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QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Understanding plant-microbe interactions requires examination of root exudation under nutrient stress using standardized and reproducible experimental systems. We grew Brachypodium distachyon hydroponically in fabricated ecosystem devices (EcoFAB 2.0) under three inorganic nitrogen forms (nitrate, ammonium, and ammonium nitrate), followed by nitrogen starvation. Analyses of exudates with liquid chromatography-tandem mass spectrometry, biomass, medium pH, and nitrogen uptake showed EcoFAB 2.0's low intratreatment data variability. Furthermore, the three inorganic nitrogen forms caused differential exudation, generalized by abundant amino acids-peptides and alkaloids. Comparatively, nitrogen deficiency decreased nitrogen-containing compounds but increased shikimates-phenylpropanoids. Subsequent bioassays with two shikimates-phenylpropanoids (shikimic and p-coumaric acids) on soil bacteria or Brachypodium seedlings revealed their distinct capacity to regulate both bacterial and plant growth. Our results suggest that (i) Brachypodium alters exudation in response to nitrogen status, which can affect rhizobacterial growth, and (ii) EcoFAB 2.0 is a valuable standardized plant research tool.

Abstract As atmospheric CO2 rises, increases in photosynthesis and plant growth are routinely documented across ecosystems globally. Although primarily studied in relation to photorespiration, serine metabolism in chloroplasts may play a key role in plant CO2 fertilization responses by linking CO2 assimilation with growth. However, little information is available on the in vivo activity of the plastidic phosphorylated serine pathway. Here, we show that the serine phosphate pathway is part of a ‘photosynthetic C1 pathway’ and demonstrate its high activity in foliage of a C3 tree where it rapidly integrates photosynthesis directly with C1 metabolism contributing to new biomass via methyl transfer reactions and imparting a large natural 13C-depleted signature. Using 13CO2-labelling, we show that leaf serine, the S-methyl group of leaf methionine, pectin methyl esters, and the associated methanol released during cell wall expansion during growth, are directly produced from photosynthetically-linked C1 metabolism, unrelated to photorespiration, within minutes of light exposure. We speculate that the photosynthetic C1 pathway and its key enzyme methionine synthase is highly conserved across the photosynthetic tree of life and may have evolved with oxygenic photosynthesis by providing a mechanism of directly linking carbon assimilation with growth. Although the rise in atmospheric CO2 inhibits major metabolic pathways like photorespiration in C3 plants, our results suggest that the photosynthetic C1 pathway may accelerate and represents a ‘missing link’ between enhanced photosynthesis and growth rates of modern plants and ecosystems during terrestrial CO2 fertilization under a changing climate.

Microbial competition within plant tissues affects invading pathogens' fitness. Metabolomics is a great tool for studying their biochemical interactions by identifying accumulated metabolites. Xylella fastidiosa, a Gram-negative bacterium causing Pierce's disease (PD) in grapevines, secretes various virulence factors including cell wall-degrading enzymes, adhesion proteins, and quorum-sensing molecules. These factors, along with outer membrane vesicles, contribute to its pathogenicity. Previous studies demonstrated that co-inoculating X. fastidiosa with the Paraburkholderia phytofirmans strain PsJN suppressed PD symptoms. Here, we further investigated the interaction between the phytopathogen and the endophyte by analyzing the exometabolome of wild-type X. fastidiosa and a diffusible signaling factor (DSF) mutant lacking quorum sensing, cultivated with 20% P. phytofirmans spent media. Liquid chromatography-mass spectrometry (LC-MS) and the Method for Metabolite Annotation and Gene Integration (MAGI) were used to detect and map metabolites to genomes, revealing a total of 121 metabolites, of which 25 were further investigated. These metabolites potentially relate to host adaptation, virulence, and pathogenicity. Notably, this study presents the first comprehensive profile of X. fastidiosa in the presence of a P. phytofirmans spent media. The results highlight that P. phytofirmans and the absence of functional quorum sensing affect the ratios of glutamine to glutamate (Gln:Glu) in X. fastidiosa. Additionally, two compounds with plant metabolism and growth properties, 2-aminoisobutyric acid and gibberellic acid, were downregulated when X. fastidiosa interacted with P. phytofirmans. These findings suggest that P. phytofirmans-mediated disease suppression involves modulation of the exometabolome of X. fastidiosa, impacting plant immunity.

Abstract Although primarily studied in relation to photorespiration, serine metabolism in chloroplasts may play a key role in plant CO2 fertilization responses by linking CO2 assimilation with growth. Here, we show that the phosphorylated serine pathway is part of a ‘photosynthetic C1 pathway’ and demonstrate its high activity in foliage of a C3 tree where it rapidly integrates photosynthesis and C1 metabolism contributing to new biomass via methyl transfer reactions, imparting a large natural 13C-depleted signature. Using 13CO2-labelling, we show that leaf serine, the S-methyl group of leaf methionine, pectin methyl esters, and the associated methanol released during cell wall expansion during growth, are directly produced from photosynthetically-linked C1 metabolism, within minutes of light exposure. We speculate that the photosynthetic C1 pathway is highly conserved across the photosynthetic tree of life, is responsible for synthesis of the greenhouse gas methane, and may have evolved with oxygenic photosynthesis by providing a mechanism of directly linking carbon and ammonia assimilation with growth. Although the rise in atmospheric CO2 inhibits major metabolic pathways like photorespiration, our results suggest that the photosynthetic C1 pathway may accelerate and represents a missing link between enhanced photosynthesis and plant growth rates during CO2 fertilization under a changing climate.

