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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Genome, transcriptome and secretome analyses of the antagonistic, yeast-like fungus Aureobasidium pullulans to identify potential biocontrol genes

Aureobasidium pullulans is an extremotolerant, cosmopolitan yeast-like fungus that successfully colonises vastly different ecological niches. The species is widely used in biotechnology and successfully applied as a commercial biocontrol agent against postharvest diseases and fireblight. However, the exact mechanisms that are responsible for its antagonistic activity against diverse plant pathogens are not known at the molecular level. Thus, it is difficult to optimise and improve the biocontrol applications of this species. As a foundation for elucidating biocontrol mechanisms, we have de novo assembled a high-quality reference genome of a strongly antagonistic A. pullulans strain, performed dual RNA-seq experiments, and analysed proteins secreted during the interaction with the plant pathogen Fusarium oxysporum. Based on the genome annotation, potential biocontrol genes were predicted to encode secreted hydrolases or to be part of secondary metabolite clusters (e.g., NRPS-like, NRPS, T1PKS, terpene, and β-lactone clusters). Transcriptome and secretome analyses defined a subset of 79 A. pullulans genes (among the 10,925 annotated genes) that were transcriptionally upregulated or exclusively detected at the protein level during the competition with F. oxysporum. These potential biocontrol genes comprised predicted secreted hydrolases such as glycosylases, esterases, and proteases, as well as genes encoding enzymes, which are predicted to be involved in the synthesis of secondary metabolites. This study highlights the value of a sequential approach starting with genome mining and consecutive transcriptome and secretome analyses in order to identify a limited number of potential target genes for detailed, functional analyses.

transcriptome↗
DOE OSTI2023

Identifying Fungal Secondary Metabolites and Their Role in Plant Pathogenesis

Pathogenic fungi are the main infectious agents of plants. Secondary metabolites produced by these fungi, also recognized as natural products, are key mediators of plant-fungal interactions. Knowledge on the biosynthesis of these metabolites, the accessibility to fungal genome sequences, and the development of gene disruption techniques open up opportunities to identify many more of these metabolites both in vitro and in planta. This methodology chapter gives a detailed systematic approach aiming to discover new natural products from phytopathogenic fungi and characterize their role in triggering plant cell death and plant disease. This approach takes advantage of the global regulation of fungal secondary metabolite production by regulatory proteins reported in various fungal species.

Tannous, Joanna↗
DOE OSTI2026

CRAGE-RB-PI-seq reveals transcriptional dynamics of plant-associated bacteria during root colonization

Plant roots release a wide array of metabolites into the rhizosphere, shaping microbial communities and their functions. While metagenomics has expanded our understanding of these communities, little is known about the physiology of their members in host environments. Transcriptome analysis via RNA sequencing is a common approach to learning more, but its use has been challenging because of low bacterial biomass and interference from plant RNA. To overcome this, we developed a randomly-barcoded promoter-library insertion sequencing (RB-PI-seq) combined with chassis-independent recombinase-assisted genome engineering (CRAGE). Using Pseudomonas simiae WCS417 as a model rhizobacterium, this method enabled targeted amplification of barcoded transcripts, bypassing plant RNA interference and allowing measurement of thousands of promoter activities during Arabidopsis root colonization. Our analysis revealed temporally resolved transcriptional regulation, including those associated with cell growth, chemotaxis, plant immune suppression, biofilm formation, and stress responses, reflecting the coordinated physiological adaptation to the root environment. Additionally, we discovered that transcriptional activation of xanthine dehydrogenase and a lysozyme inhibitor is crucial for evading plant immune systems. This framework is scalable to other bacterial species and provides new opportunities for understanding rhizobacterial gene regulation in native environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Genomic Analysis Enlightens Agaricales Lifestyle Evolution and Increasing Peroxidase Diversity

