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At least 379 records · Page 21
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

The Transcriptional Response of Soil Bacteria to Long-Term Warming and Short-Term Seasonal Fluctuations in a Terrestrial Forest

Terrestrial ecosystems are an important carbon store, and this carbon is vulnerable to microbial degradation with climate warming. After 30 years of experimental warming, carbon stocks in a temperate mixed deciduous forest were observed to be reduced by 30% in the heated plots relative to the controls. In addition, soil respiration was seasonal, as was the warming treatment effect. We therefore hypothesized that long-term warming will have higher expressions of genes related to carbohydrate and lipid metabolism due to increased utilization of recalcitrant carbon pools compared to controls. Because of the seasonal effect of soil respiration and the warming treatment, we further hypothesized that these patterns will be seasonal. We used RNA sequencing to show how the microbial community responds to long-term warming (~30 years) in Harvard Forest, MA. Total RNA was extracted from mineral and organic soil types from two treatment plots (+5°C heated and ambient control), at two time points (June and October) and sequenced using Illumina NextSeq technology. Treatment had a larger effect size on KEGG annotated transcripts than on CAZymes, while soil types more strongly affected CAZymes than KEGG annotated transcripts, though effect sizes overall were small. Although, warming showed a small effect on overall CAZymes expression, several carbohydrate-associated enzymes showed increased expression in heated soils (~68% of all differentially expressed transcripts). Further, exploratory analysis using an unconstrained method showed increased abundances of enzymes related to polysaccharide and lipid metabolism and decomposition in heated soils. Compared to long-term warming, we detected a relatively small effect of seasonal variation on community gene expression. Together, these results indicate that the higher carbohydrate degrading potential of bacteria in heated plots can possibly accelerate a self-reinforcing carbon cycle-temperature feedback in a warming climate.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

A Surface Exposed, Two-Domain Lipoprotein Cargo of a Type XI Secretion System Promotes Colonization of Host Intestinal Epithelia Expressing Glycans

The only known required component of the newly described Type XI secretion system (TXISS) is an outer membrane protein (OMP) of the DUF560 family. TXISS OMPs are broadly distributed across proteobacteria, but properties of the cargo proteins they secrete are largely unexplored. We report biophysical, histochemical, and phenotypic evidence that Xenorhabdus nematophila NilC is surface exposed. Biophysical data and structure predictions indicate that NilC is a two-domain protein with a C-terminal, 8-stranded β-barrel. This structure has been noted as a common feature of TXISS effectors and may be important for interactions with the TXISS OMP . The NilC N-terminal domain is more enigmatic, but our results indicate it is ordered and forms a β-sheet structure, and bioinformatics suggest structural similarities to carbohydrate-binding proteins. X. nematophila NilC and its presumptive TXISS OMP partner NilB are required for colonizing the anterior intestine of Steinernema carpocapsae nematodes: the receptacle of free-living, infective juveniles and the anterior intestinal cecum (AIC) in juveniles and adults. We show that, in adult nematodes, the AIC expresses a Wheat Germ Agglutinin (WGA)-reactive material, indicating the presence of N-acetylglucosamine or N-acetylneuraminic acid sugars on the AIC surface. A role for this material in colonization is supported by the fact that exogenous addition of WGA can inhibit AIC colonization by X. nematophila. Conversely, the addition of exogenous purified NilC increases the frequency with which X. nematophila is observed at the AIC, demonstrating that abundant extracellular NilC can enhance colonization. NilC may facilitate X. nematophila adherence to the nematode intestinal surface by binding to host glycans, it might support X. nematophila nutrition by cleaving sugars from the host surface, or it might help protect X. nematophila from nematode host immunity. Proteomic and metabolomic analyses of wild type X. nematophila compared to those lacking nilB and nilC revealed differences in cell wall and secreted polysaccharide metabolic pathways. Additionally, purified NilC is capable of binding peptidoglycan, suggesting that periplasmic NilC may interact with the bacterial cell wall. Overall, these findings support a model that NilB-regulated surface exposure of NilC mediates interactions between X. nematophila and host surface glycans during colonization. This is a previously unknown function for a TXISS.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Mechanism of dielectric barrier discharge plasma technology to improve the quantity of short-chain fatty acids in anaerobic fermentation of waste active sludge