Biocrusts are phototroph-driven communities inhabiting arid soil surfaces. Like plants, most photoautotrophs (largely cyanobacteria) in biocrusts are thought to exchange fixed carbon for essential nutrients like nitrogen with cyanosphere bacteria. Here, we aim to compare beneficial interactions in rhizosphere and cyanosphere environments, including finding growth-promoting strains for hosts from both environments. To examine this, we performed a retrospective analysis of 16S rRNA gene sequencing datasets, host-microbe co-culture experiments between biocrust communities/biocrust isolates and a model grass (Brachypodium distachyon) or a dominant biocrust cyanobacterium (Microcoleus vaginatus), and metabolomic analysis. All 18 microbial phyla in the cyanosphere were also present in the rhizosphere, with additional 17 phyla uniquely found in the rhizosphere. The biocrust microbes promoted the growth of the model grass, and three biocrust isolates (Bosea sp._L1B56, Pseudarthrobacter sp._L1D14 and Pseudarthrobacter picheli_L1D33) significantly promoted the growth of both hosts. Moreover, pantothenic acid was produced by Pseudarthrobacter sp._L1D14 when grown on B. distachyon exudates, and supplementation of plant growth medium with this metabolite increased B. distachyon biomass by over 60%. These findings suggest that cyanobacteria and other diverse photoautotrophic hosts can be a source for new plant growth-promoting microbes and metabolites.

Roles of different ecological classes of algal exometabolites in regulating microbial community composition are not well understood. Here, we identify exometabolites from the model diatom Phaeodactylum tricornutum and demonstrate their potential to influence bacterial abundances. We profiled exometabolites across a time course of axenic algal growth using liquid chromatography-tandem mass spectrometry. We then investigated growth of 12 bacterial isolates on individual-identified exometabolites. Lastly, we compared responses of a P. tricornutum-adapted enrichment community to additions of two contrasting metabolites: selective growth substrate 4-hydroxybenzoic acid and putative signaling/facilitator molecule lumichrome. We identified 50 P. tricornutum metabolites and found distinct temporal accumulation patterns. Two exometabolites (of 12 tested) supported growth of distinct subsets of bacterial isolates. While algal exudates and algal presence drove similar changes in community composition compared with controls, exogenous 4-hydroxybenzoic acid addition promoted increased abundances of taxa that utilized it in isolation, and also revealed the importance of factors relating to algal presence in regulating community composition. This work demonstrates that secretion of selective bacterial growth substrates represents one mechanism by which algal exometabolites can influence bacterial community composition and illustrates how the algal exometabolome has the potential to modulate bacterial communities as a function of algal growth.

The terrestrial green alga Chromochloris zofingiensis is an emerging model species with potential applications including production of triacylglycerol or astaxanthin. How C. zofingiensis interacts with the diverse substrates during trophic transitions is unknown. To characterize its substrate utilization and secretion dynamics, we cultivated the alga in a soil-based defined medium in transition between conditions with and without glucose supplementation. Then, we examined its exometabolite and endometabolite profiles. This analysis revealed that regardless of trophic modes, C. zofingiensis preferentially uptakes exogenous lysine, arginine, and purines, while secreting orotic acid. Here, we obtained metabolomic evidences that C. zofingiensis may use arginine for putrescine synthesis when in transition to heterotrophy, and for the TCA cycle during transition to photoautotrophy. We also report that glucose and fructose most effectively inhibited photosynthesis among thirteen different sugars. The utilized or secreted metabolites identified in this study provide important information to improve C. zofingiensis cultivation, and to expand its potential industrial and pharmaceutical applications.

Soil organic matter (SOM) is comprised of a diverse array of reactive carbon molecules, including hydrophilic and hydrophobic compounds, that impact rates of SOM formation and persistence. Despite clear importance to ecosystem science, little is known about broad-scale controls on SOM diversity and variability in soil. Here, we show that microbial decomposition drives significant variability in the molecular richness and diversity of SOM between soil horizons and across a continental-scale gradient in climate and ecosystem type (arid shrubs, coniferous, deciduous, and mixed forests, grasslands, and tundra sedges). The molecular dissimilarity of SOM was strongly influenced by ecosystem type (hydrophilic compounds: 17%, P < 0.001; hydrophobic compounds: 10% P < 0.001) and soil horizon (hydrophilic compounds: 17%, P < 0.001; hydrophobic compounds: 21%, P < 0.001), as assessed using metabolomic analysis of hydrophilic and hydrophobic metabolites. While the proportion of shared molecular features was significantly higher in the litter layer than subsoil C horizons across ecosystems (12 times and 4 times higher for hydrophilic and hydrophobic compounds, respectively), the proportion of site-specific molecular features nearly doubled from the litter layer to the subsoil horizon, suggesting greater differentiation of compounds after microbial decomposition within each ecosystem. Together, these results suggest that microbial decomposition of plant litter leads to a decrease in SOM α-molecular diversity, yet an increase in β-molecular diversity across ecosystems. The degree of microbial degradation, determined by the position in the soil profile, exerts a greater control on SOM molecular diversity than environmental factors, such as soil texture, moisture, and ecosystem type.

