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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 289 records · Page 16
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Delayed access to feed early post-hatch affects the development and maturation of gastrointestinal tract microbiota in broiler chickens

Background: The first two weeks of post-hatch (PH) growth in broilers (meat-type birds) are critical for gut development and microbiota colonization. In the current broiler production system, chicks may not receive feed and water for 24 to 72 h due to variations in hatching time and hatchery management. Post-hatch feed delay affects body weight, feed efficiency, mortality, and gut development. The goal of this study was to investigate changes in the microbiome in broiler chickens early PH and the effect of delayed access to feed on the microbiota. Results: Chicks either received feed and water immediately after hatch or access to feed was delayed for 48 h to mimic commercial hatchery settings (treatment, TRT). Both groups were sampled (n = 6) at -48, 0, 4 h, and 1 (24 h), 2 (48 h), 3 (72 h), 4 (96 h), 6 (144 h), 8 (192 h), 10 (240 h), 12 (288 h) and 14 (336 h) days PH. Ileal (IL) and cecal (CE) epithelial scrapings (mucosal bacteria, M) and digesta (luminal bacteria, L) were collected for microbiota analysis. Microbiota was determined by sequencing the V3-V4 region of bacterial 16S rRNA and analyzed using QIIME2. The microbiota of early ileal and cecal samples were characterized by high abundance of unclassified bacteria. Among four bacterial populations (IL-L, IL-M, CE-L, CE-M), IL-M was the least affected by delayed access to feed early PH. Both alpha and beta diversities were affected by delayed access to feed PH in IL-L, CE-M and CE-L. However, the development effect was more pronounced. In all four bacterial populations, significant changes due to developmental effect (time relative to hatch) was observed in taxonomic composition, with transient changes of bacterial taxa during the first two weeks PH. Delayed access to feed has limited influence on bacterial composition with only a few genera and species affected in all four bacterial populations. Predicted function based on 16S rRNA was also affected by delayed access to feed PH with most changes in metabolic pathway richness observed in IL-L, CE-L and CE-M. Conclusions: These results show transient changes in chicken microbiota biodiversity during the first two weeks PH and indicate that delayed access to feed affects microbiota development. Proper microbiota development could be an important factor in disease prevention and antibiotic use in broiler chickens. Moreover, significant differences in response to delayed access to feed PH between luminal and mucosal bacterial populations strongly suggests the need for separate analysis of these two populations.

16S↗
DOE OSTI2021

Species- and site-specific genome editing in complex bacterial communities

Knowledge of microbial gene functions comes from manipulating the DNA of individual strains in isolation from their natural communities. While this approach to microbial genetics has been foundational, its requirement for culturable microorganisms has left the majority of microbes and their interactions genetically unexplored. Here, we describe a generalizable strategy for editing the genomes of specific organisms within microbial communities. We identified genetically tractable bacteria within a community using Environmental Transformation Sequencing (ET-Seq), an approach in which non-targeted transposon integrations are mapped and quantified following community delivery. We next developed and used DNA-editing All-in-one RNA-guided CRISPR-Cas Transposase (DART) systems for targeted DNA insertion into organisms identified as tractable by ET-Seq, enabling organism- and locus-specific genetic manipulation within the community context. To illustrate the utility of our approach, we selectively edited closely related strains, measured gene fitness, and enriched targeted members within soil and infant gut microbiota. These results establish a new paradigm for targeted community editing relevant to research and applications on medical, agricultural, and industrial microbiomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

DNA transfer between two different species mediated by heterologous cell fusion in Clostridium coculture

Investigations of natural multispecies microbiomes and synthetic microbial cocultures are attracting renewed interest for their potential application in biotechnology, ecology, and medical fields. Previously, we have shown the syntrophic coculture of C. acetobutylicum and C. ljungdahlii undergoes heterologous cell-to-cell fusion, which facilitates the exchange of cytoplasmic protein and RNA between the two organisms. We now show that heterologous cell fusion between the two Clostridium organisms can facilitate the exchange of DNA. By applying selective pressures to this coculture system, we isolated clones of wild-type C. acetobutylicum which acquired the erythromycin resistance (erm) gene from the C. ljungdahlii strain carrying a plasmid with the erm gene. Single-molecule real-time sequencing revealed that the erm gene was integrated into the genome in a mosaic fashion. Our data also support the persistence of hybrid C. acetobutylicum / C. ljungdahlii cells displaying hybrid DNA-methylation patterns.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Model Soil Consortium 2 (MSC-2) Bacterial Isolate Genomes

