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DOE OSTI2021

Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II

Abstract Background In plants, a large diversity of polysaccharides comprise the cell wall. Each major type of plant cell wall polysaccharide, including cellulose, hemicellulose, and pectin, has distinct structures and functions that contribute to wall mechanics and influence plant morphogenesis. In recent years, pectin valorization has attracted much attention due to its expanding roles in biomass deconstruction, food and material science, and environmental remediation. However, pectin utilization has been limited by our incomplete knowledge of its structure. Herein, we present a workflow of principles relevant for the characterization of polysaccharide primary structure using nature’s most complex polysaccharide, rhamnogalacturonan-II (RG-II), as a model. Results We outline how to isolate RG-II from celery and duckweed cell walls and from red wine using chemical or enzymatic treatments coupled with size-exclusion chromatography. From there, we applied mass spectrometry (MS)-based techniques to determine the glycosyl residue and linkage compositions of the intact RG-II and derived oligosaccharides including special considerations for labile monosaccharides. In doing so, we demonstrated that in the duckweed Wolffiella repanda the arabinopyranosyl (Ara p ) residue of side chain B is substituted at O- 2 with rhamnose. We used electrospray-MS techniques to identify non-glycosyl modifications including methyl-ethers, methyl-esters, and acetyl-esters on RG-II-derived oligosaccharides. We then showed the utility of proton nuclear magnetic resonance spectroscopy ( 1 H-NMR) to investigate the structure of intact RG-II and to complement the RG-II dimerization studies performed using size-exclusion chromatography. Conclusions The complexity of pectic polysaccharide structures has hampered efforts aimed at their valorization. In this work, we used RG-II as a model to demonstrate the steps necessary to isolate and characterize polysaccharides using chromatographic, MS, and NMR techniques. The principles can be applied to the characterization of other saccharide structures and will help inform researchers on how saccharide structure relates to functional properties in the future.

09 BIOMASS FUELS↗

NASA NTRS1993

Biochemical And Genetic Modification Of Polysaccharides

Bacteriophages producing endopolysaccharase-type enzymes used to produce, isolate, and purify high yields of modified polysaccharides from polysaccharides produced by, and incorporated into capsules of, certain bacteria. Bacteriophages used in conversion of native polysaccharide materials into polymers of nearly uniform high molecular weight or, alternatively, into highly pure oligosaccharides. Also used in genetic selection of families of polysaccharides structurally related to native polysaccharide materials, but having altered properties. Resulting new polysaccharides and oligosaccharides prove useful in variety of products, including pharmaceutical chemicals, coating materials, biologically active carbohydrates, and drag-reducing additives for fluids.

Kern, Roger G.↗

DOE OSTI2020

An acidic polysaccharide (AGC3) isolated from North American ginseng ( Panax quinquefolius ) suspension culture as a potential immunomodulatory nutraceutical

Polysaccharides isolated from Panax quinquefolius roots are widely used as nutraceuticals due to their immunomodulatory properties. Despite their popularity, several challenges exist in isolating ginseng root polysaccharides such as batch-to-batch structural inconsistencies and bacterial endotoxin contamination. A plant tissue culture-based platform offers a potential solution to isolate natural polysaccharide fractions with consistent chemical characteristics and reduced endotoxin content. In this study, an acidic polysaccharide fraction (AGC3) with immunomodulatory properties was isolated from Panax quinquefolius suspension cultures. The heterogeneous fraction (molecular weight: 4.81 and 32.14 kDa), purified by anion exchange chromatography, was predominantly composed of galactose (>60%) along with the presence of rhamnose, arabinose, glucose, glucuronic acid and galacturonic acid. The major glycosidic linkages were found to be t-Galp (47.7%), 4-Galp (15.6%), 2,4-Rhap (8.1%), 6-Galp (8.1%) and 4-GalAp (6.8%). Structural analyses indicated the presence of a pectic rhamnogalacturonan I polysaccharide in AGC3. AGC3 significantly (p < 0.05) stimulated RAW 264.7 murine macrophage cells and primary murine splenocytes by enhancing the production of several immunomodulatory mediators such as IL-6, TNF-α, GM-CSF and MCP-1. The results also indicated the putative roles of NF-κB (p65/RelA) and MAPK (p38) signaling pathways in the immunostimulatory response. Additionally, AGC3 induced murine splenocyte proliferation, another major indicator of immunostimulation. Overall, AGC3 has the potential to be used as an immunostimulatory nutraceutical.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Unifying themes and distinct features of carbon and nitrogen assimilation by polysaccharide-degrading bacteria: a summary of four model systems