Root exudates are plant-derived, exported metabolites likely shaping root-associated microbiomes by acting as nutrients and signals. However, root exudation dynamics are unclear and thus also, if changes in exudation are reflected in changes in microbiome structure. Here, we assess commonalities and differences between exudates of different plant species, diurnal exudation dynamics, as well as the accompanying methodological aspects of exudate sampling. We find that exudates should be collected for hours rather than days as many metabolite abundances saturate over time. Plant growth in sterile, nonsterile, or sugar-supplemented environments significantly alters exudate profiles. A comparison of Arabidopsis thaliana, Brachypodium distachyon, and Medicago truncatula shoot, root, and root exudate metabolite profiles reveals clear differences between these species, but also a core metabolome for tissues and exudates. Exudate profiles also exhibit a diurnal signature. These findings add to the methodological and conceptual groundwork for future exudate studies to improve understanding of plant-microbe interactions.

Chemical signalling in the plant microbiome can have drastic effects on microbial community structure, and on host growth and development. Previously, we demonstrated that the auxin metabolic signal interference performed by the bacterial genus Variovorax via an auxin degradation locus was essential for maintaining stereotypic root development in an ecologically relevant bacterial synthetic community. Here, we dissect the Variovorax auxin degradation locus to define the genes iadDE as necessary and sufficient for indole-3-acetic acid (IAA) degradation and signal interference. We determine the crystal structures and binding properties of the operon's MarR-family repressor with IAA and other auxins. Auxin degradation operons were identified across the bacterial tree of life and we define two distinct types on the basis of gene content and metabolic products: iac-like and iad-like. The structures of MarRs from representatives of each auxin degradation operon type establish that each has distinct IAA-binding pockets. Comparison of representative IAA-degrading strains from diverse bacterial genera colonizing Arabidopsis plants show that while all degrade IAA, only strains containing iad-like auxin-degrading operons interfere with auxin signalling in a complex synthetic community context. This suggests that iad-like operon-containing bacterial strains, including Variovorax species, play a key ecological role in modulating auxins in the plant microbiome.

With the advent of genome sequencing and mining technologies, secondary metabolite biosynthetic gene clusters (BGCs) within bacterial genomes are becoming easier to predict. For subsequent BGC characterization, clustered regularly interspaced short palindromic repeats (CRISPR) has contributed to knocking out target genes and/or modulating their expression; however, CRISPR is limited to strains for which robust genetic tools are available. Here we present a strategy that combines CRISPR with chassis-independent recombinase-assisted genome engineering (CRAGE), which enables CRISPR systems in diverse bacteria. To demonstrate CRAGE-CRISPR, we select 10 polyketide/non-ribosomal peptide BGCs in Photorhabdus luminescens as models and create their deletion and activation mutants. Subsequent loss- and gain-of-function studies confirm 22 secondary metabolites associated with the BGCs, including a metabolite from a previously uncharacterized BGC. These results demonstrate that the CRAGE-CRISPR system is a simple yet powerful approach to rapidly perturb expression of defined BGCs and to profile genotype-phenotype relationships in bacteria.

Domestication and breeding have affected interactions between plants and their microbiomes in ways that are only beginning to be understood but may have important implications for recruitment of rhizosphere microorganisms, particularly under stress conditions. We investigated the responses of a modern maize (Zea mays subsp. mays) cultivar and its wild relative, teosinte (Z. mays subsp. parviglumis), to different phosphate availabilities. We appraised responses of the plant-microbial holobiont to phosphate stresses by profiling root exudate metabolomes, and microbial communities in the root endosphere and rhizosphere. We also performed plate assays to quantify phosphate-solubilizing microorganisms from the rhizosphere. Although root exudate metabolite profiles were distinct between the teosinte and modern maize under high phosphate, both plants shifted exudate compositions in response to phosphate stress toward a common metabolite profile. Root and rhizosphere microbial communities also responded significantly to both plant type and the phosphate availability. A subset of bacterial and fungal taxa were differentially abundant under the different phosphate conditions, with each of the three conditions favoring different taxa. Both teosinte and maize rhizospheres harbored phosphate-solubilizing microorganisms under all growth conditions. These results suggest that the root exudation response to phosphate stress was conserved through the domestication of maize from teosinte, shifting exudation levels of specific metabolites. Although microbial communities also shifted, plate-based assays did not detect selective recruitment of phosphate solubilizers in response to phosphate availability.

Microorganisms have evolved various life-history strategies to survive fluctuating resource conditions in soils. However, it remains elusive how the life-history strategies of microorganisms influence their processing of organic carbon, which may affect microbial interactions and carbon cycling in soils. Here, we characterized the genomic traits, exometabolite profiles, and interactions of soil bacteria representing copiotrophic and oligotrophic strategists. Isolates were selected based on differences in ribosomal RNA operon (rrn) copy number, as a proxy for life-history strategies, with pairs of "high" and "low" rrn copy number isolates represented within the Micrococcales, Corynebacteriales, and Bacillales. We found that high rrn isolates consumed a greater diversity and amount of substrates than low rrn isolates in a defined growth medium containing common soil metabolites. We estimated overlap in substrate utilization profiles to predict the potential for resource competition and found that high rrn isolates tended to have a greater potential for competitive interactions. The predicted interactions positively correlated with the measured interactions that were dominated by negative interactions as determined through sequential growth experiments. This suggests that resource competition was a major force governing interactions among isolates, while cross-feeding of metabolic secretion likely contributed to the relatively rare positive interactions observed. By connecting bacterial life-history strategies, genomic features, and metabolism, our study advances the understanding of the links between bacterial community composition and the transformation of carbon in soils.