Model Soil Consortium 2 (MSC-2) bacterial isolate genome collections are derived from the soil consortium MSC-1 multi-species cultured isolate genome collections from a previously reported WA-IsoC_MSC1.1.0 collection of a naturally evolved, model soil consortia (10.25584/WAIsoCMSC1/1635272). This collection contains 8 different isolate species cultivated under variable carbon and nitrogen sources, originating from the IAREC grassland soil field site located in Prosser, WA, USA. Genomic sequencing of one or more organisms, or genomes in general, such as meta-information on genomes, genome projects, gene names of a given organism within a natural environment. The version described here is the first version.

Soil microbiome, defined community, chitin↗
DOE OSTI2024

An expanded transcriptome atlas for Bacteroides thetaiotaomicron reveals a small RNA that modulates tetracycline sensitivity

Plasticity in gene expression allows bacteria to adapt to diverse environments. This is particularly relevant in the dynamic niche of the human intestinal tract; however, transcriptional networks remain largely unknown for gut-resident bacteria. Here we apply differential RNA sequencing (RNA-seq) and conventional RNA-seq to the model gut bacterium Bacteroides thetaiotaomicron to map transcriptional units and profile their expression levels across 15 in vivo-relevant growth conditions. We infer stress- and carbon source-specific transcriptional regulons and expand the annotation of small RNAs (sRNAs). Integrating this expression atlas with published transposon mutant fitness data, we predict conditionally important sRNAs. These include MasB, which downregulates tetracycline tolerance. Using MS2 affinity purification and RNA-seq, we identify a putative MasB target and assess its role in the context of the MasB-associated phenotype. These data—publicly available through the Theta-Base web browser (http://micromix.helmholtz-hiri.de/bacteroides/)—constitute a valuable resource for the microbiome community.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Oxic–anoxic cycling promotes coupling between complex carbon metabolism and denitrification in woodchip bioreactors

Denitrifying woodchip bioreactors (WBRs) are increasingly used to manage the release of non-point source nitrogen (N) by stimulating microbial denitrification. Woodchips serve as a renewable organic carbon (C) source, yet the recalcitrance of organic C in lignocellulosic biomass causes many WBRs to be C-limited. Prior studies have observed that oxic–anoxic cycling increased the mobilization of organic C, increased nitrate (NO 3 – ) removal rates, and attenuated production of nitrous oxide (N 2 O). Here, in this work, we use multi-omics approaches and amplicon sequencing of fungal 5.8S-ITS2 and prokaryotic 16S rRNA genes to elucidate the microbial drivers for enhanced NO 3 – removal and attenuated N 2 O production under redox-dynamic conditions. Transient oxic periods stimulated the expression of fungal ligninolytic enzymes, increasing the bioavailability of woodchip-derived C and stimulating the expression of denitrification genes. Nitrous oxide reductase (nosZ) genes were primarily clade II, and the ratio of clade II/clade I nosZ transcripts during the oxic–anoxic transition was strongly correlated with the N 2 O yield. Analysis of metagenome-assembled genomes revealed that many of the denitrifying microorganisms also have a genotypic ability to degrade complex polysaccharides like cellulose and hemicellulose, highlighting the adaptation of the WBR microbiome to the ecophysiological niche of the woodchip matrix.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

MicroFisher: Fungal taxonomic classification for metatranscriptomic and metagenomic data using multiple short hypervariable markers

AbstractProfiling the taxonomic and functional composition of microbes using metagenomic (MG) and metatranscriptomic (MT) sequencing is advancing our understanding of microbial functions. However, the sensitivity and accuracy of microbial classification using genome– or core protein-based approaches, especially the classification of eukaryotic organisms, is limited by the availability of genomes and the resolution of sequence databases. To address this, we propose the MicroFisher, a novel approach that applies multiple hypervariable marker genes to profile fungal communities from MGs and MTs. This approach utilizes the hypervariable regions of ITS and large subunit (LSU) rRNA genes for fungal identification with high sensitivity and resolution. Simultaneously, we propose a computational pipeline (MicroFisher) to optimize and integrate the results from classifications using multiple hypervariable markers. To test the performance of our method, we applied MicroFisher to the synthetic community profiling and found high performance in fungal prediction and abundance estimation. In addition, we also used MGs from forest soil and MTs of root eukaryotic microbes to test our method and the results showed that MicroFisher provided more accurate profiling of environmental microbiomes compared to other classification tools. Overall, MicroFisher serves as a novel pipeline for classification of fungal communities from MGs and MTs.

