Cynipid wasps systematically reprogram host metabolism and restructure cell walls in developing galls.

Many insects have evolved the ability to manipulate plant growth to generate extraordinary structures called galls, in which insect larva can develop while being sheltered and feeding on the plant. In particular, cynipid (Hymenoptera: Cynipidae) wasps have evolved to form morphologically complex galls and generate an astonishing array of gall shapes, colors, and sizes. However, the biochemical basis underlying these remarkable cellular and developmental transformations remains poorly understood. A key determinant in plant cellular development is cell wall deposition that dictates the physical form and physiological function of newly developing cells, tissues, and organs. However, it is unclear to what degree cell walls are restructured to initiate and support the formation of new gall tissue. Here, we characterize the molecular alterations underlying gall development using a combination of metabolomic, histological, and biochemical techniques to elucidate how valley oak (Quercus lobata) leaf cells are reprogrammed to form galls. Strikingly, gall development involves an exceptionally coordinated spatial deposition of lignin and xylan to form de novo gall vasculature. Our results highlight how cynipid wasps can radically change the metabolite profile and restructure the cell wall to enable the formation of galls, providing insights into the mechanism of gall induction and the extent to which plants can be entirely reprogrammed to form unique structures and organs.

Information theory and machine learning illuminate large-scale metabolomic responses of Brachypodium distachyon to environmental change.

Plant responses to environmental change are mediated via changes in cellular metabolomes. However, <5% of signals obtained from liquid chromatography tandem mass spectrometry (LC-MS/MS) can be identified, limiting our understanding of how metabolomes change under biotic/abiotic stress. To address this challenge, we performed untargeted LC-MS/MS of leaves, roots, and other organs of Brachypodium distachyon (Poaceae) under 17 organ-condition combinations, including copper deficiency, heat stress, low phosphate, and arbuscular mycorrhizal symbiosis. We found that both leaf and root metabolomes were significantly affected by the growth medium. Leaf metabolomes were more diverse than root metabolomes, but the latter were more specialized and more responsive to environmental change. We found that 1 week of copper deficiency shielded the root, but not the leaf metabolome, from perturbation due to heat stress. Machine learning (ML)-based analysis annotated approximately 81% of the fragmented peaks versus approximately 6% using spectral matches alone. We performed one of the most extensive validations of ML-based peak annotations in plants using thousands of authentic standards, and analyzed approximately 37% of the annotated peaks based on these assessments. Analyzing responsiveness of each predicted metabolite class to environmental change revealed significant perturbations of glycerophospholipids, sphingolipids, and flavonoids. Co-accumulation analysis further identified condition-specific biomarkers. To make these results accessible, we developed a visualization platform on the Bio-Analytic Resource for Plant Biology website (https://bar.utoronto.ca/efp_brachypodium_metabolites/cgi-bin/efpWeb.cgi), where perturbed metabolite classes can be readily visualized. Overall, our study illustrates how emerging chemoinformatic methods can be applied to reveal novel insights into the dynamic plant metabolome and stress adaptation.

Expression in poplar of dehydroshikimate dehydratase induces transcriptional and metabolic changes in the phenylpropanoid pathway.

Modification of lignin in feedstocks via genetic engineering aims to reduce biomass recalcitrance to facilitate efficient conversion processes. These improvements can be achieved by expressing exogenous enzymes that interfere with native biosynthetic pathways responsible for the production of the lignin precursors. In-planta expression of a 3-dehydroshikimate dehydratase (QsuB) in poplar trees reduced lignin content and altered their monomer composition, which enabled higher yields of sugars after cell wall polysaccharide hydrolysis. Understanding how plants respond to such genetic modifications at the transcriptional and metabolic levels is needed to facilitate further improvement and field deployment. In this work, we amassed fundamental knowledge on lignin-modified QsuB poplar using RNA-seq and metabolomics. The data clearly demonstrate that changes in gene expression and metabolite abundance can occur in a strict spatiotemporal fashion, revealing tissue-specific responses in the xylem, phloem, or periderm. In the poplar line that exhibits the strongest reduction in lignin, we found that 3% of the transcripts had altered expression levels and ~19% of the detected metabolites had differential abundance in the xylem from older stems. Changes affect predominantly the shikimate and phenylpropanoid pathways as wells as secondary cell wall metabolism, and result in significant accumulation of hydroxybenzoates derived from protocatechuate and salicylate.