Exometabolomics is an approach to assess how microorganisms alter, or react to their environments through the depletion and production of metabolites. It allows the examination of how soil microbes transform the small molecule metabolites within their environment, which can be used to study resource competition and cross-feeding. This approach is most powerful when used with defined media that enable tracking of all metabolites. However, microbial growth media have traditionally been developed for the isolation and growth of microorganisms but not metabolite utilization profiling through Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Here, we describe the construction of a defined medium, the Northen Lab Defined Medium (NLDM), that not only supports the growth of diverse soil bacteria but also is defined and therefore suited for exometabolomic experiments. Metabolites included in NLDM were selected based on their presence in R2A medium and soil, elemental stoichiometry requirements, as well as knowledge of metabolite usage by different bacteria. We found that NLDM supported the growth of 108 of the 110 phylogenetically diverse (spanning 36 different families) soil bacterial isolates tested and all of its metabolites were trackable through LC-MS/MS analysis. These results demonstrate the viability and utility of the constructed NLDM medium for growing and characterizing diverse microbial isolates and communities.

Different electrical and thermoelectric properties of a Si-based thermoelectric generator (TEG) are described based on the Kubo–Greenwood formalism. Temperature and doping dependence, phonon scattering (acoustic and optical phonons), and scattering on impurities are included. Comparisons with experimentally verified data confirm the validity of the model. Experimental studies were carried out on a micromechanically fabricated TEG. Devices were realized using a standard CMOS SOI technology in a lateral geometry. All thermopiles are located on a thin membrane to reduce the heat flow. The thickness of the membrane was adjusted between 20 and 30 µm ensuring also sufficient mechanical stability. Measurements on individual devices confirm the results of the theoretical model. The Seebeck coefficient was calculated and experimentally measured as S = 0.5 mV/K at an acceptor level of 1019 cm−3 at room temperature. The power factor is S2 · σ = 0.0073 W/mK2.

SummaryExometabolomics is an approach to assess how microorganisms alter their environments through the depletion and secretion of chemical compounds. Comparisons of inoculated with uninoculated media can be used to provide direct biochemical observations on depleted and secreted metabolites which can be used to predict resource competition, cross-feeding and secondary metabolite production in microbial isolates and communities. This approach is most powerful when used with defined media that enable tracking of all depleted metabolites. However, microbial growth media have traditionally been developed for the isolation and growth of microorganisms but not metabolite utilization profiling through LC-MS/MS. Here, we describe the construction of a defined medium, the Northen Lab Defined Medium (NLDM), that not only supports the growth of diverse bacteria but is defined and therefore suited for exometabolomic experiments. Metabolites included in NLDM were selected based on their presence in R2A medium and soil, elemental stoichiometry requirements, as well as knowledge of metabolite usage by different bacteria. We found that NLDM supported the growth of 53 phylogenetically diverse soil bacterial isolates and all of its metabolites were trackable through LC–MS/MS analysis. These results demonstrate the viability and utility of the constructed NLDM medium for cultivating and characterizing diverse microbial isolates and communities.Originality-Significance StatementWe build a defined medium based on the metabolite composition of R2A medium and soil, elemental stoichiometry requirements, and knowledge of metabolite usage by different bacteria. The newly formulated defined medium was evaluated on its ability to support the growth of soil isolates and its application for metabolite utilization profiling. We found that of 53 phylogenetically diverse soil bacterial isolates grew on the defined medium and all of its metabolites were trackable through LC–MS/MS analysis. This demonstrates the viability and utility of the constructed defined medium for cultivating and characterizing diverse microbial isolates and communities.

Microorganisms play vital roles in modulating organic matter decomposition and nutrient cycling in soil ecosystems. The enzyme latch paradigm posits microbial degradation of polyphenols is hindered in anoxic peat leading to polyphenol accumulation, and consequently diminished microbial activity. This model assumes that polyphenols are microbially unavailable under anoxia, a supposition that has not been thoroughly investigated in any soil type. Here, we use anoxic soil reactors amended with and without a chemically defined polyphenol to test this hypothesis, employing metabolomics and genome-resolved metaproteomics to interrogate soil microbial polyphenol metabolism. Challenging the idea that polyphenols are not bioavailable under anoxia, we provide metabolite evidence that polyphenols are depolymerized, resulting in monomer accumulation, followed by the generation of small phenolic degradation products. Further, we show that soil microbiome function is maintained, and possibly enhanced, with polyphenol addition. In summary, this study provides chemical and enzymatic evidence that some soil microbiota can degrade polyphenols under anoxia and subvert the assumed polyphenol lock on soil microbial metabolism.