Our current understanding of enzymatic polysaccharide degradation has come from a huge number of in vitro studies with purified enzymes. While this vast body of work has been invaluable in identifying and characterizing novel mechanisms of action and engineering desirable traits into these enzymes, a comprehensive picture of how these enzymes work as part of a native in vivo system is less clear. Recently, several model bacteria have emerged with genetic systems that allow for a more nuanced study of carbohydrate active enzymes (CAZymes) and how their activity affects bacterial carbon metabolism. With these bacterial model systems it is now possible to not only study a single nutrient system in isolation (i.e. carbohydrate degradation and carbon metabolism), but also how multiple systems are integrated. Given that most environmental polysaccharides are carbon rich but nitrogen poor (e.g. lignocellulose), the interplay between carbon and nitrogen metabolism in polysaccharide-degrading bacteria can now be studied in a physiologically relevant manner. Therefore, in this review we have summarized what has been experimentally determined for CAZyme regulation, production, and export in relation to nitrogen metabolism for two Gram-positive (Caldicellulosiruptor bescii and Clostridium thermocellum) and two Gram-negative (Bacteroides thetaiotaomicron and Cellvibrio japonicus) polysaccharide-degrading bacteria. By comparing and contrasting these four bacteria we have highlighted the shared and unique features of each, with a focus on in vivo studies, in regard to carbon and nitrogen assimilation. Finally, we conclude with what we believe are two important questions that can act as guideposts for future work to better understand the integration of carbon and nitrogen metabolism in polysaccharide-degrading bacteria.

Cellvibrio japonicus↗

DOE OSTI2025

Evaluating size exclusion chromatography for nucleic acid removal in Klebsiella pneumoniae cell surface polysaccharide purification

Cell surface-associated polysaccharides in Klebsiella are major virulence determinants and crucial targets for developing vaccines. Traditionally, the purification of these cell surface polysaccharides from Klebsiella pneumoniae involves a multi-step process comprising phenol extraction, nuclease digestion, ultracentrifugation, and repeated ethanol extractions. In this study, we evaluated size exclusion chromatography for effectively eliminating nucleic acid contamination while purifying high molecular weight cell surface-associated polysaccharides. Post-initial extraction, the nucleic acid content remains significantly elevated, and kinetic analysis reveals that DNase I and RNase A digestion is neither economically viable nor effective for removing these contaminants. Employing an appropriate size exclusion resin removes over 99 % of nucleic acid contamination, as confirmed by nucleic acid content analysis and agarose gel electrophoresis. Purity and structural analysis using 1H 1D-NMR and 2D-NMR demonstrate that the cell surface-associated polysaccharide purified with this study is highly homogeneous and identified as antigenic O-polysaccharide. This approach streamlines the purification process by removing the need for nuclease digestion and additional ethanol precipitation steps.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2026

In situ transmission electron microscopy observations of CaCO 3 crystallization onto polysaccharide-coated nanoparticles

Polysaccharides and proteoglycans are widely associated with the organic matrix at sites of CaCO 3 biomineralization, and previous studies indicate that these macromolecules may confer greater roles in mineral nucleation than previously recognized. This investigation uses in situ liquid-phase transmission electron microscopy (LP-TEM) to observe CaCO 3 nucleation onto aminated silica (SiO 2 –NH 3 + ) nanoparticles treated with a layer of chitosan (near-neutral derivative of chitin) or heparin (a carboxylated and highly sulfated glycosaminoglycan). In the absence of polysaccharides, few CaCO 3 particles formed and exhibited mobility. However, the SiO 2 –NH 3 + nanoparticles were enveloped in a region of higher mass density relative to the bulk solution, suggesting the development of a local solute-rich environment that surrounds the charged NH 3 + groups. The heparin- or chitosan-coated silica particles also exhibited regions of higher mass density around the nanoparticles. In the presence of these polysaccharide coatings, we observed the nucleation of abundant CaCO 3 particles whereby the polyanionic heparin promoted more nucleation than the weakly cationic chitosan. Many crystallites appeared to form at the polysaccharide–TEM cell membrane–solution interface, further indicating interfacial and macromolecule-specific control on crystallization. The combined results demonstrate that chitosan and heparin have an appreciable effect on the timing, size, and location of CaCO 3 nucleation compared to the polysaccharide-free nanoparticles.