Anaerobic gut fungi are an untapped reservoir of natural products.

Anaerobic fungi (class Neocallimastigomycetes) thrive as low-abundance members of the herbivore digestive tract. The genomes of anaerobic gut fungi are poorly characterized and have not been extensively mined for the biosynthetic enzymes of natural products such as antibiotics. Here, we investigate the potential of anaerobic gut fungi to synthesize natural products that could regulate membership within the gut microbiome. Complementary 'omics' approaches were combined to catalog the natural products of anaerobic gut fungi from four different representative species: Anaeromyces robustus (Arobustus), Caecomyces churrovis (Cchurrovis), Neocallimastix californiae (Ncaliforniae), and Piromyces finnis (Pfinnis). In total, 146 genes were identified that encode biosynthetic enzymes for diverse types of natural products, including nonribosomal peptide synthetases and polyketide synthases. In addition, N. californiae and C. churrovis genomes encoded seven putative bacteriocins, a class of antimicrobial peptides typically produced by bacteria. During standard laboratory growth on plant biomass or soluble substrates, 26% of total core biosynthetic genes in all four strains were transcribed. Across all four fungal strains, 30% of total biosynthetic gene products were detected via proteomics when grown on cellobiose. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of fungal supernatants detected 72 likely natural products from A. robustus alone. A compound produced by all four strains of anaerobic fungi was putatively identified as the polyketide-related styrylpyrone baumin. Molecular networking quantified similarities between tandem mass spectrometry (MS/MS) spectra among these fungi, enabling three groups of natural products to be identified that are unique to anaerobic fungi. Overall, these results support the finding that anaerobic gut fungi synthesize natural products, which could be harnessed as a source of antimicrobials, therapeutics, and other bioactive compounds.

Establishing a versatile toolkit of flux enhanced strains and cell extracts for pathway prototyping.

Building and optimizing biosynthetic pathways in engineered cells holds promise to address societal needs in energy, materials, and medicine, but it is often time-consuming. Cell-free synthetic biology has emerged as a powerful tool to accelerate design-build-test-learn cycles for pathway engineering with increased tolerance to toxic compounds. However, most cell-free pathway prototyping to date has been performed in extracts from wildtype cells which often do not have sufficient flux towards the pathways of interest, which can be enhanced by engineering. Here, to address this gap, we create a set of engineered Escherichia coli and Saccharomyces cerevisiae strains rewired via CRISPR-dCas9 to achieve high-flux toward key metabolic precursors; namely, acetyl-CoA, shikimate, triose-phosphate, oxaloacetate, α-ketoglutarate, and glucose-6-phosphate. Cell-free extracts generated from these strains are used for targeted enzyme screening in vitro. As model systems, we assess in vivo and in vitro production of triacetic acid lactone from acetyl-CoA and muconic acid from the shikimate pathway. The need for these platforms is exemplified by the fact that muconic acid cannot be detected in wildtype extracts provided with the same biosynthetic enzymes. We also perform metabolomic comparison to understand biochemical differences between the cellular and cell-free muconic acid synthesis systems (E. coli and S. cerevisiae cells and cell extracts with and without metabolic rewiring). While any given pathway has different interfaces with metabolism, we anticipate that this set of pre-optimized, flux enhanced cell extracts will enable prototyping efforts for new biosynthetic pathways and the discovery of biochemical functions of enzymes.

Substrate availability and not thermal acclimation controls microbial temperature sensitivity response to long-term warming.

Microbes are responsible for cycling carbon (C) through soils, and predicted changes in soil C stocks under climate change are highly sensitive to shifts in the mechanisms assumed to control the microbial physiological response to warming. Two mechanisms have been suggested to explain the long-term warming impact on microbial physiology: microbial thermal acclimation and changes in the quantity and quality of substrates available for microbial metabolism. Yet studies disentangling these two mechanisms are lacking. To resolve the drivers of changes in microbial physiology in response to long-term warming, we sampled soils from 13- and 28-year-old soil warming experiments in different seasons. We performed short-term laboratory incubations across a range of temperatures to measure the relationships between temperature sensitivity of physiology (growth, respiration, carbon use efficiency, and extracellular enzyme activity) and the chemical composition of soil organic matter. We observed apparent thermal acclimation of microbial respiration, but only in summer, when warming had exacerbated the seasonally-induced, already small dissolved organic matter pools. Irrespective of warming, greater quantity and quality of soil carbon increased the extracellular enzymatic pool and its temperature sensitivity. We propose that fresh litter input into the system seasonally cancels apparent thermal acclimation of C-cycling processes to decadal warming. Our findings reveal that long-term warming has indirectly affected microbial physiology via reduced C availability in this system, implying that earth system models including these negative feedbacks may be best suited to describe long-term warming effects on these soils.