Pseudomonas species are ubiquitous in nature and include numerous medically, agriculturally and technologically beneficial strains of which the interspecific interactions are of great interest for biotechnologies. Specifically, co-cultures containing Pseudomonas stutzeri have been used for bioremediation, biocontrol, aquaculture management and wastewater denitrification. Furthermore, the use of P. stutzeri biofilms, in combination with consortia-based approaches, may offer advantages for these processes. Understanding the interspecific interaction within biofilm co-cultures or consortia provides a means for improvement of current technologies. However, the investigation of biofilm-based consortia has been limited. We present an adaptable and scalable method for the analysis of macroscopic interactions (colony morphology, inhibition, and invasion) between colony-forming bacterial strains using an automated printing method followed by analysis of the genes and metabolites involved in the interactions. Using Biofilm Interaction Mapping and Analysis (BIMA), these interactions were investigated between P. stutzeri strain RCH2, a denitrifier isolated from chromium (VI) contaminated soil, and 13 other species of pseudomonas isolated from non-contaminated soil. One interaction partner, Pseudomonas fluorescens N1B4 was selected for mutant fitness profiling of a DNA-barcoded mutant library; with this approach four genes of importance were identified and the effects on interactions were evaluated with deletion mutants and mass spectrometry based metabolomics.

Root morphology and exudation define a plants' sphere of influence in soils. In turn, soil characteristics influence plant growth, morphology, root microbiome, and rhizosphere chemistry. Collectively, all these parameters have significant implications on the major biogeochemical cycles, crop yield, and ecosystem health. However, how plants are shaped by the physiochemistry of soil particles is still not well understood. We explored how particle size and chemistry of growth substrates affect root morphology and exudation of a model grass. We grew Brachypodium distachyon in glass beads with various sizes (0.5, 1, 2, 3 mm), as well as in sand (0.005, 0.25, 4 mm) and in clay (4 mm) particles and in particle-free hydroponic medium. Plant morphology, root weight, and shoot weight were measured. We found that particle size significantly influenced root fresh weight and root length, whereas root number and shoot weight remained constant. Next, plant exudation profiles were analyzed with mass spectrometry imaging and liquid chromatography-mass spectrometry. Mass spectrometry imaging suggested that both, root length and number shape root exudation. Exudate profiles were comparable for plants growing in glass beads or sand with various particles sizes, but distinct for plants growing in clay for in situ exudate collection. Clay particles were found to sorb 20% of compounds exuded by clay-grown plants, and 70% of compounds from a defined exudate medium. The sorbed compounds belonged to a range of chemical classes, among them nucleosides, organic acids, sugars, and amino acids. Some of the sorbed compounds could be desorbed by a rhizobacterium (Pseudomonas fluorescens WCS415), supporting its growth. This study demonstrates the effect of different characteristics of particles on root morphology, plant exudation and availability of nutrients to microorganisms. These findings further support the critical importance of the physiochemical properties of soils when investigating plant morphology, plant chemistry, and plant-microbe interactions.

Root exudates display a diurnal signature, change with growth environment, and can be divided into a core metabolome common to multiple plant species, and specialized exudates produced by distinct species.

Microbial competition within plant tissues affects invading pathogens’ fitness. Metabolomics is a great tool for studying their biochemical interactions by identifying accumulated metabolites. Xylella fastidiosa, a Gram-negative bacterium causing Pierce’s disease (PD) in grapevines, secretes various virulence factors including cell wall-degrading enzymes, adhesion proteins, and quorum-sensing molecules. These factors, along with outer membrane vesicles, contribute to its pathogenicity. Previous studies demonstrated that co-inoculating X. fastidiosa with the Paraburkholderia phytofirmans strain PsJN suppressed PD symptoms. Here, we further investigated the interaction between the phytopathogen and the endophyte by analyzing the exometabolome of wild-type X. fastidiosa and a diffusible signaling factor (DSF) mutant lacking quorum sensing, cultivated with 20% P. phytofirmans spent media. Liquid chromatography–mass spectrometry (LC-MS) and the Method for Metabolite Annotation and Gene Integration (MAGI) were used to detect and map metabolites to genomes, revealing a total of 121 metabolites, of which 25 were further investigated. These metabolites potentially relate to host adaptation, virulence, and pathogenicity. Notably, this study presents the first comprehensive profile of X. fastidiosa in the presence of a P. phytofirmans spent media. The results highlight that P. phytofirmans and the absence of functional quorum sensing affect the ratios of glutamine to glutamate (Gln:Glu) in X. fastidiosa. Additionally, two compounds with plant metabolism and growth properties, 2-aminoisobutyric acid and gibberellic acid, were downregulated when X. fastidiosa interacted with P. phytofirmans. These findings suggest that P. phytofirmans-mediated disease suppression involves modulation of the exometabolome of X. fastidiosa, impacting plant immunity.