15 GEOTHERMAL ENERGY↗

DOE OSTI2025

Author Correction: Genome-guided isolation of the hyperthermophilic aerobe Fervidibacter sacchari reveals conserved polysaccharide metabolism in the Armatimonadota

Correction to: Nature Communicationshttps://doi.org/10.1038/s41467-024-53784-3, published online 4 November 2024 In the version of this article initially published, Table 1 did not include the properties of the taxa being proposed or refer directly to another location in the main manuscript describing the properties. As such, the original manuscript did not comply with Rule 27 (2)(c) of the ICNP. Also, Table 1 listed the order Fervidibacterales as the nomenclatural type for the class Fervidibacteria, which violates latest emended version of Rule 15 stating that the nomenclatural type for a class must be a genus. Below we provide a modification of Table 1 containing protologues with these errors corrected. We have also changed the order of the taxa in the table to meet the most common ordering. (Table presented.) Taxon names proposed under the ICNP Proposed taxon Etymology Description Genus Fervidibacter Fer.vi.di.bac’ter. L. masc. adj. fervidus, hot, steaming; N.L. masc. n. bacter, a rod; N.L. masc. n. Fervidibacter, a hot rod Thermophilic or hyperthermophilic inhabitants of freshwater thermal environments. All members are likely polysaccharide-degrading chemoheterotrophs with numerous carbohydrate-active enzymes encoded in their genomes. Aerobic, with high-affinity and/or low-affinity terminal oxidases present in the genomes. The oxidative pentose phosphate pathway and the tricarboxylic acid cycle are complete in genomes belonging to the genus. Gram-stain-negative and diderm cell envelope structure. Ovoid- to rod-shaped morphology. Spores are not formed. The genus is a distinct phylogenetic lineage in the family Fervidibacteraceae, the order Fervidibacterales, and the class Fervidibacteria in the phylum Armatimonadota. The type species is Fervidibacter sacchariT. Species Fervidibacter sacchari sac’cha.ri. N.L. gen. n. sacchari, of sugar Hyperthermophilic, microaerophilic, facultatively anaerobic, and grows chemoheterotrophically on monosaccharides and polysaccharides. Cells are ovoid- to rod-shaped, Gram-stain negative, and are 0.9–1.3 µm in width and 1.6–3.6 µm in length. Grows between 65 and 87.5 °C and an optimum temperature of 80 °C, and a pH range of 6.5–8.6 with an optimum pH of 7.5. Grows at an optimum O2 concentration of 5–10%. Grows on D-arabinose, D-galactose, D-glucose, D-rhamnose, D-ribose, D-xylose, chondroitin sulfate, colloidal chitin, galactan, gellan gum, guar gum, karaya gum, locust bean gum, xantham gum, xyloglucan, β-glucan, glycogen, starch, AFEX-pretreated corn stover, miscanthus, sugarcane bagasse, acetate and casamino acids. Grows weakly on xyloglucan under fermentation conditions. The major fatty acids (>10%) are C16:0, C18:0 and/or cyclo-C17:0, and iso-C16:0. The major respiratory quinones (>10%) are MK-8 and MK-9. The isolate and genomes of the species have been recovered from geothermal springs in the Great Basin, Nevada, USA. GC content of genomes range between 51–52%. Subunits for both the high-affinity and low-affinity terminal oxidases are encoded in the genomes. Genomes also encode a Group 3d [NiFe] hydrogenase, which produces hydrogen as an electron sink for NAD+ regeneration. The type strain PD1T (= JCM 39283T = DSM 113467T) was isolated from Great Boiling Spring in Nevada, USA. Family Fervidibacteraceae Fer.vi.di.bac.te.ra’ce.ae. N.L. masc. n. Fervidibacter type genus of the family; L. suff. -aceae ending to denote a family; N.L. fem. pl. n. Fervidibacteraceae the family of the genus Fervidibacter Thermophilic or hyperthermophilic inhabitants of freshwater thermal environments. All members are likely polysaccharide-degrading chemoheterotrophs with numerous carbohydrate-active enzymes encoded in their genomes. Aerobic, with high-affinity and/or low-affinity terminal oxidases present in the genomes. The oxidative pentose phosphate pathway and the tricarboxylic acid cycle are complete in genomes belonging to the family. The family is a distinct phylogenetic lineage in the order Fervidibacterales and the class Fervidibacteria in the phylum Armatimonadota. The type genus is Fervidibacter. Order Fervidibacterales Fer.vi.di.bac.te.ra’les. N.L. masc. n. Fervidibacter type genus of the order; L. suff. -ales ending to denote an order; N.L. fem. pl. n. Fervidibacterales the order of the genus Fervidibacter Thermophilic or hyperthermophilic inhabitants of freshwater thermal environments. All members are likely polysaccharide-degrading chemoheterotrophs with numerous carbohydrate-active enzymes encoded in their genomes. Aerobic or strictly anaerobic. Phylogenomic placement of this lineage within the Fervidibacteria and relative evolutionary divergence supports delineation of this lineage as an order within the class Fervidibacteria and phylum Armatimonadota. The type genus is Fervidibacter. Class Fervidibacteria Fer.vi.di.bac.te’ri.a. N.L. masc. n. Fervidibacter type genus of the type order of the class; L. suff. -ia ending to denote a class; N.L. neut. pl. n. Fervidibacteria the class of the order Fervidibacterales Thermophilic or hyperthermophilic inhabitants of freshwater thermal environments. All members are likely polysaccharide-degrading chemoheterotrophs with numerous carbohydrate-active enzymes encoded in their genomes. Aerobic or strictly anaerobic. Phylogenomic placement of this lineage within the Armatimonadota and relative evolutionary divergence supports delineation of this lineage as a class within the Armatimonadota. The type genus is Fervidibacter. The error has not been corrected in the PDF or HTML versions of the Article.