Development of platforms for functional characterization and production of phenazines using a multi-chassis approach via CRAGE.

Phenazines (Phzs), a family of chemicals with a phenazine backbone, are secondary metabolites with diverse properties such as antibacterial, anti-fungal, or anticancer activity. The core derivatives of phenazine, phenazine-1-carboxylic acid (PCA) and phenazine-1,6-dicarboxylic acid (PDC), are themselves precursors for various other derivatives. Recent advances in genome mining tools have enabled researchers to identify many biosynthetic gene clusters (BGCs) that might produce novel Phzs. To characterize the function of these BGCs efficiently, we performed modular construct assembly and subsequent multi-chassis heterologous expression using chassis-independent recombinase-assisted genome engineering (CRAGE). CRAGE allowed rapid integration of a PCA BGC into 23 diverse γ-proteobacteria species and allowed us to identify top PCA producers. We then used the top five chassis hosts to express four partially refactored PDC BGCs. A few of these platforms produced high levels of PDC. Specifically, Xenorhabdus doucetiae and Pseudomonas simiae produced PDC at a titer of 293 mg/L and 373 mg/L, respectively, in minimal media. These titers are significantly higher than those previously reported. Furthermore, selectivity toward PDC production over PCA production was improved by up to 9-fold. The results show that these strains are promising chassis for production of PCA, PDC, and their derivatives, as well as for function characterization of Phz BGCs identified via bioinformatics mining.

Ecological generalism drives hyperdiversity of secondary metabolite gene clusters in xylarialean endophytes.

Although secondary metabolites are typically associated with competitive or pathogenic interactions, the high bioactivity of endophytic fungi in the Xylariales, coupled with their abundance and broad host ranges spanning all lineages of land plants and lichens, suggests that enhanced secondary metabolism might facilitate symbioses with phylogenetically diverse hosts. Here, we examined secondary metabolite gene clusters (SMGCs) across 96 Xylariales genomes in two clades (Xylariaceae s.l. and Hypoxylaceae), including 88 newly sequenced genomes of endophytes and closely related saprotrophs and pathogens. We paired genomic data with extensive metadata on endophyte hosts and substrates, enabling us to examine genomic factors related to the breadth of symbiotic interactions and ecological roles. All genomes contain hyperabundant SMGCs; however, Xylariaceae have increased numbers of gene duplications, horizontal gene transfers (HGTs) and SMGCs. Enhanced metabolic diversity of endophytes is associated with a greater diversity of hosts and increased capacity for lignocellulose decomposition. Our results suggest that, as host and substrate generalists, Xylariaceae endophytes experience greater selection to diversify SMGCs compared with more ecologically specialised Hypoxylaceae species. Overall, our results provide new evidence that SMGCs may facilitate symbiosis with phylogenetically diverse hosts, highlighting the importance of microbial symbioses to drive fungal metabolic diversity.

Regulation of Oxygenic Photosynthesis during Trophic Transitions in the Green Alga Chromochloris zofingiensis.

Light and nutrients are critical regulators of photosynthesis and metabolism in plants and algae. Many algae have the metabolic flexibility to grow photoautotrophically, heterotrophically, or mixotrophically. Here, we describe reversible Glc-dependent repression/activation of oxygenic photosynthesis in the unicellular green alga Chromochloris zofingiensis. We observed rapid and reversible changes in photosynthesis, in the photosynthetic apparatus, in thylakoid ultrastructure, and in energy stores including lipids and starch. Following Glc addition in the light, C. zofingiensis shuts off photosynthesis within days and accumulates large amounts of commercially relevant bioproducts, including triacylglycerols and the high-value nutraceutical ketocarotenoid astaxanthin, while increasing culture biomass. RNA sequencing reveals reversible changes in the transcriptome that form the basis of this metabolic regulation. Functional enrichment analyses show that Glc represses photosynthetic pathways while ketocarotenoid biosynthesis and heterotrophic carbon metabolism are upregulated. Because sugars play fundamental regulatory roles in gene expression, physiology, metabolism, and growth in both plants and animals, we have developed a simple algal model system to investigate conserved eukaryotic sugar responses as well as mechanisms of thylakoid breakdown and biogenesis in chloroplasts. Understanding regulation of photosynthesis and metabolism in algae could enable bioengineering to reroute metabolism toward beneficial bioproducts for energy, food, pharmaceuticals, and human health.