It is generally believed that exchange of secondary metabolite biosynthetic gene clusters (BGCs) among closely related bacteria is an important driver of BGC evolution and diversification. Applying this idea may help researchers efficiently connect many BGCs to their products and characterize the products' roles in various environments. However, existing genetic tools support only a small fraction of these efforts. Here, we present the development of chassis-independent recombinase-assisted genome engineering (CRAGE), which enables single-step integration of large, complex BGC constructs directly into the chromosomes of diverse bacteria with high accuracy and efficiency. To demonstrate the efficacy of CRAGE, we expressed three known and six previously identified but experimentally elusive non-ribosomal peptide synthetase (NRPS) and NRPS-polyketide synthase (PKS) hybrid BGCs from Photorhabdus luminescens in 25 diverse γ-Proteobacteria species. Successful activation of six BGCs identified 22 products for which diversity and yield were greater when the BGCs were expressed in strains closely related to the native strain than when they were expressed in either native or more distantly related strains. Activation of these BGCs demonstrates the feasibility of exploiting their underlying catalytic activity and plasticity, and provides evidence that systematic approaches based on CRAGE will be useful for discovering and identifying previously uncharacterized metabolites.

There is a dynamic reciprocity between plants and their environment: soil physiochemical properties influence plant morphology and metabolism, and root morphology and exudates shape the environment surrounding roots. Here, we investigate the reproducibility of plant trait changes in response to three growth environments. We utilized fabricated ecosystem (EcoFAB) devices to grow the model grass Brachypodium distachyon in three distinct media across four laboratories: phosphate-sufficient and -deficient mineral media allowed assessment of the effects of phosphate starvation, and a complex, sterile soil extract represented a more natural environment with yet uncharacterized effects on plant growth and metabolism. Tissue weight and phosphate content, total root length, and root tissue and exudate metabolic profiles were consistent across laboratories and distinct between experimental treatments. Plants grown in soil extract were morphologically and metabolically distinct, with root hairs four times longer than with other growth conditions. Further, plants depleted half of the metabolites investigated from the soil extract. To interact with their environment, plants not only adapt morphology and release complex metabolite mixtures, but also selectively deplete a range of soil-derived metabolites. The EcoFABs utilized here generated high interlaboratory reproducibility, demonstrating their value in standardized investigations of plant traits.

Abstract Background The prophylactic administration of tranexamic acid reduces blood loss during procedures at high risk of perioperative bleeding. Several studies in cardiac surgery and orthopedics confirmed this finding. The aim of this prospective, double-blind, randomized study is to evaluate the effect of tranexamic acid on peri-and postoperative blood loss and on the incidence and severity of complications. Methods/design Based on the results of our pilot study, we decided to conduct this prospective, double-blind, randomized trial to confirm the preliminary data. The primary endpoint is to analyze the effect of tranexamic acid on perioperative and postoperative blood loss (decrease in hemoglobin levels) in robotic-assisted radical prostatectomy. The additional endpoint is to analyze the effect of tranexamic acid on postoperative complications and confirm the safety of tranexamic acid in robotic-assisted radical prostatectomy. Discussion No study to date has tested the prophylactic administration of tranexamic acid at the beginning of robotic-assisted radical prostatectomy. This study is designed to answer the question of whether the administration of tranexamic acid might lower the blood loss after the procedure or increase the rate and severity of complications. Trial registration ClinicalTrials.gov NCT04319614. Registered on 25 March 2020

Domestication and breeding have affected interactions between plants and their microbiomes in ways that are only beginning to be understood but may have important implications for recruitment of rhizosphere microorganisms, particularly under stress conditions. We investigated the responses of a modern maize (Zea mays subsp. mays) cultivar and its wild relative, teosinte (Z. mays subsp. parviglumis), to different phosphate availabilities. We appraised responses of the plant-microbial holobiont to phosphate stresses by profiling root exudate metabolomes, and microbial communities in the root endosphere and rhizosphere. We also performed plate assays to quantify phosphate-solubilizing microorganisms from the rhizosphere. Although root exudate metabolite profiles were distinct between the teosinte and modern maize under high phosphate, both plants shifted exudate compositions in response to phosphate stress toward a common metabolite profile. Root and rhizosphere microbial communities also responded significantly to both plant type and the phosphate availability. A subset of bacterial and fungal taxa were differentially abundant under the different phosphate conditions, with each of the three conditions favoring different taxa. Both teosinte and maize rhizospheres harbored phosphate-solubilizing microorganisms under all growth conditions. These results suggest that the root exudation response to phosphate stress was conserved through the domestication of maize from teosinte, shifting exudation levels of specific metabolites. Although microbial communities also shifted, plate-based assays did not detect selective recruitment of phosphate solubilizers in response to phosphate availability.

This study examined the metabolism of flavonolignans and taxifolin in 33 healthy adult male volunteers, who received 200 mg of silymarin orally twice daily for three months. First-morning urine and feces collected on days 10 and 90 were analyzed by UHPLC-MS and bacterial composition of feces was investigated by Next Generation Sequencing. The analyses revealed 5/4 types of flavonolignan/taxifolin metabolites in urine and 13/10 types of metabolites in feces, with large differences in total metabolite levels in both urine and feces observed among individual participants. In urine, 21 and 23 participants showed some increase in the total amount of flavonolignan and taxifolin metabolites, respectively, on day 90 compared with day 10. In feces, some correlation was found between the amount of metabolites and the composition of the intestinal microbiota. In conclusion, silymarin flavonolignans and taxifolin undergo multiple biotransformation reactions, and the metabolic profiles of the constituents considerably vary among individuals.