Nou, Nancy O↗

DOE OSTI2022

Golgi-localized putative S-adenosyl methionine transporters required for plant cell wall polysaccharide methylation

Polysaccharide methylation, especially that of pectin, is a common and important feature of land plant cell walls. Polysaccharide methylation takes place in the Golgi apparatus and therefore relies on the import of S-adenosyl methionine (SAM) from the cytosol into the Golgi. However, so far, no Golgi SAM transporter has been identified in plants. Here we studied major facilitator superfamily members in Arabidopsis that we identified as putative Golgi SAM transporters (GoSAMTs). Knockout of the two most highly expressed GoSAMTs led to a strong reduction in Golgi-synthesized polysaccharide methylation. Furthermore, solid-state NMR experiments revealed that reduced methylation changed cell wall polysaccharide conformations, interactions and mobilities. Notably, NMR revealed the existence of pectin ‘egg-box’ structures in intact cell walls and showed that their formation is enhanced by reduced methyl esterification. Here, these changes in wall architecture were linked to substantial growth and developmental phenotypes. In particular, anisotropic growth was strongly impaired in the double mutant. The identification of putative transporters involved in import of SAM into the Golgi lumen in plants provides new insights into the paramount importance of polysaccharide methylation for plant cell wall structure and function.

36 MATERIALS SCIENCE↗

DOE OSTI2020

Chemical structure and genetic organization of the E. coli O6:K15 capsular polysaccharide

Capsular polysaccharides are important virulence factors in pathogenic bacteria. Characterizing the structural components and biosynthetic pathways for these polysaccharides is key to our ability to design vaccines and other preventative therapies that target encapsulated pathogens. Many gramnegative pathogens such as Neisseria meningitidis and Escherichia coli express acidic capsules. The E. coli K15 serotype has been identifed as both an enterotoxigenic and uropathogenic pathogen. Despite its relevance as a disease-causing serotype, the associated capsular polysaccharide remains poorly characterized. We describe in this report the chemical structure of the K15 polysaccharide, based on chemical analysis and nuclear magnetic resonance (NMR) data. The repeating structure of the K15 polysaccharide consists of 4)-α-GlcpNAc-(1→5)-α-KDOp-(2→partially O-acetylated at 3-hydroxyl of GlcNAc. We also report, the organization of the gene cluster responsible for capsule biosynthesis. We identify genes in this cluster that potentially encode an O-acetyltransferase, an N-acetylglucosamine transferase, and a KDO transferase consistent with the structure we report.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2003