Metabolomic Patterns of Septoria Canker Resistant and Susceptible Populus trichocarpa Genotypes 24 Hours Postinoculation.

Sphaerulina musiva is an economically and ecologically important fungal pathogen that causes Septoria stem canker and leaf spot disease of Populus species. To bridge the gap between genetic markers and structural barriers previously found to be linked to Septoria canker disease resistance in poplar, we used hydrophilic interaction liquid chromatography and tandem mass spectrometry to identify and quantify metabolites involved with signaling and cell wall remodeling. Fluctuations in signaling molecules, organic acids, amino acids, sterols, phenolics, and saccharides in resistant and susceptible P. trichocarpa inoculated with S. musiva were observed. The patterns of 222 metabolites in the resistant host implicate systemic acquired resistance (SAR), cell wall apposition, and lignin deposition as modes of resistance to this hemibiotrophic pathogen. This pattern is consistent with the expected response to the biotrophic phase of S. musiva colonization during the first 24 h postinoculation. The fungal pathogen metabolized key regulatory signals of SAR, other phenolics, and precursors of lignin biosynthesis that were depleted in the susceptible host. This is the first study to characterize metabolites associated with the response to initial colonization by S. musiva between resistant and susceptible hosts.

An integrated workflow for phenazine-modifying enzyme characterization.

Increasing availability of new genomes and putative biosynthetic gene clusters (BGCs) has extended the opportunity to access novel chemical diversity for agriculture, medicine, environmental and industrial purposes. However, functional characterization of BGCs through heterologous expression is limited because expression may require complex regulatory mechanisms, specific folding or activation. We developed an integrated workflow for BGC characterization that integrates pathway identification, modular design, DNA synthesis, assembly and characterization. This workflow was applied to characterize multiple phenazine-modifying enzymes. Phenazine pathways are useful for this workflow because all phenazines are derived from a core scaffold for modification by diverse modifying enzymes (PhzM, PhzS, PhzH, and PhzO) that produce characterized compounds. We expressed refactored synthetic modules of previously uncharacterized phenazine BGCs heterologously in Escherichia coli and were able to identify metabolic intermediates they produced, including a previously unidentified metabolite. These results demonstrate how this approach can accelerate functional characterization of BGCs.

Morphology-Driven Control of Metabolite Selectivity Using Nanostructure-Initiator Mass Spectrometry.

Nanostructure-initiator mass spectrometry (NIMS) is a laser desorption/ionization analysis technique based on the vaporization of a nanostructure-trapped liquid "initiator" phase. Here we report an intriguing relationship between NIMS surface morphology and analyte selectivity. Scanning electron microscopy and spectroscopic ellipsometry were used to characterize the surface morphologies of a series of NIMS substrates generated by anodic electrochemical etching. Mass spectrometry imaging was applied to compare NIMS sensitivity of these various surfaces toward the analysis of diverse analytes. The porosity of NIMS surfaces was found to increase linearly with etching time where the pore size ranged from 4 to 12 nm with corresponding porosities estimated to be 7-70%. Surface morphology was found to significantly and selectively alter NIMS sensitivity. The small molecule (<2k Da) sensitivity was found to increase with increased porosity, whereas low porosity had the highest sensitivity for the largest molecules examined. Estimation of molecular sizes showed that this transition occurs when the pore size is <3× the maximum of molecular dimensions. While the origins of selectivity are unclear, increased signal from small molecules with increased surface area is consistent with a surface area restructuring-driven desorption/ionization process where signal intensity increases with porosity. In contrast, large molecules show highest signal for the low-porosity and small-pore-size surfaces. We attribute this to strong interactions between the initiator-coated pore structures and large molecules that hinder desorption/ionization by trapping large molecules. This finding may enable us to design NIMS surfaces with increased specificity to molecules of interest.