Abstract As actors of global carbon cycle, Agaricomycetes (Basidiomycota) have developed complex enzymatic machineries that allow them to decompose all plant polymers, including lignin. Among them, saprotrophic Agaricales are characterized by an unparalleled diversity of habitats and lifestyles. Comparative analysis of 52 Agaricomycetes genomes (14 of them sequenced de novo) reveals that Agaricales possess a large diversity of hydrolytic and oxidative enzymes for lignocellulose decay. Based on the gene families with the predicted highest evolutionary rates—namely cellulose-binding CBM1, glycoside hydrolase GH43, lytic polysaccharide monooxygenase AA9, class-II peroxidases, glucose–methanol–choline oxidase/dehydrogenases, laccases, and unspecific peroxygenases—we reconstructed the lifestyles of the ancestors that led to the extant lignocellulose-decomposing Agaricomycetes. The changes in the enzymatic toolkit of ancestral Agaricales are correlated with the evolution of their ability to grow not only on wood but also on leaf litter and decayed wood, with grass-litter decomposers as the most recent eco-physiological group. In this context, the above families were analyzed in detail in connection with lifestyle diversity. Peroxidases appear as a central component of the enzymatic toolkit of saprotrophic Agaricomycetes, consistent with their essential role in lignin degradation and high evolutionary rates. This includes not only expansions/losses in peroxidase genes common to other basidiomycetes but also the widespread presence in Agaricales (and Russulales) of new peroxidases types not found in wood-rotting Polyporales, and other Agaricomycetes orders. Therefore, we analyzed the peroxidase evolution in Agaricomycetes by ancestral-sequence reconstruction revealing several major evolutionary pathways and mapped the appearance of the different enzyme types in a time-calibrated species tree.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Photosynthetic adaptation to polar life: Energy balance, photoprotection and genetic redundancy

The persistent low temperature that characterize polar habitats combined with the requirement for light for all photoautotrophs creates a conundrum. The absorption of too much light at low temperature can cause an energy imbalance that decreases photosynthetic performance that has a negative impact on growth and can affect long-term survival. The goal of this review is to survey the mechanism(s) by which polar photoautotrophs maintain cellular energy balance, that is, photostasis to overcome the potential for cellular energy imbalance in their low temperature environments. Photopsychrophiles are photosynthetic organisms that are obligately adapted to low temperature (0⁰- 15 °C) but usually die at higher temperatures (≥20 °C). In contrast, photopsychrotolerant species can usually tolerate and survive a broad range of temperatures (5⁰- 40 °C). First, we summarize the basic concepts of excess excitation energy, energy balance, photoprotection and photostasis and their importance to survival in polar habitats. Second, we compare the photoprotective mechanisms that underlie photostasis and survival in aquatic cyanobacteria and green algae as well as terrestrial Antarctic and Arctic plants. We show that polar photopsychrophilic and photopsychrotolerant organisms attain energy balance at low temperature either through a regulated reduction in the efficiency of light absorption or through enhanced capacity to consume photosynthetic electrons by the induction of O 2 as an alternative electron acceptor. Finally, we compare the published genomes of three photopsychrophilic and one photopsychrotolerant alga with five mesophilic green algae including the model green alga, Chlamydomonas reinhardtii. We relate our genomic analyses to photoprotective mechanisms that contribute to the potential attainment of photostasis. Finally, we discuss how the observed genomic redundancy in photopsychrophilic genomes may confer energy balance, photoprotection and resilience to their harsh polar environment. Primary production in aquatic, Antarctic and Arctic environments is dependent on diverse algal and cyanobacterial communities. Although mosses and lichens dominate the Antarctic terrestrial landscape, only two extant angiosperms exist in the Antarctic. Furthermore, the identification of a single ‘molecular key’ to unravel adaptation of photopsychrophily and photopsychrotolerance remains elusive. Since these photoautotrophs represent excellent biomarkers to assess the impact of global warming on polar ecosystems, increased study of these polar photoautotrophs remains essential.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Reconfiguring Plant Metabolism for Biodegradable Plastic Production