Temperature modulates the cell wall mechanical properties of rice coleoptiles by altering the molecular mass of hemicellulosic polysaccharides

The present study was conducted to investigate the mechanism inducing the difference in the cell wall extensibility of rice (Oryza sativa L. cv. Koshihikari) coleoptiles grown under various temperature (10-50 degrees C) conditions. The growth rate and the cell wall extensibility of rice coleoptiles exhibited the maximum value at 30-40 degrees C, and became smaller as the growth temperature rose or dropped from this temperature range. The amounts of cell wall polysaccharides per unit length of coleoptile increased in coleoptiles grown at 40 degrees C, but not at other temperature conditions. On the other hand, the molecular size of hemicellulosic polysaccharides was small at temperatures where the cell wall extensibility was high (30-40 degrees C). The autolytic activities of cell walls obtained from coleoptiles grown at 30 and 40 degrees C were substantially higher than those grown at 10, 20 and 50 degrees C. Furthermore, the activities of (1-->3),(1-->4)-beta-glucanases extracted from coleoptile cell walls showed a similar tendency. When oat (1-->3),(1-->4)-beta-glucans with high molecular mass were incubated with the cell wall enzyme preparations from coleoptiles grown at various temperature conditions, the extensive molecular mass downshifts were brought about only by the cell wall enzymes obtained from coleoptiles grown at 30-40 degrees C. There were close correlations between the cell wall extensibility and the molecular mass of hemicellulosic polysaccharides or the activity of beta -glucanases. These results suggest that the environmental temperature regulates the cell wall extensibility of rice coleoptiles by modifying mainly the molecular mass of hemicellulosic polysaccharides. Modulation of the activity of beta-glucanases under various temperature conditions may be involved in the alteration of the molecular size of hemicellulosic polysaccharides.

Cellulose/chemistry/metabolism↗

NASA NTRS1995

Structurally altered capsular polysaccharides produced by mutant bacteria

Structurally altered capsular polysaccharides are produced by mutant bacteria. These polysaccharides are isolated by selecting a wild type bacterial strain and a phage producing degradative enzymes that have substrate specificity for the capsular polysaccharides produced by the wild type bacteria. Phage-resistant mutants producing capsular polysaccharides are selected and the structurally altered capsular polysaccharide is isolated therefrom.

Kern, Roger G.↗

NASA NTRS1994

Method for producing capsular polysaccharides

Kern, Roger G.↗

DOE OSTI2024

Polyproline peptide targets Klebsiella pneumoniae polysaccharides to collapse biofilms

Hypervirulent Klebsiella pneumoniae is known for its increased extracellular polysaccharide production. Biofilm matrices of hyper- virulent K. pneumoniae have increased polysaccharide abundance and are uniquely susceptible to disruption by peptide bactenecin 7 (bac7 (1–35)). Here, using confocal microscopy, we show that poly- saccharides within the biofilm matrix collapse following bac7 (1–35) treatment. This collapse led to the release of cells from the biofilm, which were then killed by the peptide. Characterization of truncated peptide analogs revealed that their interactions with polysaccharide were responsible for the biofilm matrix changes that accompany bac7 (1–35) treatment. Ultraviolet photodissociation mass spec- trometry with the parental peptide or a truncated analog bac7 (10–35) reveal the important regions for bac7 (1–35) complexing with polysaccharides. Finally, we tested bac7 (1–35) using a murine skin abscess model and observed a significant decrease in the bacte- rial burden. These findings unveil the potential of bac7 (1–35) poly- saccharide interactions to collapse K. pneumoniae biofilms.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Polysaccharide niche partitioning of distinct Polaribacter clades during North Sea spring algal blooms

