulose production and CSC velocity at the plasma membrane.
ABSTRACT
Cellulosic microfibrils in plant cell walls are largely ensheathed and probably tethered by hydrogen-bonded hemicelluloses. Ensheathing may vary developmentally as hemicelluloses are peeled to enable cell expansion. We characterised a simple method to quantify ensheathed versus naked cellulosic surfaces based on the ability to adsorb a radiolabelled 'cellulose-complementary oligosaccharide', [3H]cellopentaitol. Filter-paper (cellulose) adsorbed 40% and >80% of aqueous 5nM [3H]cellopentaitol within ~1h and ~20h respectively. When [3H]cellopentaitol was rapidly dried onto filter-paper, ~50% of it was desorbable by water, whereas after ~1d annealing in aqueous medium the adsorption became too strong to be reversible in water. 'Strongly' adsorbed [3H]cellopentaitol was, however, ~98% desorbed by 6M NaOH, ~50% by 0.2M cellobiose, and ~30% by 8M urea, indicating a role for hydrogen-bonding reinforced by complementarity of shape. Gradual adsorption was promoted by kosmotropes (1.4M Na2SO4 or 30% methanol), and inhibited by chaotropes (8M urea), supporting a role for hydrogen-bonding. [3H]Cellopentaitol adsorption was strongly competed by non-radioactive cello-oligosaccharides (Cell2-6), the IC50 (half-inhibitory concentration) being highly size-dependent: Cell2, ~70 mM; Cell3, ~7 mM; and Cell4-6, ~0.05 mM. Malto-oligosaccharides (400mM) had no effect, confirming the role of complementarity. The quantity of adsorbed [3H]cellopentaitol was proportional to mass of cellulose. Of seven cottons tested, wild-type Gossypium arboreum fibres were least capable of adsorbing [3H]cellopentaitol, indicating ensheathment of their microfibrillar surfaces, confirmed by their resistance to cellulase digestion, and potentially attributable to a high glucuronoarabinoxylan content. In conclusion, [3H]cellopentaitol adsorption is a simple, sensitive and quantitative way of titrating 'naked' cellulose surfaces.
ABSTRACT
Cellulose biosynthesis in sessile bacterial colonies originates in the membrane-integrated bacterial cellulose synthase (Bcs) AB complex. We utilize optical tweezers to measure single-strand cellulose biosynthesis by BcsAB from Rhodobacter sphaeroides. Synthesis depends on uridine diphosphate glucose, Mg2+, and cyclic diguanosine monophosphate, with the last displaying a retention time of â¼80 min. Below a stall force of 12.7 pN, biosynthesis is relatively insensitive to force and proceeds at a rate of one glucose addition every 2.5 s at room temperature, increasing to two additions per second at 37°. At low forces, conformational hopping is observed. Single-strand cellulose stretching unveiled a persistence length of 6.2 nm, an axial stiffness of 40.7 pN, and an ability for complexes to maintain a tight grip, with forces nearing 100 pN. Stretching experiments exhibited hysteresis, suggesting that cellulose microstructure underpinning robust biofilms begins to form during synthesis. Cellohexaose spontaneously binds to nascent single cellulose strands, impacting polymer mechanical properties and increasing BcsAB activity.
Subject(s)
Rhodobacter sphaeroides , Uridine Diphosphate Glucose , Carbohydrate Metabolism , Cellulose/metabolism , Glucose/metabolism , Rhodobacter sphaeroides/metabolism , Uridine Diphosphate Glucose/metabolismABSTRACT
SignificanceWe propose a printable structural color ink composed of cholesteric cellulose liquid crystals together with gelatin and a thermal-responsive hydrogel. The ink is endowed with vivid structural colors and printability due to its constituents. Based on this, we print a series of graphics and three-dimensional (3D) objects with vivid color appearances. Moreover, the printed objects possess dual thermal responsiveness, which results in visible color change around body temperature. These performances, together with the biocompatibility of the constituents, indicate that the present ink represents a leap forward to the next-generation 3D printing and would unlock a wide range of real-life applications.
ABSTRACT
BACKGROUND: The tunicates form a group of filter-feeding marine animals closely related to vertebrates. They share with them a number of features such as a notochord and a dorsal neural tube in the tadpole larvae of ascidians, one of the three groups that make tunicates. However, a number of typical chordate characters have been lost in different branches of tunicates, a diverse and fast-evolving phylum. Consequently, the tunic, a sort of exoskeleton made of extracellular material including cellulose secreted by the epidermis, is the unifying character defining the tunicate phylum. In the larva of ascidians, the tunic differentiates in the tail into a median fin (with dorsal and ventral extended blades) and a caudal fin. RESULTS: Here we have performed experiments in the ascidian Phallusia mammillata to address the molecular control of tunic 3D morphogenesis. We have demonstrated that the tail epidermis medio-lateral patterning essential for peripheral nervous system specification also controls tunic elongation into fins. More specifically, when tail epidermis midline identity was abolished by BMP signaling inhibition, or CRISPR/Cas9 inactivation of the transcription factor coding genes Msx or Klf1/2/4/17, median fin did not form. We postulated that this genetic program should regulate effectors of tunic secretion. We thus analyzed the expression and regulation in different ascidian species of two genes acquired by horizontal gene transfer (HGT) from bacteria, CesA coding for a cellulose synthase and Gh6 coding for a cellulase. We have uncovered an unexpected dynamic history of these genes in tunicates and high levels of variability in gene expression and regulation among ascidians. Although, in Phallusia, Gh6 has a regionalized expression in the epidermis compatible with an involvement in fin elongation, our functional studies indicate a minor function during caudal fin formation only. CONCLUSIONS: Our study constitutes an important step in the study of the integration of HGT-acquired genes into developmental networks and a cellulose-based morphogenesis of extracellular material in animals.