BackgroundAs microbiome research becomes increasingly prevalent in the fields of human health, agriculture and biotechnology, there exists a need for a resource to better link organisms and environmental chemistries. Exometabolomics experiments now provide assertions of the metabolites present within specific environments and how the production and depletion of metabolites is linked to specific microbes. This information could be broadly useful, from comparing metabolites across environments, to predicting competition and exchange of metabolites between microbes, and to designing stable microbial consortia. Here, we introduce Web of Microbes (WoM; freely available at: http://webofmicrobes.org ), the first exometabolomics data repository and visualization tool.DescriptionWoM provides manually curated, direct biochemical observations on the changes to metabolites in an environment after exposure to microorganisms. The web interface displays a number of key features: (1) the metabolites present in a control environment prior to inoculation or microbial activation, (2) heatmap-like displays showing metabolite increases or decreases resulting from microbial activities, (3) a metabolic web displaying the actions of multiple organisms on a specified metabolite pool, (4) metabolite interaction scores indicating an organism's interaction level with its environment, potential for metabolite exchange with other organisms and potential for competition with other organisms, and (5) downloadable datasets for integration with other types of -omics datasets.ConclusionWe anticipate that Web of Microbes will be a useful tool for the greater research community by making available manually curated exometabolomics results that can be used to improve genome annotations and aid in the interpretation and construction of microbial communities.

There is a dynamic reciprocity between plants and their environment: On one hand, the physiochemical properties of soil influence plant morphology and metabolism, while on the other, root morphology and exudates shape the environment surrounding roots. Here, we investigate both of these aspects as well as the reproducibility of these responses across laboratories. The model grass Brachypodium distachyon was grown in phosphate-sufficient and phosphate-deficient mineral media, as well as in sterile soil extract, within fabricated ecosystem (EcoFAB) devices across four laboratories. Tissue weight and phosphate content, total root length, root tissue and exudate metabolic profiles were found to be consistent across laboratories and distinct between experimental treatments. Plants grown in soil extract were morphologically and metabolically distinct in all laboratories, with root hairs four times longer compared to other growth conditions. Further, plants depleted half of the investigated metabolites from the soil extract. To interact with their environment, plants not only adapt morphology and release complex metabolite mixtures; they also selectively deplete a range of soil-derived metabolites. The EcoFABs utilized here generated high inter-laboratory reproducibility, demonstrating that their value in standardized investigations of plant traits.

Solar panels can be found practically all over the world and represent a standard surface that can be colonized by microbial communities that are resistant to harsh environmental conditions, including high irradiation, temperature fluctuations and desiccation. These properties make them not only ideal sources of stress-resistant bacteria, but also standard devices to study the microbial communities and their colonization process from different areas of Earth. We report here a comprehensive description of the microbial communities associated with solar panels in Berkeley, CA, United States. Cultivable bacteria were isolated to characterize their adhesive capabilities, and UV- and desiccation-resistance properties. Furthermore, a parallel culture-independent metagenomic and metabolomic approach has allowed us to gain insight on the taxonomic and functional nature of these communities. Metagenomic analysis was performed using the Illumina HiSeq2500 sequencing platform, revealing that the bacterial population of the Berkeley solar panels is composed mainly of Actinobacteria, Bacteroidetes and Proteobacteria, as well as lower amounts of Deinococcus-Thermus and Firmicutes. Furthermore, a clear predominance of Hymenobacter sp. was also observed. A functional analysis revealed that pathways involved in the persistence of microbes on solar panels (i.e., stress response, capsule development, and metabolite repair) and genes assigned to carotenoid biosynthesis were common to all metagenomes. On the other hand, genes involved in photosynthetic pathways and general autotrophic subsystems were rare, suggesting that these pathways are not critical for persistence on solar panels. Metabolomics was performed using a liquid chromatography tandem mass spectrometry (LC-MS/MS) approach. When comparing the metabolome of the solar panels from Berkeley and from Valencia (Spain), a very similar composition in polar metabolites could be observed, although some metabolites appeared to be differentially represented (for example, trigonelline, pantolactone and 5-valerolactone were more abundant in the samples from Valencia than in the ones from Berkeley). Furthermore, triglyceride metabolites were highly abundant in all the solar panel samples, and both locations displayed similar profiles. The comparison of the taxonomic profile of the Californian solar panels with those previously described in Spain revealed striking similarities, highlighting the central role of both selective pressures and the ubiquity of microbial populations in the colonization and establishment of microbial communities.

BackgroundMixed cultures of different microbial species are increasingly being used to carry out a specific biochemical function in lieu of engineering a single microbe to do the same task. However, knowing how different species' metabolisms will integrate to reach a desired outcome is a difficult problem that has been studied in great detail using steady-state models. However, many biotechnological processes, as well as natural habitats, represent a more dynamic system. Examining how individual species use resources in their growth medium or environment (exometabolomics) over time in batch culture conditions can provide rich phenotypic data that encompasses regulation and transporters, creating an opportunity to integrate the data into a predictive model of resource use by a mixed community.ResultsHere we use exometabolomic profiling to examine the time-varying substrate depletion from a mixture of 19 amino acids and glucose by two Pseudomonas and one Bacillus species isolated from ground water. Contrary to studies in model organisms, we found surprisingly few correlations between resource preferences and maximal growth rate or biomass composition. We then modeled patterns of substrate depletion, and used these models to examine if substrate usage preferences and substrate depletion kinetics of individual isolates can be used to predict the metabolism of a co-culture of the isolates. We found that most of the substrates fit the model predictions, except for glucose and histidine, which were depleted more slowly than predicted, and proline, glycine, glutamate, lysine and arginine, which were all consumed significantly faster.ConclusionsOur results indicate that a significant portion of a model community's overall metabolism can be predicted based on the metabolism of the individuals. Based on the nature of our model, the resources that significantly deviate from the prediction highlight potential metabolic pathways affected by species-species interactions, which when further studied can potentially be used to modulate microbial community structure and/or function.