For decades, plants have been the subject of genetic engineering to synthesize novel, value-added compounds. Polyhydroxyalkanoates (PHAs), a large class of biodegradable biopolymers naturally synthesized in eubacteria, are among the novel products that have been introduced to make use of plant acetyl-CoA metabolic pathways. It was hoped that renewable PHA production would help address environmental issues associated with the accumulation of nondegradable plastic wastes. However, after three decades of effort synthesizing PHAs, and in particular the simplest form polyhydroxybutyrate (PHB), and seeking to improve their production in plants, it has proven very difficult to reach a commercially profitable rate in a normally growing plant. This seems to be due to the growth defects associated with PHA production and accumulation in plant cells. Here, we review major breakthroughs that have been made in plant-based PHA synthesis using traditional genetic engineering approaches and discuss challenges that have been encountered. Then, from the point of view of plant synthetic biology, we provide perspectives on reprograming plant acetyl-CoA pathways for PHA production, with the goal of maximizing PHA yield while minimizing growth inhibition. Specifically, we suggest genetic elements that can be considered in genetic circuit design, approaches for nuclear genome and plastome modification, and the use of multiomics and mathematical modeling in understanding and restructuring plant metabolic pathways.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Knocking Out the Wall: Revised Protocols for Gene Targeting in Physcomitrella patens

The moss Physcomitrella patens has become established as a model for investigating plant gene function due to the feasibility of gene targeting. The chemical composition of the P. patens cell wall is similar to that of vascular plants and phylogenetic analyses of glycosyltransferase sequences from the P. patens genome have identified genes that putatively encode cell wall biosynthetic enzymes, providing a basis for investigating the evolution of cell wall polysaccharides and the enzymes that synthesize them. Here, the protocols described in this chapter provide methods for targeted gene knockout in P. patens, from constructing vectors and maintaining cultures to transforming protoplasts and analysing the genotypes and phenotypes of the resulting transformed lines.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A rapid thioacidolysis method for biomass lignin composition and tricin analysis

Abstract Background Biomass composition varies from plant to plant and greatly affects biomass utilization. Lignin is a heterogeneous phenolic polymer derived mainly from p -coumaryl, coniferyl, and sinapyl alcohols and makes up to 10–25% of lignocellulosic biomass. Recently, tricin, an O -methylated flavone, was identified as a lignin monomer in many grass species. Tricin may function as a nucleation site for lignification and is advocated as a novel target for lignin engineering to reduce lignin content and improve biomass digestibility in grasses. Thioacidolysis is an analytical method that can be adapted to analyze both lignin monomeric composition and tricin content in the lignin polymer. However, the original thioacidolysis procedure is complex, laborious, and time consuming, making it difficult to be adopted for large-scale screening in biomass research. In this study, a modified, rapid higher throughput thioacidolysis method was developed. Results In combination with gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS), the modified thioacidolysis method can be used to simultaneously characterize the lignin composition and tricin content using 2–5 mg of dry samples. The modified method eliminates the solvent extraction and drastically improves the throughput; 80 samples can be processed in one day per person. Our results indicate that there is no significant difference in the determination of lignin S/G ratio and tricin content between the original and modified methods. Conclusions A modified thioacidolysis protocol was established. The results demonstrate that the modified method can be used for rapid, high-throughput, and reliable lignin composition and tricin content analyses for screening transgenic plants for cell wall modifications or in large-scale genome-wide association studies (GWAS).

09 BIOMASS FUELS↗
DOE OSTI2021

Glacier ice archives nearly 15,000-year-old microbes and phages

Background Glacier ice archives information, including microbiology, that helps reveal paleoclimate histories and predict future climate change. Though glacier-ice microbes are studied using culture or amplicon approaches, more challenging metagenomic approaches, which provide access to functional, genome-resolved information and viruses, are under-utilized, partly due to low biomass and potential contamination. Results We expand existing clean sampling procedures using controlled artificial ice-core experiments and adapted previously established low-biomass metagenomic approaches to study glacier-ice viruses. Controlled sampling experiments drastically reduced mock contaminants including bacteria, viruses, and free DNA to background levels. Amplicon sequencing from eight depths of two Tibetan Plateau ice cores revealed common glacier-ice lineages including Janthinobacterium, Polaromonas, Herminiimonas, Flavobacterium, Sphingomonas, and Methylobacterium as the dominant genera, while microbial communities were significantly different between two ice cores, associating with different climate conditions during deposition. Separately, ~355- and ~14,400-year-old ice were subject to viral enrichment and low-input quantitative sequencing, yielding genomic sequences for 33 vOTUs. These were virtually all unique to this study, representing 28 novel genera and not a single species shared with 225 environmentally diverse viromes. Further, 42.4% of the vOTUs were identifiable temperate, which is significantly higher than that in gut, soil, and marine viromes, and indicates that temperate phages are possibly favored in glacier-ice environments before being frozen. In silico host predictions linked 18 vOTUs to co-occurring abundant bacteria (Methylobacterium, Sphingomonas, and Janthinobacterium), indicating that these phages infected ice-abundant bacterial groups before being archived. Functional genome annotation revealed four virus-encoded auxiliary metabolic genes, particularly two motility genes suggest viruses potentially facilitate nutrient acquisition for their hosts. Finally, given their possible importance to methane cycling in ice, we focused on Methylobacterium viruses by contextualizing our ice-observed viruses against 123 viromes and prophages extracted from 131 Methylobacterium genomes, revealing that the archived viruses might originate from soil or plants. Conclusions Together, these efforts further microbial and viral sampling procedures for glacier ice and provide a first window into viral communities and functions in ancient glacier environments. Such methods and datasets can potentially enable researchers to contextualize new discoveries and begin to incorporate glacier-ice microbes and their viruses relative to past and present climate change in geographically diverse regions globally.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Data for Transformation and Gene Editing in the Bioenergy Grass Miscanthus