The mechanism of improving the anaerobic fermentation performance of waste active sludge by using dielectric barrier discharge (DBD) plasma pretreatment technology was investigated. The maximum accumulation of short-chain fatty acids (SCFAs) was observed on the 7th day of anaerobic fermentation when the DBD power was 76.50 W, which was 1726.70 mg COD/L, 1.50 times of the control group. The ratio of acetic acid in DBD group was 9.30% higher than that in the control. Further mechanism research indicated that DBD pretreatment can destroy the structure of extracellular polymer substances and release organic substances such as protein and polysaccharide. The dissolved organic matter analysis indicated that the DBD technique could increase the release of biodegradable organics (eg., tyrosine proteins, soluble microbial by-products), thus accelerate the biotransformation of organic substance. Bacterial community structure analysis showed that the increase in the abundance of Firmicutes and Bacteroidetes and the decrease in the abundance of Proteobacteria in DBD group were beneficial to the accumulation of SCFAs. Besides, further archaeal analysis indicated that the decrease of Methanosaeta sp. and Methanosarcina sp . abundance in the DBD group facilitate acetic acid accumulation. This study demonstrated that the DBD technique can be used as an effective and potential pretreatment method to improve sludge anaerobic fermentation performance.

Wang, Jie↗
DOE OSTI2023

Whither the genus Caldicellulosiruptor and the order Thermoanaerobacterales: phylogeny, taxonomy, ecology, and phenotype

The order Thermoanaerobacterales currently consists of fermentative anaerobic bacteria, including the genus Caldicellulosiruptor. Caldicellulosiruptor are represented by thirteen species; all, but one, have closed genome sequences. Interest in these extreme thermophiles has been motivated not only by their high optimal growth temperatures (≥70°C), but also by their ability to hydrolyze polysaccharides including, for some species, both xylan and microcrystalline cellulose. Caldicellulosiruptor species have been isolated from geographically diverse thermal terrestrial environments located in New Zealand, China, Russia, Iceland and North America. Evidence of their presence in other terrestrial locations is apparent from metagenomic signatures, including volcanic ash in permafrost. Here, phylogeny and taxonomy of the genus Caldicellulosiruptor was re-examined in light of new genome sequences. Based on genome analysis of 15 strains, a new order, Caldicellulosiruptorales, is proposed containing the family Caldicellulosiruptoraceae, consisting of two genera, Caldicellulosiruptor and Anaerocellum. Furthermore, the order Thermoanaerobacterales also was re-assessed, using 91 genome-sequenced strains, and should now include the family Thermoanaerobacteraceae containing the genera Thermoanaerobacter, Thermoanaerobacterium, Caldanaerobacter, the family Caldanaerobiaceae containing the genus Caldanaerobius, and the family Calorimonaceae containing the genus Calorimonas. A main outcome of ANI/AAI analysis indicates the need to reclassify several previously designated species in the Thermoanaerobacterales and Caldicellulosiruptorales by condensing them into strains of single species. Comparative genomics of carbohydrate-active enzyme inventories suggested differentiating phenotypic features, even among strains of the same species, reflecting available nutrients and ecological roles in their native biotopes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Biochemical characterization of Fsa16295Glu from “Fervidibacter sacchari,” the first hyperthermophilic GH50 with β-1,3-endoglucanase activity and founding member of the subfamily GH50_3

The aerobic hyperthermophile “Fervidibacter sacchari” catabolizes diverse polysaccharides and is the only cultivated member of the class “Fervidibacteria” within the phylum Armatimonadota. It encodes 117 putative glycoside hydrolases (GHs), including two from GH family 50 (GH50). In this study, we expressed, purified, and functionally characterized one of these GH50 enzymes, Fsa16295Glu. We show that Fsa16295Glu is a β-1,3-endoglucanase with optimal activity on carboxymethyl curdlan (CM-curdlan) and only weak agarase activity, despite most GH50 enzymes being described as β-agarases. The purified enzyme has a wide temperature range of 4–95°C (optimal 80°C), making it the first characterized hyperthermophilic representative of GH50. The enzyme is also active at a broad pH range of at least 5.5–11 (optimal 6.5–10). Fsa16295Glu possesses a relatively high k cat /K M of 1.82 × 10 7 s-1 M-1 with CM-curdlan and degrades CM-curdlan nearly completely to sugar monomers, indicating preferential hydrolysis of glucans containing β-1,3 linkages. Finally, a phylogenetic analysis of Fsa16295Glu and all other GH50 enzymes revealed that Fsa16295Glu is distant from other characterized enzymes but phylogenetically related to enzymes from thermophilic archaea that were likely acquired horizontally from “Fervidibacteria.” Given its functional and phylogenetic novelty, we propose that Fsa16295Glu represents a new enzyme subfamily, GH50_3.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Towards Elucidating Structure–Spectra Relationships in Rhamnogalacturonan II: Computational Protocols for Accurate 13C and 1H Shifts for Apiose and Its Borate Esters