Massive releases of organic substrates during marine algal blooms trigger growth of many clades of heterotrophic bacteria. Algal polysaccharides represent the most diverse and structurally complex class of these substrates, yet their role in shaping the microbial community composition is poorly understood. We investigated, whether polysaccharide utilization capabilities contribute to niche differentiation of Polaribacter spp. (class Flavobacteriia; known to include relevant polysaccharidedegraders) that were abundant during 2009–2012 spring algal blooms in the southern North Sea. We identified six distinct Polaribacter clades using phylogenetic and phylogenomic analyses, quantified their abundances via fluorescence in situ hybridization, compared metagenome-assembled genomes, and assessed in situ gene expression using metaproteomics. Four clades with distinct polysaccharide niches were dominating. Polaribacter 2-a comprised typical first responders featuring small genomes with limited polysaccharide utilization capacities. Polaribacter 3-a were abundant only in 2010 and possessed a distinct sulfated α-glucoronomannan degradation potential. Polaribacter 3-b responded late in blooms and had the capacity to utilize sulfated xylan. Polaribacter 1-a featured high numbers of glycan degradation genes and were particularly abundant following Chattonella algae blooms. These results support the hypothesis that sympatric Polaribacter clades occupy distinct glycan niches during North Sea spring algal blooms.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Ancient Origin of Acetyltransferases Catalyzing O -acetylation of Plant Cell Wall Polysaccharides

Abstract Members of the domain of unknown function 231/trichome birefringence–like (TBL) family have been shown to be O-acetyltransferases catalyzing the acetylation of plant cell wall polysaccharides, including pectins, mannan, xyloglucan and xylan. However, little is known about the origin and evolution of plant cell wall polysaccharide acetyltransferases. Here, we investigated the biochemical functions of TBL homologs from Klebsormidium nitens, a representative of an early divergent class of charophyte green algae that are considered to be the closest living relatives of land plants, and Marchantia polymorpha, a liverwort that is an extant representative of an ancient lineage of land plants. The genomes of K. nitens and Marchantia polymorpha harbor two and six TBL homologs, respectively. Biochemical characterization of their recombinant proteins expressed in human embryonic kidney 293 cells demonstrated that the two K. nitens TBLs exhibited acetyltransferase activities acetylating the pectin homogalacturonan (HG) and hence were named KnPOAT1 and KnPOAT2. Among the six M. polymorpha TBLs, five (MpPOAT1 to 5) possessed acetyltransferase activities toward pectins and the remaining one (MpMOAT1) catalyzed 2-O- and 3-O-acetylation of mannan. While MpPOAT1,2 specifically acetylated HG, MpPOAT3,4,5 could acetylate both HG and rhamnogalacturonan-I. Consistent with the acetyltransferase activities of these TBLs, pectins isolated from K. nitens and both pectins and mannan from M. polymorpha were shown to be acetylated. These findings indicate that the TBL genes were recruited as cell wall polysaccharide O-acetyltransferases as early as in charophyte green algae with activities toward pectins and they underwent expansion and functional diversification to acetylate various cell wall polysaccharides during evolution of land plants.

Cell Biology↗

DOE OSTI2023

In planta single-molecule imaging and holographic force spectroscopy to study real-time, multimodal turnover dynamics of polysaccharides and associated carbohydrate metabolites

Cellular production of polysaccharides from simple sugar molecules serves critical roles in a variety of metabolic processes essential to the survival of every living organism. In the context of photosynthetically fixed carbon, cell wall polysaccharides synthesis is particularly of interest for increased biomass accumulation for human food, animal feed, and bioenergy related applications. Despite a long history of research in plant cell biology, our understanding of in planta cell wall biosynthesis and its regulation is far from complete due to lack of a comprehensive microscopy toolkit that encompass the multiple spatial and temporal scales in which cell wall polysaccharides fibrils are synthesized and assembled into intricate cell wall networks. In this project, a multidisciplinary team of scientists from Rutgers University, Vanderbilt University, and Oak Ridge National Laboratory carried out innovative multimodal, multiscale microscopy studies of cell wall synthesis using single-molecule force-spectroscopy, super-resolution fluorescence microscopy, and in vivo live cell imaging. Our novel multimodal and holistic imaging approach revealed plant cell wall polysaccharides synthesis processes in unprecedented detail across multiple spatiotemporal scales, from a single-molecule to a single-cell. Besides, the team has made technical and scientific innovations across multiple research fields—microscopy, bioengineering, single-molecule biophysics, and plant biology, etc.—to accomplish the goals. The results from this project will greatly advance the mechanistic and holistic understanding of in planta cell wall synthesis, which will accelerate the development of better transgenic crops for bioenergy applications. Moreover, the new toolbox, combining powerful advanced microscopy assays with cell/protein engineering, will have broader impacts on molecular and cellular biology fields by paving the way for studying cellular processes occurring across multiple physical scales with multimodal microscopy methodologies.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