Miscanthus, a C4 member of the family Poaceae, is a promising perennial crop for bioenergy, renewable bioproducts, and carbon sequestration. Species of interest include nothospecies Miscanthus x giganteus and its parental species M. sacchariflorus and M. sinensis . Use of biotechnology-based procedures to genetically improve miscanthus, to date, have only included plant transformation procedures for introduction of exogenous genes into the host genome at random, non-targeted sites.

Biomass Analytics↗
DOE OSTI2024

DOE BSSD Performance Management Metrics Report Q1

Microbes play key roles in our biosphere, from driving global nutrient cycling to impacting plant, animal and human health and disease. Complex data from microbial genomes, proteins, and metabolites provide a window into these tiny engines that drive life on our planet. Yet these data are dispersed among researchers’ laboratories and various repositories, making it difficult to access. This calls for new ways of managing data, improving data interoperability, advancing community standards, and creating an infrastructure where data are shared efficiently. We have built the National Microbiome Data Collaborative (NMDC) to advance how scientists create, use, and reuse data to redefine the way we understand and harness the power of microbes. The vision of the National Microbiome Data Collaborative (NMDC) is to drive a microbiome data sharing network connecting data, people, and ideas to advance microbiome innovation and discovery. The NMDC was launched in 2019 and brought together DOE National Laboratories to collaborate across resources, capabilities, and expertise. The NMDC team was strategically assembled to include software developers, microbial researchers, metadata experts, and multi-omics specialists. The diversity of the NMDC team reflects the inherently interdisciplinary nature of microbiome science, and we leverage the strengths of the DOE National Laboratory system. Towards BER’s goal of advancing an iterative systems biology approach to the understanding of microbial genomes, the NMDC serves as a foundation for infrastructure, data standards, and community building. Together with the flagship DOE User Facilities, the Joint Genome Institute (JGI) and the Environmental Molecular Sciences Laboratory (EMSL), we are developing core capabilities in metadata standards for environmental descriptors and sample handling and processing; standardized bioinformatic workflows; an interface for data search and access; and robust community engagement activities. The NMDC production platform supports long-term data infrastructure and community building for BER’s bioenergy and environmental research goals. Our approach leverages lessons learned and an ambitious framework for collaborative, interdisciplinary data infrastructure to support microbiome research. The NMDC supports data, information, and knowledge access through three defined software tools – the Submission Portal, NMDC EDGE, and the Data Portal – driven by community needs. Herein, we describe the value proposition for the microbiome research community, our overarching strategy, and challenges and opportunities for developing the NMDC as both an infrastructure and community engagement program.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The Genetic Architecture of Nitrogen Use Efficiency in Switchgrass ( Panicum virgatum L.)