Apiose is a naturally occurring, uncommon branched-chain pentose found in plant cell walls as part of the complex polysaccharide Rhamnogalacturonan II (RG-II). The structural elucidation of the three-dimensional structure of RG-II by nuclear magnetic resonance (NMR) spectroscopy is significantly complicated by the ability of apiose to cross-link via borate ester linkages to form RG-II dimers. Here, we developed a computational approach to gain insight into the structure–spectra relationships of apio–borate complexes in an effort to complement experimental assignments of NMR signals in RG-II. Our protocol involved structure optimizations using density functional theory (DFT) followed by isotropic magnetic shielding constant calculations using the gauge-invariant atomic orbital (GIAO) approach to predict chemical shifts. We evaluated the accuracy of 23 different functional–basis set (FBS) combinations with and without implicit solvation for predicting the experimental 1 H and 13 C shifts of a methyl apioside and its three borate derivatives. The computed NMR predictions were evaluated on the basis of the overall shift accuracy, relative shift ordering, and the ability to distinguish between dimers and monomers. We demonstrate that the consideration of implicit solvation during geometry optimizations in addition to the magnetic shielding constant calculations greatly increases the accuracy of NMR chemical shift predictions and can correctly reproduce the ordering of the 13 C shifts and yield predictions that are, on average, within 1.50 ppm for 13 C and 0.12 ppm for 1 H shifts for apio–borate compounds.

BASIC BIOLOGICAL SCIENCES,BIOMASS FUELS↗
DOE OSTI2020

Insights of Molecular Mechanism of Xylem Development in Five Black Poplar Cultivars

Black poplar (Populus deltoides, P. nigra, and their hybrids) is the main poplar cultivars in China. It offers interesting options of large-scale biomass production for bioenergy due to its rapid growth and high yield. Poplar wood properties were associated with chemical components and physical structures during wood formation. In this study, five poplar cultivars, P. euramericana ‘Zhonglin46’ (Pe1), P. euramericana ‘Guariento’ (Pe2), P. nigra ‘N179’ (Pn1), P. deltoides ‘Danhong’ (Pd1), and P. deltoides ‘Nanyang’ (Pd2), were used to explore the molecular mechanism of xylem development. We analyzed the structural differences of developing xylem in the five cultivars and profiled the transcriptome-wide gene expression patterns through RNA sequencing. The cross sections of the developing xylem showed that the cell wall thickness of developed fiber in Pd1 was thickest and the number of xylem vessels of Pn1 was the least. A total of 10,331 differentially expressed genes were identified among 10 pairwise comparisons of the five cultivars, most of them were related to programmed cell death and secondary cell wall thickening. K-means cluster analysis and Gene Ontology enrichment analysis showed that the genes highly expressed in Pd1 were related to nucleotide decomposition, metabolic process, transferase, and microtubule cytoskeleton; whereas the genes highly expressed in Pn1 were involved in cell wall macromolecule decomposition and polysaccharide binding processes. Based on a weighted gene co-expression network analysis, a large number of candidate regulators for xylem development were identified. And their potential regulatory roles to cell wall biosynthesis genes were validated by a transient overexpression system. This study provides a set of promising candidate regulators for genetic engineering to improve feedstock and enhance biofuel conversion in the bioenergy crop Populus.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A t FUT4 and A t FUT6 Are Arabinofuranose-Specific Fucosyltransferases