Wang, Haihua↗
DOE OSTI2023

A global perspective on bacterial diversity in the terrestrial deep subsurface

While recent efforts to catalogue Earth’s microbial diversity have focused upon surface and marine habitats, 12–20% of Earth’s biomass is suggested to exist in the terrestrial deep subsurface, compared to ~1.8% in the deep subseafloor. Metagenomic studies of the terrestrial deep subsurface have yielded a trove of divergent and functionally important microbiomes from a range of localities. However, a wider perspective of microbial diversity and its relationship to environmental conditions within the terrestrial deep subsurface is still required. Our meta-analysis reveals that terrestrial deep subsurface microbiota are dominated by Betaproteobacteria, Gammaproteobacteria and Firmicutes, probably as a function of the diverse metabolic strategies of these taxa. Evidence was also found for a common small consortium of prevalent Betaproteobacteria and Gammaproteobacteria operational taxonomic units across the localities. This implies a core terrestrial deep subsurface community, irrespective of aquifer lithology, depth and other variables, that may play an important role in colonizing and sustaining microbial habitats in the deep terrestrial subsurface. An in silico contamination-aware approach to analysing this dataset underscores the importance of downstream methods for assuring that robust conclusions can be reached from deep subsurface-derived sequencing data. Understanding the global panorama of microbial diversity and ecological dynamics in the deep terrestrial subsurface provides a first step towards understanding the role of microbes in global subsurface element and nutrient cycling.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Previously uncharacterized rectangular bacterial structures in the dolphin mouth

Much remains to be explored regarding the diversity of uncultured, host-associated microbes. Here, we describe rectangular bacterial structures (RBSs) in the mouths of bottlenose dolphins. DNA staining revealed multiple paired bands within RBSs, suggesting the presence of cells dividing along the longitudinal axis. Cryogenic transmission electron microscopy and tomography showed parallel membrane-bound segments that are likely cells, encapsulated by an S-layer-like periodic surface covering. RBSs displayed unusual pilus-like appendages with bundles of threads splayed at the tips. We present multiple lines of evidence, including genomic DNA sequencing of micromanipulated RBSs, 16S rRNA gene sequencing, and fluorescence in situ hybridization, suggesting that RBSs are bacterial and distinct from the genera Simonsiella and Conchiformibius (family Neisseriaceae), with which they share similar morphology and division patterning. Our findings highlight the diversity of novel microbial forms and lifestyles that await characterization using tools complementary to genomics such as microscopy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Age over sex: evaluating gut microbiota differences in healthy Chinese populations

Age and gender have been recognized as two pivotal covariates affecting the composition of the gut microbiota. However, their mediated variations in microbiota seem to be inconsistent across different countries and races. In this study, 613 individuals, whom we referred to as the “healthy” population, were selected from 1,018 volunteers through rigorous selection using 16S rRNA sequencing. Three enterotypes were identified, namely, Escherichia–Shigella , mixture ( Bacteroides and Faecalibacterium ), and Prevotella . Moreover, 11 covariates that explain the differences in microbiota were determined, with age being the predominant factor. Furthermore, age-related differences in alpha diversity, beta diversity, and core genera were observed in our cohort. Remarkably, after adjusting for 10 covariates other than age, abundant genera that differed between age groups were demonstrated. In contrast, minimal differences in alpha diversity, beta diversity, and differentially abundant genera were observed between male and female individuals. Furthermore, we also demonstrated the age trajectories of several well-known beneficial genera, lipopolysaccharide (LPS)-producing genera, and short-chain fatty acids (SCFAs)-producing genera. Overall, our study further elucidated the effects mediated by age and gender on microbiota differences, which are of significant importance for a comprehensive understanding of the gut microbiome spectrum in healthy individuals.