It is thought that polyphenols inhibit organic matter decomposition in soils devoid of oxygen. Here the authors use metabolomics and genome-resolved metaproteomics to provide experimental evidence of polyphenol biodegradation and maintained soil microbial community metabolism despite anoxia.

Microorganisms have evolved various life-history strategies to survive fluctuating resource conditions in soils. However, it remains elusive how the life-history strategies of microorganisms influence their processing of organic carbon, which may affect microbial interactions and carbon cycling in soils. Here, we characterized the genomic traits, exometabolite profiles, and interactions of soil bacteria representing copiotrophic and oligotrophic strategists. Isolates were selected based on differences in ribosomal RNA operon (rrn) copy number, as a proxy for life-history strategies, with pairs of “high” and “low” rrn copy number isolates represented within the Micrococcales, Corynebacteriales, and Bacillales. We found that high rrn isolates consumed a greater diversity and amount of substrates than low rrn isolates in a defined growth medium containing common soil metabolites. We estimated overlap in substrate utilization profiles to predict the potential for resource competition and found that high rrn isolates tended to have a greater potential for competitive interactions. The predicted interactions positively correlated with the measured interactions that were dominated by negative interactions as determined through sequential growth experiments. This suggests that resource competition was a major force governing interactions among isolates, while cross-feeding of metabolic secretion likely contributed to the relatively rare positive interactions observed. By connecting bacterial life-history strategies, genomic features, and metabolism, our study advances the understanding of the links between bacterial community composition and the transformation of carbon in soils.

Exometabolomics is an approach to assess how microorganisms alter, or react to their environments through the depletion and production of metabolites. It allows the examination of how soil microbes transform the small molecule metabolites within their environment, which can be used to study resource competition and cross-feeding. This approach is most powerful when used with defined media that enable tracking of all metabolites. However, microbial growth media have traditionally been developed for the isolation and growth of microorganisms but not metabolite utilization profiling through Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Here, we describe the construction of a defined medium, the Northen Lab Defined Medium (NLDM), that not only supports the growth of diverse soil bacteria but also is defined and therefore suited for exometabolomic experiments. Metabolites included in NLDM were selected based on their presence in R2A medium and soil, elemental stoichiometry requirements, as well as knowledge of metabolite usage by different bacteria. We found that NLDM supported the growth of 108 of the 110 phylogenetically diverse (spanning 36 different families) soil bacterial isolates tested and all of its metabolites were trackable through LC–MS/MS analysis. These results demonstrate the viability and utility of the constructed NLDM medium for growing and characterizing diverse microbial isolates and communities.

BackgroundHealthcare associated infections (HAI) with multidrug-resistant (MDR) bacteria continue to be a global threat, highlighting an urgent need for novel antibiotics. In this study, we assessed the potential of free fatty acids and cholesteryl esters that form part of the innate host defense as novel antibacterial agents for use against MDR bacteria.MethodsLiposomes of six different phospholipid mixtures were employed as carrier for six different fatty acids and four different cholesteryl esters. Using a modified MIC assay based on DNA quantification with the fluoroprobe Syto9, formulations were tested against Gram-positive and Gram-negative bacteria implicated in HAI. Formulations with MIC values in the low μg/mL range were further subjected to determination of minimal bactericidal activity, hemolysis assay with sheep erythrocytes, and cytotoxicity testing with the human liver cell line HepG2. The potential for synergistic activity with a standard antibiotic was also probed.ResultsPalmitic acid and stearic acid prepared in carrier 4 (PA4 and SA4, respectively) were identified as most active lipids (MIC against MDR Staphylococcus epidermidis was 0.5 and 0.25 μg/mL, respectively; MIC against vancomycin resistant Enterococcus faecalis (VRE) was 2 and 0.5 μg/mL, respectively). Cholesteryl linoleate formulated with carrier 3 (CL3) exhibited activity against the S. epidermidis strain (MIC 1 μg/mL) and a Pseudomonas aeruginosa strain (MIC 8 μg/mL) and lowered the vancomycin MIC for VRE from 32-64 μg/mL to as low as 4 μg/mL. At 90 μg/mL PA4, SA4, and CL3 effected less than 5 % hemolysis over 3 h and PA4 and CL3 did not exhibit significant cytotoxic activity against HepG2 cells when applied at 100 μg/mL over 48 h.ConclusionsOur results showed that selected fatty acids and cholesteryl esters packaged with phospholipids exhibit antibacterial activity against Gram-positive and Gram-negative bacteria and may augment the activity of antibiotics. Bactericidal activity could be unlinked from hemolytic and cytotoxic activity and the type of phospholipid carrier greatly influenced the activity. Thus, fatty acids and cholesteryl esters packaged in liposomes may have potential as novel lipophilic antimicrobial agents.