The bulk of plant biomass is comprised of plant cell walls, which are complex polymeric networks, composed of diverse polysaccharides, proteins, polyphenolics, and hydroxyproline-rich glycoproteins (HRGPs). Glycosyltransferases (GTs) work together to synthesize the saccharide components of the plant cell wall. The Arabidopsis thaliana fucosyltransferases (FUTs), At FUT4, and At FUT6, are members of the plant-specific GT family 37 (GT37). At FUT4 and At FUT6 transfer fucose (Fuc) onto arabinose (Ara) residues of arabinogalactan (AG) proteins (AGPs) and have been postulated to be non-redundant AGP-specific FUTs. At FUT4 and At FUT6 were recombinantly expressed in mammalian HEK293 cells and purified for biochemical analysis. We report an updated understanding on the specificities of At FUT4 and At FUT6 that are involved in the synthesis of wall localized AGPs. Our findings suggest that they are selective enzymes that can utilize various arabinogalactan (AG)-like and non-AG-like oligosaccharide acceptors, and only require a free, terminal arabinofuranose. We also report with GUS promoter-reporter gene studies that AtFUT4 and AtFUT6 gene expression is sub-localized in different parts of developing A. thaliana roots.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Salt Stress Induces Paramylon Accumulation and Fine-Tuning of the Macro-Organization of Thylakoid Membranes in Euglena gracilis Cells

The effects of salt stress condition on the growth, morphology, photosynthetic performance, and paramylon content were examined in the mixotrophic, unicellular, flagellate Euglena gracilis. We found that salt stress negatively influenced cell growth, accompanied by a decrease in chlorophyll (Chl) content. Circular dichroism (CD) spectroscopy revealed the changes in the macro-organization of pigment-protein complexes due to salt treatment, while the small-angle neutron scattering (SANS) investigations suggested a reduction in the thylakoid stacking, an effect confirmed by the transmission electron microscopy (TEM). At the same time, the analysis of the thylakoid membrane complexes using native-polyacrylamide gel electrophoresis (PAGE) revealed no significant change in the composition of supercomplexes of the photosynthetic apparatus. Salt stress did not substantially affect the photosynthetic activity, as reflected by the fact that Chl fluorescence yield, electron transport rate (ETR), and energy transfer between the photosystems did not change considerably in the salt-grown cells. We have observed notable increases in the carotenoid-to-Chl ratio and the accumulation of paramylon in the salt-treated cells. We propose that the accumulation of storage polysaccharides and changes in the pigment composition and thylakoid membrane organization help the adaptation of E. gracilis cells to salt stress and contribute to the maintenance of cellular processes under stress conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Cell Wall Compositions of Sorghum bicolor Leaves and Roots Remain Relatively Constant Under Drought Conditions

Renewable fuels are needed to replace fossil fuels in the immediate future. Lignocellulosic bioenergy crops provide a renewable alternative that sequesters atmospheric carbon. To prevent displacement of food crops, it would be advantageous to grow biofuel crops on marginal lands. These lands will likely face more frequent and extreme drought conditions than conventional agricultural land, so it is crucial to see how proposed bioenergy crops fare under these conditions and how that may affect lignocellulosic biomass composition and saccharification properties. We found that while drought impacts the plant cell wall of Sorghum bicolor differently according to tissue and timing of drought induction, drought-induced cell wall compositional modifications are relatively minor and produce no negative effect on biomass conversion. This contrasts with the cell wall-related transcriptome, which had a varied range of highly variable genes (HVGs) within four cell wall-related GO categories, depending on the tissues surveyed and time of drought induction. Further, many HVGs had expression changes in which putative impacts were not seen in the physical cell wall or which were in opposition to their putative impacts. Interestingly, most pre-flowering drought-induced cell wall changes occurred in the leaf, with matrix and lignin compositional changes that did not persist after recovery from drought. Most measurable physical post-flowering cell wall changes occurred in the root, affecting mainly polysaccharide composition and cross-linking. This study couples transcriptomics to cell wall chemical analyses of a C4 grass experiencing progressive and differing drought stresses in the field. As such, we can analyze the cell wall-specific response to agriculturally relevant drought stresses on the transcriptomic level and see whether those changes translate to compositional or biomass conversion differences. Our results bolster the conclusion that drought stress does not substantially affect the cell wall composition of specific aerial and subterranean biomass nor impede enzymatic hydrolysis of leaf biomass, a positive result for biorefinery processes. Coupled with previously reported results on the root microbiome and rhizosphere and whole transcriptome analyses of this study, we can formulate and test hypotheses on individual gene candidates’ function in mediating drought stress in the grass cell wall, as demonstrated in sorghum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Using CRISPR-Cas9 Technology to Eliminate Xyloglucan in Tobacco Cell Walls and Change the Uptake and Translocation of Inorganic Arsenic