Wu, Jiacheng↗
DOE OSTI2022

Illuminating the pathways to carbon liberation: a systems approach to characterizing the consequential unknowns of carbon transformation and loss from thawing permafrost peatlands (Final Report)

The IsoGenie3 Project delivered new systems-level insights into carbon cycling in thawing permafrost landscapes, with an emphasis on methane and carbon dioxide emissions. From >200 samples from the site collected over a decade, co-analyzed for geochemistry and microbiology, the team recovered ~1,500 assembled microbial genomes and ~1,900 viral population genomes, revealing appreciable genetic novelty - from a new highly abundant bacterial phylum, to novel methane consumers and their activities, to rampant viral novelty. IsoGenie3 linked these organisms to carbon compound transformations (which define the cycling of organic matter in soils, and the loss of the greenhouse gases carbon dioxide and methane), and saw that the microbes at each stage of permafrost thaw had different genetic potential to degrade categories of compounds, expressed that genetic potential differently, and actually transformed carbon compounds into greenhouse gases in different ways. IsoGenie 3 identified that some of the thaw-stage differences were due to plant-microbiome relationships; the plant species across the thaw gradient contributed different carbon compounds into the soil, and hosted distinct microbiota (differing among parts of plants as well as species). Lastly, microbes in the saturated post-thaw conditions appeared likely to contribute to the mobilization and toxification of mercury released during thaw. In parallel with ongoing field sampling and analysis, hypotheses arising from field observations were tested via lab incubation experiments. When communities are taken out of their native habitats, they behave differently, and the team first rigorously quantified the magnitude of this effect on microbiome composition and functional capacity, organic matter composition, and gas production; overall the main system processes were maintained in the lab incubations under the conditions tested. Further, the microbial data could inform geochemical reaction network models of those processes. Then, the team ran experiments with additions of compounds, varying temperature, and “live” vs. “dead” peat (the latter having been gamma irradiated, with a few additional variants to control for methodological artifacts). From these, we (a) determined the importance of plant-derived soluble phenolic compounds in bogs’ extraordinary recalcitrance of organic matter, and carbon gas emissions skewed to carbon dioxide; (b) proposed an abiotic ‘tanning’ mechanism, which could contribute to Sphagnum’s inhibitory effect on anaerobic decomposition through alteration of N availability. IsoGenie3 illuminated longer-term and landscape-scale interactions of permafrost thaw and carbon cycling, advancing knowledge of the drivers of methane dynamics not only across in the permafrost-associated peatland (where hydrology and plant communities dictate microbiomes) but also their interconnected lakes (where sediment carbon quality and resident microbiota are determined by position within lake, and lake features). By leveraging observations of site methane dynamics extending well before this project, the team was able to construct a 44-year portrait of the interplay of permafrost thaw, hydrology, vegetation dynamics, and carbon gas emissions, and the doubling of the fully-thawed fens over this time. From the detailed study of this focal site, IsoGenie3 also aimed to improve model representation of these kinds of sites and processes. To improve predictions of methane transformations, we incorporated acetate and isotope dynamics into the ‘DNDC’ biogeochemistry model. In addition, recovered genomes were grouped into ‘functional groups’, i.e. the genomes that perform a specific function of interest, then used to parameterize maximum growth rate and optimum growth temperature (via signatures in their sequence composition) for the BioCrunch model. The BioCrunch model was then in turn used to test the impact of increasing functional resolution of the microbes, on the carbon gas emissions. Lastly for modeling, the ecosys model was parameterized from the microbial and other data, and used to evaluate drivers of e.g. change in methane emissions. Finally, this project also led to the development of a range of new methods and tools, a new metric of organic matter decomposability, as well as a graph-database solution to multidisciplinary data storage and querying. This project’s ongoing analyses at our focal site also contributed to broader advancements in understanding elements of genetic plasticity and methane metabolism, climate change microbiology and community assembly, global peatland geochemistry and Arctic lakes’ roles in climate feedbacks.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron

Ordered transposon-insertion collections, in which specific transposon-insertion mutants are stored as monocultures in a genome-scale collection, represent a promising tool for genetic dissection of human gut microbiota members. However, publicly available collections are scarce and the construction methodology remains in early stages of development. Here, we describe the assembly of a genome-scale ordered collection of transposon-insertion mutants in the model gut anaerobe Bacteroides thetaiotaomicron VPI-5482 that we created as a resource for the research community. We used flow cytometry to sort single cells from a pooled library, located mutants within this initial progenitor collection by applying a pooling strategy with barcode sequencing, and re-arrayed specific mutants to create a condensed collection with single-insertion strains covering >2500 genes. To demonstrate the potential of the condensed collection for phenotypic screening, we analyzed growth dynamics and cell morphology. We identified both growth defects and altered cell shape in mutants disrupting sphingolipid synthesis and thiamine scavenging. Finally, we analyzed the process of assembling the B. theta condensed collection to identify inefficiencies that limited coverage. We demonstrate as part of this analysis that the process of assembling an ordered collection can be accurately modeled using barcode sequencing data. We expect that utilization of this ordered collection will accelerate research into B. theta physiology and that lessons learned while assembling the collection will inform future efforts to assemble ordered mutant collections for an increasing number of gut microbiota members.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Clades of huge phages from across Earth’s ecosystems

Bacteriophages typically have small genomes and depend on their bacterial hosts for replication. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is—to our knowledge—the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR–Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR–Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR–Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth’s ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Geochemistry, RNA radiocarbon signature, and metatranscriptome data from Rifle, Colorado groundwater well

Understanding the different sources of carbon subsurface microbes use is highly informative yet remains challenging. The goal of this work was to develop and test a cultivation-independent method that analyzes both the radiocarbon and ribosomal RNA (rRNA) signature of unamplified RNA from the same environmental sample, and that collectively characterizes community carbon utilization under in situ conditions. The method was applied to a filtered groundwater sample from a geochemically reduced zone of a shallow alluvial aquifer near Rife, Colorado (CO), where multiple potential carbon sources co-occur. The radiocarbon and RNA-Seq results, while addressing diverse sample properties, indicated a strikingly similar conclusion about the quantitative role of autotrophy in the subsurface microbial community.This data package provides (1) ribosomal RNA (rRNA) sequences for groundwater microbial populations, (2) the taxonomic identity and relative abundance of groundwater microbial populations based on these rRNA sequences, (3) radiocarbon signatures of the rRNA indicating the source of carbon used by the groundwater microbial populations (provided in both Word docx file and pdf file), (4) geochemical measurements for the monitoring well corresponding to these measurements.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

A Single Application of Compost Can Leave Lasting Impacts on Soil Microbial Community Structure and Alter Cross-Domain Interaction Networks

Our current understanding suggests that nutrient management strategies applied to agricultural soils over multiple years are required to cause major and stable shifts in soil microbial communities. However, some studies suggest that agricultural soils can benefit even from sporadic, single additions of organic matter. Here we investigate how single additions of high-quality organic matter can cause significant shifts in microbial soil communities over multiple cropping cycles. We grew radishes in a tropical Oxisol soil for six crop cycles after a single application of a high-nitrogen compost or urea. At planting and before biomass harvest, we sampled soils influenced by the radish rhizosphere and sequenced bacterial and archaeal 16S and fungal ITS rDNA marker genes. We measured microbial richness and diversity, community composition and structure, and constructed correlation networks to predict cross-domain microbial interactions. We found that a single application of compost, compared to urea or control, resulted in a persistent improved plant biomass response and led to sustained changes in the soil microbial community throughout the duration of the 227-day study. Compost altered the structure of both the fungal and prokaryotic microbial communities, introduced new microorganisms that persisted in the resident soil system, and altered soil microbial correlation network structure and hub taxa. In contrast, fertilization with urea did not significantly alter the structure of soil microbial communities compared to the control but reduced network complexity and altered hub taxa. This study highlights the significant impacts that high-quality organic matter fertilization can exert on agricultural soil microbiomes and adds to the growing body of knowledge on using organic fertilizers as a way to steer the soil microbiome toward a healthier soil.

Heisey, Steven↗
DOE OSTI2024

Genetic and enzymatic characterization of Amy13E from Cellvibrio japonicus reclassifies it as a cyclodextrinase also capable of α-diglucoside degradation