A red algal polysaccharide influences the multicellular development of the choanoflagellate Salpingoeca rosetta

The choanoflagellate Salpingoeca rosetta exemplifies the capacity of marine microeukaryotes to integrate environmental cues into their life histories. Some of the cues that S. rosetta detects are lipids from their bacterial prey that induce the development of multicellular colonies called rosettes. In the aquatic environments where S. rosetta would encounter bacterial cues, microbial communities gain refuge and food from algae. For example, microbes in coastal environments benefit from macroalgae that annually release ∼10 14 g of carbon, primarily in the form of polysaccharides that comprise ∼80% of dissolved organic carbon in surface waters. Despite the ecological impact of algae and historical descriptions of choanoflagellates attached to algae, the influence of algae on choanoflagellate life history transitions has remained unexplored. Here, we report that porphyran, a polysaccharide produced by the red macroalga Porphyra umbilicalis, induces multicellular development in S. rosetta. We first noticed this response when S. rosetta formed multicellular rosette colonies while growing in media prepared from P. umbilicalis, even though rosette-inducing bacteria were absent. By biochemically purifying extracts of P. umbilicalis, we identified porphyran as the rosette-inducing factor. This response provides a biochemical insight that indicates algal polysaccharides may serve as cues to mediate associations between choanoflagellates and algae. Moreover, this observation complements evidence from environmental and geochemical studies that show the impact that algae have exerted on the ecology and evolution of eukaryotes, including a rise in algal productivity during the origin of animals, the closest living relatives of choanoflagellates.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Thermodynamics of calcium binding to heparin: Implications of solvation and water structuring for polysaccharide biofunctions

Heparan sulfates are found in all animal tissues and have essential roles in living systems. This family of biomacromolecules modulates binding to calcium ions (Ca 2+ ) in low free energy reactions that influence biochemical processes from cell signaling and anticoagulant efficacy to biomineralization. Despite their ubiquity, the thermodynamic basis for how heparans and similarly functionalized biomolecules regulate Ca 2+ interactions is not yet established. Using heparosan (Control) and heparins with different positions of sulfate groups, we quantify how SO 3 − and COO − content and SO 3 − position modulate Ca 2+ binding by isothermal titration calorimetry. The free energy of all heparin-Ca2+ interactions (ΔG rxn ) is dominated by entropic contributions due to favorable water release from polar, hydrophilic groups. Heparin with both sulfate esters (O-SO 3 − ) and sulfamides (N-SO 3 − ) has the strongest binding to Ca 2+ compared to heparosan and to heparin with only O-SO 3 − groups (~3X). By linking Ca 2+ binding thermodynamics to measurements of the interfacial energy for calcite (CaCO 3 ) crystallization onto polysaccharides, we show molecule-specific differences in nucleation rate can be explained by differences in water structuring during Ca 2+ interactions. A large entropic term (-TΔS rxn ) upon Ca 2+ –polysaccharide binding correlates with high interfacial energy to CaCO 3 nucleation. Combining our measurements with literature values indicates many Ca 2+ –polysaccharide interactions have a shared thermodynamic signature. The resulting enthalpy–entropy compensation relationship suggests these interactions are generally dominated by water restructuring involving few conformational changes, distinct from Ca 2+ –protein binding. Our findings quantify the thermodynamic origins of heparin-specific interactions with Ca 2+ and demonstrate the contributions of solvation and functional group position during biomacromolecule-mediated ion regulation.

15 GEOTHERMAL ENERGY↗