Xyloglucan is a quantitatively major polysaccharide in the primary cell walls of flowering plants and has been reported to affect plants’ ability to tolerate toxic elements. However, it is not known if altering the amounts of xyloglucan in the wall influences the uptake and translocation of inorganic arsenic (As). Here, we identified two Nicotiana tabacum genes that encode xyloglucan-specific xylosyltransferases (XXT), which we named NtXXT1 and NtXXT2. We used CRISPR-Cas9 technology to generate ntxxt1 , ntxxt2 , and ntxxt1/2 mutant tobacco plants to determine if preventing xyloglucan synthesis affects plant growth and their ability to accumulate As. We show that NtXXT1 and NtXXT2 are required for xyloglucan biosynthesis because no discernible amounts of xyloglucan were present in the cell walls of the ntxxt1/2 double mutant. The tobacco double mutant ( ntxxt1/2 ) and the corresponding Arabidopsis mutant ( atxxt1/2 ) do not have severe growth defects but do have a short root hair phenotype and a slow growth rate. This phenotype is rescued by overexpressing NtXXT1 or NtXXT2 in atxxt1/2 . Growing ntxxt mutants in the presence of AsIII or AsV showed that the absence of cell wall xyloglucan affects the accumulation and translocation of As. Most notably, root retention of As increased substantially and the amounts of As translocated to the shoots decreased in ntxxt1/2 . Our results suggest that xyloglucan-deficient plants provide a strategy for the phytoremediation of As contaminated soils.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Engineering Curcumin Biosynthesis in Poplar Affects Lignification and Biomass Yield

Lignocellulosic biomass is recalcitrant toward deconstruction into simple sugars mainly due to the presence of lignin. By engineering plants to partially replace traditional lignin monomers with alternative ones, lignin degradability and extractability can be enhanced. Previously, the alternative monomer curcumin has been successfully produced and incorporated into lignified cell walls of Arabidopsis by the heterologous expression of DIKETIDE-CoA SYNTHASE (DCS) and CURCUMIN SYNTHASE2 (CURS2). The resulting transgenic plants did not suffer from yield penalties and had an increased saccharification yield after alkaline pretreatment. Here, we translated this strategy into the bio-energy crop poplar. Via the heterologous expression of DCS and CURS2 under the control of the secondary cell wall CELLULOSE SYNTHASE A8-B promoter (ProCesA8-B), curcumin was also produced and incorporated into the lignified cell walls of poplar. ProCesA8-B:DCS_CURS2 transgenic poplars, however, suffered from shoot-tip necrosis and yield penalties. Compared to that of the wild-type (WT), the wood of transgenic poplars had 21% less cellulose, 28% more matrix polysaccharides, 23% more lignin and a significantly altered lignin composition. More specifically, ProCesA8-B:DCS_CURS2 lignin had a reduced syringyl/guaiacyl unit (S/G) ratio, an increased frequency of p-hydroxyphenyl (H) units, a decreased frequency of p-hydroxybenzoates and a higher fraction of phenylcoumaran units. Without, or with alkaline or hot water pretreatment, the saccharification efficiency of the transgenic lines was equal to that of the WT. These differences in (growth) phenotype illustrate that translational research in crops is essential to assess the value of an engineering strategy for applications. Further fine-tuning of this research strategy (e.g., by using more specific promoters or by translating this strategy to other crops such as maize) might lead to transgenic bio-energy crops with cell walls more amenable to deconstruction without settling in yield.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genetic diversity, population structure and anthracnose resistance response in a novel sweet sorghum diversity panel