ABSTRACT Cyclodextrinases are carbohydrate-active enzymes involved in the linearization of circular amylose oligosaccharides. Primarily thought to function as part of starch metabolism, there have been previous reports of bacterial cyclodextrinases also having additional enzymatic activities on linear malto-oligosaccharides. This substrate class also includes environmentally rare α-diglucosides such as kojibiose (α−1,2), nigerose (α−1,3), and isomaltose (α−1,6), all of which have valuable properties as prebiotics or low-glycemic index sweeteners. Previous genome sequencing of threeCellvibrio japonicusstrains adapted to utilize these α-diglucosides identified multiple, but uncharacterized, mutations in each strain. One of the mutations identified was in theamy13Egene, which was annotated to encode a neopullulanase. In this report, we functionally characterized this gene and determined that it in fact encodes a cyclodextrinase with additional activities on α-diglucosides. Deletion analysis ofamy13Efound that this gene was essential for kojibiose and isomaltose metabolism inC. japonicus. Interestingly, a Δamy13Emutant was not deficient for cyclodextrin or pullulan utilization inC. japonicus; however, heterologous expression of the gene inE. coliwas sufficient for cyclodextrin-dependent growth. Biochemical analyses found thatCjAmy13E cleaved multiple substrates but preferred cyclodextrins and maltose, but had no activity on pullulan. Our characterization of theCjAmy13E cyclodextrinase is useful for refining functional enzyme predictions in related bacteria and for engineering enzymes for biotechnology or biomedical applications. IMPORTANCE Understanding the bacterial metabolism of cyclodextrins and rare α-diglucosides is increasingly important, as these sugars are becoming prevalent in the foods, supplements, and medicines humans consume that subsequently feed the human gut microbiome. Our analysis of a cyclomaltodextrinase with an expanded substrate range is significant because it broadens the potential applications of the GH13 family of carbohydrate active enzymes (CAZymes) in biotechnology and biomedicine. Specifically, this study provides a workflow for the discovery and characterization of novel activities in bacteria that possess a high number of CAZymes that otherwise would be missed due to complications with functional redundancy. Furthermore, this study provides a model from which predictions can be made why certain bacteria in crowded niches are able to robustly utilize rare carbon sources, possibly to gain a competitive growth advantage.

Biotechnology & Applied Microbiology↗
DOE OSTI2020

Combinatorial Glycomic Analyses to Direct CAZyme Discovery for the Tailored Degradation of Canola Meal Non-Starch Dietary Polysaccharides

Canola meal (CM), the protein-rich by-product of canola oil extraction, has shown promise as an alternative feedstuff and protein supplement in poultry diets, yet its use has been limited due to the abundance of plant cell wall fibre, specifically non-starch polysaccharides (NSP) and lignin. The addition of exogenous enzymes to promote the digestion of CM NSP in chickens has potential to increase the metabolizable energy of CM. We isolated chicken cecal bacteria from a continuous-flow mini-bioreactor system and selected for those with the ability to metabolize CM NSP. Of 100 isolates identified, Bacteroides spp. and Enterococcus spp. were the most common species with these capabilities. To identify enzymes specifically for the digestion of CM NSP, we used a combination of glycomics techniques, including enzyme-linked immunosorbent assay characterization of the plant cell wall fractions, glycosidic linkage analysis (methylation-GC-MS analysis) of CM NSP and their fractions, bacterial growth profiles using minimal media supplemented with CM NSP, and the sequencing and de novo annotation of bacterial genomes of high-efficiency CM NSP utilizing bacteria. The SACCHARIS pipeline was used to select plant cell wall active enzymes for recombinant production and characterization. This approach represents a multidisciplinary innovation platform to bioprospect endogenous CAZymes from the intestinal microbiota of herbivorous and omnivorous animals which is adaptable to a variety of applications and dietary polysaccharides.

glycome profiling↗
DOE OSTI2021

Molecular basis of C-S bond cleavage in the glycyl radical enzyme isethionate sulfite-lyase

Desulfonation of isethionate by the bacterial glycyl radical enzyme (GRE) isethionate sulfite-lyase (IslA) generates sulfite, a substrate for respiration that in turn produces the disease-associated metabolite hydrogen sulfide. Here, we present a 2.7 Å resolution X-ray structure of wild-type IslA from Bilophila wadsworthia with isethionate bound. In comparison with other GREs, alternate positioning of the active site β strands allows for distinct residue positions to contribute to substrate binding. These structural differences, combined with sequence variations, create a highly tailored active site for the binding of the negatively charged isethionate substrate. Through the kinetic analysis of 14 IslA variants and computational analyses, we probe the mechanism by which radical chemistry is used for C-S bond cleavage. This work further elucidates the structural basis of chemistry within the GRE superfamily and will inform structure-based inhibitor design of IsIA and thus of microbial hydrogen sulfide production.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