Switchgrass (Panicum virgatum L.) has immense potential as a bioenergy crop with the aim of producing biofuel as an end goal. Nitrogen (N)-related sustainability traits, such as nitrogen use efficiency (NUE) and nitrogen remobilization efficiency (NRE), are important factors affecting switchgrass quality and productivity. Hence, it is imperative to develop nitrogen use-efficient switchgrass accessions by exploring the genetic basis of NUE in switchgrass. For that, we used 331 diverse field-grown switchgrass accessions planted under low and moderate N fertility treatments. We performed a genome wide association study (GWAS) in a holistic manner where we not only considered NUE as a single trait but also used its related phenotypic traits, such as total dry biomass at low N and moderate N, and nitrogen use index, such as NRE. We have evaluated the phenotypic characterization of the NUE and the related traits, highlighted their relationship using correlation analysis, and identified the top ten nitrogen use-efficient switchgrass accessions. Our GWAS analysis identified 19 unique single nucleotide polymorphisms (SNPs) and 32 candidate genes. Two promising GWAS candidate genes, caffeoyl-CoA O-methyltransferase (CCoAOMT) and <alfin-like 6(AL6), were further supported by linkage disequilibrium (LD) analysis. Finally, we discussed the potential role of nitrogen in modulating the expression of these two genes. Our findings have opened avenues for the development of improved nitrogen use-efficient switchgrass lines.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Conservation and Expansion of Transcriptional Factor Repertoire in the Fusarium oxysporum Species Complex

The Fusarium oxysporum species complex (FOSC) includes both plant and human pathogens that cause devastating plant vascular wilt diseases and threaten public health. Each F. oxysporum genome comprises core chromosomes (CCs) for housekeeping functions and accessory chromosomes (ACs) that contribute to host-specific adaptation. This study inspects global transcription factor profiles (TFomes) and their potential roles in coordinating CC and AC functions to accomplish host-specific interactions. Remarkably, we found a clear positive correlation between the sizes of TFomes and the proteomes of an organism. With the acquisition of ACs, the FOSC TFomes were larger than the other fungal genomes included in this study. Among a total of 48 classified TF families, 14 families involved in transcription/translation regulations and cell cycle controls were highly conserved. Among the 30 FOSC expanded families, Zn2-C6 and Znf_C2H2 were most significantly expanded to 671 and 167 genes per family including well-characterized homologs of Ftf1 (Zn2-C6) and PacC (Znf_C2H2) that are involved in host-specific interactions. Manual curation of characterized TFs increased the TFome repertoires by 3% including a disordered protein Ren1. RNA-Seq revealed a steady pattern of expression for conserved TF families and specific activation for AC TFs. Functional characterization of these TFs could enhance our understanding of transcriptional regulation involved in FOSC cross-kingdom interactions, disentangle species-specific adaptation, and identify targets to combat diverse diseases caused by this group of fungal pathogens.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The essential effector SCRE1 in Ustilaginoidea virens suppresses rice immunity via a small peptide region

Summary The biotrophic fungal pathogen Ustilaginoidea virens causes rice false smut, a newly emerging plant disease that has become epidemic worldwide in recent years. The U. virens genome encodes many putative effector proteins that, based on the study of other pathosystems, could play an essential role in fungal virulence. However, few studies have been reported on virulence functions of individual U. virens effectors. Here, we report our identification and characterization of the secreted cysteine‐rich protein SCRE1, which is an essential virulence effector in U. virens . When SCRE1 was heterologously expressed in Magnaporthe oryzae , the protein was secreted and translocated into plant cells during infection. SCRE1 suppresses the immunity‐associated hypersensitive response in the nonhost plant Nicotiana benthamiana . Induced expression of SCRE1 in rice also inhibits pattern‐triggered immunity and enhances disease susceptibility to rice bacterial and fungal pathogens. The immunosuppressive activity is localized to a small peptide region that contains an important ‘cysteine‐proline‐alanine‐arginine‐serine’ motif. Furthermore, the scre1 knockout mutant generated using the CRISPR/Cas9 system is attenuated in U. virens virulence to rice, which is greatly complemented by the full‐length SCRE1 gene. Collectively, this study indicates that the effector SCRE1 is able to inhibit host immunity and is required for full virulence of U. virens .