Sweet sorghum is an attractive feedstock for the production of renewable chemicals and fuels due to the readily available fermentable sugars that can be extracted from the juice, and the additional stream of fermentable sugars that can be obtained from the cell wall polysaccharides in the bagasse. An important selection criterion for new sweet sorghum germplasm is resistance to anthracnose, a disease caused by the fungal pathogen Colletotrichum sublineolum. The identification of novel anthracnose-resistance sources present in sweet sorghum germplasm offers a fast track towards the development of new resistant sweet sorghum germplasm. We established a sweet sorghum diversity panel (SWDP) of 272 accessions from the USDA-ARS National Plant Germplasm (NPGS) collection that includes landraces from 22 countries and advanced breeding material, and that represents ~15% of the NPGS sweet sorghum collection. Genomic characterization of the SWDP identified 171,954 single nucleotide polymorphisms (SNPs) with an average of one SNP per 4,071 kb. Population structure analysis revealed that the SWDP could be stratified into four populations and one admixed group, and that this population structure could be aligned to sorghum’s racial classification. Results from a two-year replicated trial of the SWDP for anthracnose resistance response in Texas, Georgia, Florida, and Puerto Rico showed 27 accessions to be resistant across locations, while 145 accessions showed variable resistance response against local pathotypes. A genome-wide association study identified 16 novel genomic regions associated with anthracnose resistance. Four resistance loci on chromosomes 3, 6, 8 and 9 were identified against pathotypes from Puerto Rico, and two resistance loci on chromosomes 3 and 8 against pathotypes from Texas. In Georgia and Florida, three resistance loci were detected on chromosomes 4, 5, 6 and four on chromosomes 4, 5 (two loci) and 7, respectively. One resistance locus on chromosome 2 was effective against pathotypes from Texas and Puerto Rico and a genomic region of 41.6 kb at the tip of chromosome 8 was associated with resistance response observed in Georgia, Texas, and Puerto Rico. This publicly available SWDP and the extensive evaluation of anthracnose resistance represent a valuable genomic resource for the improvement of sorghum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Effects of feruloyl-CoA 6'-hydroxylase 1 overexpression on lignin and cell wall characteristics in transgenic hybrid aspen

In plant cell walls, lignin, cellulose, and the hemicelluloses form intricate three-dimensional structures. Owing to its complexity, lignin often acts as a bottleneck for the efficient utilization of polysaccharide components as biochemicals and functional materials. A promising approach to mitigate and/or overcome lignin recalcitrance is the qualitative and quantitative modification of lignin by genetic engineering. Feruloyl-CoA 6'-hydroxylase (F6'H1) is a 2-oxoglutarate-dependent dioxygenase that catalyzes the conversion of feruloyl-CoA, one of the intermediates of the lignin biosynthetic pathway, into 6'-hydroxyferuloyl-CoA, the precursor of scopoletin (7-hydroxy-6-methoxycoumarin). In a previous study with Arabidopsis thaliana, we demonstrated that overexpression of F6'H1 under a xylem-preferential promoter led to scopoletin incorporation into the cell wall. This altered the chemical structure of lignin without affecting lignin content or saccharification efficiency. In the present study, the same F6'H1 construct was introduced into hybrid aspen (Populus tremula × tremuloides T89), a model woody plant, and its effects on plant morphology, lignin chemical structure, global gene expression, and phenolic metabolism were examined. The transgenic plants successfully overproduced scopoletin while exhibiting severe growth retardation, a phenotype not previously observed in Arabidopsis. Scopoletin accumulation was most pronounced in the secondary walls of tracheary elements and the compound middle lamella, with low levels in the fiber cell walls. Overexpression of F6'H1 also affected the metabolism of aromatics, including lignin precursors. Heteronuclear single-quantum coherence (HSQC) NMR spectroscopy revealed that scopoletin in cell walls was bound to lignin, leading to a reduction in lignin content and changes in its monomeric composition and molar mass distribution. Furthermore, the enzymatic saccharification efficiency of the transgenic cell walls was more than three times higher than that of the wild-type plants, even without pretreatment. Although addressing growth inhibition remains a priority, incorporating scopoletin into lignin demonstrates significant potential for improving woody biomass utilization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Characterization of Oral Candida spp. Biofilms in Children and Adults Carriers from Eastern Europe and South America

Background: Candida albicans and non-Candida albicans Candida species (NCACs) are known to colonize and invade various tissues, including the oral mucosa. In this work, we aimed to characterize mature biofilms of several Candida spp. clinical isolates (n = 33) obtained from the oral mucosa of children, adults, and elders of Eastern Europe and South America. Methods: Each strain was evaluated for its capacity to form biofilms in terms of total biomass using the crystal violet assay and for matrix components production (proteins and carbohydrates) using the BCA and phenol-sulfuric tests, respectively. The effect of different antifungals on biofilm formation was studied. Results: in the children’s group, a predominance of C. krusei (81%) was observed, while, among adults, the main species was C. albicans (59%). Most strains showed a reduced response to antimicrobial drugs when in biofilm form (p < 0.01). Moreover, it was observed that strains isolated from children produced more matrix, with higher levels of protein and polysaccharides. Conclusions: children were more likely to be infected by NCACs than adults. More importantly, these NCACs were able to form biofilms richer in matrix components. This finding is of clinical importance, particularly in pediatric care, since stronger biofilms are highly associated with antimicrobial resistance, recurrent infections, and higher therapeutic failure.

