Complete biosynthesis of QS-21 in engineered yeast.

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.

Decomposition decreases molecular diversity and ecosystem similarity of soil organic matter.

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 ß-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.

Conservation of beneficial microbes between the rhizosphere and the cyanosphere.

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.

Dynamic Phaeodactylum tricornutum exometabolites shape surrounding bacterial communities.

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.

Multilab EcoFAB study shows highly reproducible physiology and depletion of soil metabolites by a model grass.

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.

Web of microbes (WoM): a curated microbial exometabolomics database for linking chemistry and microbes.

BACKGROUND: As 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. DESCRIPTION: WoM 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. CONCLUSION: We 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.

Dynamic substrate preferences predict metabolic properties of a simple microbial consortium.

BACKGROUND: Mixed 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. RESULTS: Here 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. CONCLUSIONS: Our 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.

The Exometabolome of Xylella fastidiosa in Contact with Paraburkholderia phytofirmans Supernatant Reveals Changes in Nicotinamide, Amino Acids, Biotin, and Plant Hormones.

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.

Reproducible growth of Brachypodium in EcoFAB 2.0 reveals that nitrogen form and starvation modulate root exudation.

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.

The core metabolome and root exudation dynamics of three phylogenetically distinct plant species.

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.

CRAGE-CRISPR facilitates rapid activation of secondary metabolite biosynthetic gene clusters in bacteria.

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.

Substrate Utilization and Competitive Interactions Among Soil Bacteria Vary With Life-History Strategies.

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.

A Defined Medium for Cultivation and Exometabolite Profiling of Soil Bacteria.

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.

Diverse MarR bacterial regulators of auxin catabolism in the plant microbiome.

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.

Decrypting bacterial polyphenol metabolism in an anoxic wetland soil.

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.

Biofilm Interaction Mapping and Analysis (BIMA) of Interspecific Interactions in Pseudomonas Co-culture Biofilms.

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 exudate availability are shaped by particle size and chemistry in Brachypodium distachyon.

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.

CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria.

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.

Microbial Ecology on Solar Panels in Berkeley, CA, United States.

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.

Exometabolomics Assisted Design and Validation of Synthetic Obligate Mutualism.

Synthetic microbial ecology has the potential to enhance the productivity and resiliency of biotechnology processes compared to approaches using single isolates. Engineering microbial consortia is challenging; however, one approach that has attracted significant attention is the creation of synthetic obligate mutualism using auxotrophic mutants that depend on each other for exchange or cross-feeding of metabolites. Here, we describe the integration of mutant library fitness profiling with mass spectrometry based exometabolomics as a method for constructing synthetic mutualism based on cross-feeding. Two industrially important species lacking known ecological interactions, Zymomonas mobilis and Escherichia coli, were selected as the test species. Amino acid exometabolites identified in the spent medium of Z. mobilis were used to select three corresponding E. coli auxotrophs (proA, pheA and IlvA), as potential E. coli counterparts for the coculture. A pooled mutant fitness assay with a Z. mobilis transposon mutant library was used to identify mutants with improved growth in the presence of E. coli. An auxotroph mutant in a gene (ZMO0748) with sequence similarity to cysteine synthase A (cysK), was selected as the Z. mobilis counterpart for the coculture. Exometabolomic analysis of spent E. coli medium identified glutathione related metabolites as potentially available for rescue of the Z. mobilis cysteine synthase mutant. Three sets of cocultures between the Z. mobilis auxotroph and each of the three E. coli auxotrophs were monitored by optical density for growth and analyzed by flow cytometry to confirm high cell counts for each species. Taken together, our methods provide a technological framework for creating synthetic mutualisms combining existing screening based methods and exometabolomics for both the selection of obligate mutualism partners and elucidation of metabolites involved in auxotroph rescue.