Zhang, Nan↗
DOE OSTI2021

Genome-wide association of the metabolic shifts underpinning dark-induced senescence in Arabidopsis

Dark-induced senescence provokes profound metabolic shifts to recycle nutrients and to guarantee plant survival. To date, research on these processes has largely focused on characterizing mutants deficient in individual pathways. Here, we adopted a time-resolved genome-wide association-based approach to characterize dark-induced senescence by evaluating the photochemical efficiency and content of primary and lipid metabolites at the beginning, or after 3 or 6 days in darkness. We discovered six patterns of metabolic shifts and identified 215 associations with 81 candidate genes being involved in this process. Among these associations, we validated the roles of four genes associated with glycine, galactinol, threonine, and ornithine levels. We also demonstrated the function of threonine and galactinol catabolism during dark-induced senescence. Intriguingly, we determined that the association between tyrosine contents and TYROSINE AMINOTRANSFERASE 1 influences enzyme activity of the encoded protein and transcriptional activity of the gene under normal and dark conditions, respectively. Moreover, the single-nucleotide polymorphisms affecting the expression of THREONINE ALDOLASE 1 and the amino acid transporter gene AVT 1 B, respectively, only underlie the variation in threonine and glycine levels in the dark. Taken together, these results allow us to present a very detailed model of the metabolic aspects of dark-induced senescence, as well as the process itself.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

SNPeffect: identifying functional roles of SNPs using metabolic networks

Genetic sources of phenotypic variation have been a focus of plant studies aimed at improving agricultural yield and understanding adaptive processes. Genome-wide association studies identify the genetic background behind a trait by examining associations between phenotypes and single-nucleotide polymorphisms (SNPs). Although such studies are common, biological interpretation of the results remains a challenge; especially due to the confounding nature of population structure and the systematic biases thus introduced. Here, we propose a complementary analysis (SNPeffect) that offers putative genotype-to-phenotype mechanistic interpretations by integrating biochemical knowledge encoded in metabolic models. SNPeffect is used to explain differential growth rate and metabolite accumulation in A. thaliana and P. trichocarpa accessions as the outcome of SNPs in enzyme-coding genes. To this end, we also constructed a genome-scale metabolic model for Populus trichocarpa, the first for a perennial woody tree. As expected, our results indicate that growth is a complex polygenic trait governed by carbon and energy partitioning. The predicted set of functional SNPs in both species are associated with experimentally characterized growth-determining genes and also suggest putative ones. Functional SNPs were found in pathways such as amino acid metabolism, nucleotide biosynthesis, and cellulose and lignin biosynthesis, in line with breeding strategies that target pathways governing carbon and energy partition.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Viruses of Nitrogen-Fixing Mesorhizobium Bacteria in Globally Distributed Chickpea Root Nodules

Legume nodules are specialized environments on plant roots that are induced and dominated by nitrogen-fixing bacteria. Bacteriophages (phages) in these nodules could potentially provide top-down controls on the population size and, therefore, the function of nitrogen-fixing symbionts. Here we sought to characterize the diversity and biogeographical patterns of phages that infect nitrogen-fixing Mesorhizobium symbionts isolated from root nodules, leveraging 266 genomes of Mesorhizobium isolated from nodules and 648 nodule metagenomes collected from three species of chickpea plants ( Cicer spp.) under different agricultural management practices, spanning eight countries on five continents. We identified 106 phage populations (viral operational taxonomic units [vOTUs]) in Mesorhizobium draft genomes, 37% of which were confirmed as likely prophages. These vOTUs were detected in 64% of the Mesorhizobium-dominated nodule metagenomes and 58% of the Mesorhizobium isolates. Per metagenome, 1 to 16 putative Mesorhizobium vOTUs were detected, with more than half of the nodules containing only one such vOTU. The majority of vOTUs were detected exclusively in Ethiopia, followed by India and Morocco, with the lowest richness of putative Mesorhizobium phages in countries that applied industrial Mesorhizobium inoculants to crops. Two vOTUs were identified in five or more countries and in nodules dominated by different strains of Mesorhizobium, suggesting infection of diverse Mesorhizobium hosts and long-term interactions. Beta-diversity of these Mesorhizobium phage assemblages was significantly correlated with the dominant Mesorhizobium strain, but not with measured environmental parameters. Our findings indicate that nitrogen-fixing nodules in chickpea plants can contain distinct viral assemblages, with potential impacts on the nodule microbiome that bear further exploration.

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↗