Alves, Anelise Maria Costa Vasconcelos (ORCID:0000↗
DOE OSTI2023

Harnessing Plant Sugar Metabolism for Glycoengineering

Plants possess an innate ability to generate vast amounts of sugar and produce a range of sugar-derived compounds that can be utilized for applications in industry, health, and agriculture. Nucleotide sugars lie at the unique intersection of primary and specialized metabolism, enabling the biosynthesis of numerous molecules ranging from small glycosides to complex polysaccharides. Plants are tolerant to perturbations to their balance of nucleotide sugars, allowing for the overproduction of endogenous nucleotide sugars to push flux towards a particular product without necessitating the re-engineering of upstream pathways. Pathways to produce even non-native nucleotide sugars may be introduced to synthesize entirely novel products. Heterologously expressed glycosyltransferases capable of unique sugar chemistries can further widen the synthetic repertoire of a plant, and transporters can increase the amount of nucleotide sugars available to glycosyltransferases. In this opinion piece, we examine recent successes and potential future uses of engineered nucleotide sugar biosynthetic, transport, and utilization pathways to improve the production of target compounds. Additionally, we highlight current efforts to engineer glycosyltransferases. Ultimately, the robust nature of plant sugar biochemistry renders plants a powerful chassis for the production of target glycoconjugates and glycans.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structure of Lipopolysaccharide from Liberibacter crescens Is Low Molecular Weight and Offers Insight into Candidatus Liberibacter Biology

Huanglongbing (HLB) disease, also known as citrus greening disease, was first reported in the US in 2005. Since then, the disease has decimated the citrus industry in Florida, resulting in billions of dollars in crop losses and the destruction of thousands of acres of citrus groves. The causative agent of citrus greening disease is the phloem limited pathogen Candidatus Liberibacter asiaticus. As it has not been cultured, very little is known about the structural biology of the organism. Liberibacter are part of the Rhizobiaceae family, which includes nitrogen-fixing symbionts of legumes as well as the Agrobacterium plant pathogens. To better understand the Liberibacter genus, a closely related culturable bacterium (Liberibacter crescens or Lcr) has attracted attention as a model organism for structural and functional genomics of Liberibacters. Given that the structure of lipopolysaccharides (LPS) from Gram-negative bacteria plays a crucial role in mediating host-pathogen interactions, we sought to characterize the LPS from Lcr. We found that the major lipid A component of the LPS consisted of a pentaacylated molecule with a β-6-GlcN disaccharide backbone lacking phosphate. The polysaccharide portion of the LPS was unusual compared to previously described members of the Rhizobiaceae family in that it contained ribofuranosyl residues. The LPS structure presented here allows us to extrapolate known LPS structure/function relationships to members of the Liberibacter genus which cannot yet be cultured. It also offers insights into the biology of the organism and how they manage to effectively attack citrus trees.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Genomic Analysis of Aspergillus Section Terrei Reveals a High Potential in Secondary Metabolite Production and Plant Biomass Degradation

Aspergillus terreus has attracted interest due to its application in industrial biotechnology, particularly for the production of itaconic acid and bioactive secondary metabolites. As related species also seem to possess a prosperous secondary metabolism, they are of high interest for genome mining and exploitation. Here, we present draft genome sequences for six species from Aspergillus section Terrei and one species from Aspergillus section Nidulantes. Whole-genome phylogeny confirmed that section Terrei is monophyletic. Genome analyses identified between 70 and 108 key secondary metabolism genes in each of the genomes of section Terrei, the highest rate found in the genus Aspergillus so far. The respective enzymes fall into 167 distinct families with most of them corresponding to potentially unique compounds or compound families. Moreover, 53% of the families were only found in a single species, which supports the suitability of species from section Terrei for further genome mining. Intriguingly, this analysis, combined with heterologous gene expression and metabolite identification, suggested that species from section Terrei use a strategy for UV protection different to other species from the genus Aspergillus. Section Terrei contains a complete plant polysaccharide degrading potential and an even higher cellulolytic potential than other Aspergilli, possibly facilitating additional applications for these species in biotechnology.

60 APPLIED LIFE SCIENCES↗