An integrated metagenomic, metabolomic and transcriptomic survey of Populus across genotypes and environments.

Bridging molecular information to ecosystem-level processes would provide the capacity to understand system vulnerability and, potentially, a means for assessing ecosystem health. Here, we present an integrated dataset containing environmental and metagenomic information from plant-associated microbial communities, plant transcriptomics, plant and soil metabolomics, and soil chemistry and activity characterization measurements derived from the model tree species Populus trichocarpa. Soil, rhizosphere, root endosphere, and leaf samples were collected from 27 different P. trichocarpa genotypes grown in two different environments leading to an integrated dataset of 318 metagenomes, 98 plant transcriptomes, and 314 metabolomic profiles that are supported by diverse soil measurements. This expansive dataset will provide insights into causal linkages that relate genomic features and molecular level events to system-level properties and their environmental influences.

OpenMSI: a high-performance web-based platform for mass spectrometry imaging.

Mass spectrometry imaging (MSI) enables researchers to directly probe endogenous molecules directly within the architecture of the biological matrix. Unfortunately, efficient access, management, and analysis of the data generated by MSI approaches remain major challenges to this rapidly developing field. Despite the availability of numerous dedicated file formats and software packages, it is a widely held viewpoint that the biggest challenge is simply opening, sharing, and analyzing a file without loss of information. Here we present OpenMSI, a software framework and platform that addresses these challenges via an advanced, high-performance, extensible file format and Web API for remote data access (http://openmsi.nersc.gov). The OpenMSI file format supports storage of raw MSI data, metadata, and derived analyses in a single, self-describing format based on HDF5 and is supported by a large range of analysis software (e.g., Matlab and R) and programming languages (e.g., C++, Fortran, and Python). Careful optimization of the storage layout of MSI data sets using chunking, compression, and data replication accelerates common, selective data access operations while minimizing data storage requirements and are critical enablers of rapid data I/O. The OpenMSI file format has shown to provide >2000-fold improvement for image access operations, enabling spectrum and image retrieval in less than 0.3 s across the Internet even for 50 GB MSI data sets. To make remote high-performance compute resources accessible for analysis and to facilitate data sharing and collaboration, we describe an easy-to-use yet powerful Web API, enabling fast and convenient access to MSI data, metadata, and derived analysis results stored remotely to facilitate high-performance data analysis and enable implementation of Web based data sharing, visualization, and analysis.

"Replica-extraction-transfer" nanostructure-initiator mass spectrometry imaging of acoustically printed bacteria.

Traditionally, microbes are studied under controlled laboratory conditions as isolates in planktonic culture. However, this is a vast extrapolation from their natural state; development of new techniques is required to decipher the largely unknown world of microbial chemical interactions in more realistic environments. The field of mass spectrometry imaging has made significant progress in localizing metabolites in and around bacterial colonies, primarily by using MALDI and ESI-based techniques that interrogate the top surface of the sample. Unfortunately, surface-based laser-desorption techniques, such as nanostructure-initiator mass spectrometry (NIMS), which has advantages in detection of small metabolite compounds and low background, has not been suitable for direct microbe imaging because desorption/ionization occurs on the bottom of the sample. Here, we describe a "replica-extraction-transfer" (REX) technique that overcomes this barrier by transferring biomolecules from agar cultures of spatially arrayed bacterial colonies onto NIMS surfaces; further, we demonstrate that acoustic printing of bacteria can be used to create complex colony geometries to probe microbial interactions with NIMS imaging. REX uses a solvent-laden semisolid (e.g., gel) to first extract metabolites from a microbial sample, such as a biofilm or agar culture; the metabolites are then replica "stamped" onto the NIMS surface. Using analytical standards we show that REX-NIMS effectively transfers and detects a range of small molecule compounds including amino acids and polyamines. This approach is then used to analyze the metabolite composition of streaked Shewanella oneidensis MR1 and Pseudomonas stutzeri RCH2 colonies and further resolve complex patterns produced by acoustic printing of liquid microbial cultures. Applying multivariate statistical analysis of the NIMS imaging data identified ions that were localized to different regions between and within colonies, as well as to the agar gel. Subsequent high-resolution tandem mass spectrometry was used to characterize two species-specific lipids that correlated with the spatial location of each microbial species and were found to be highly abundant in cell extracts. Overall, the use of acoustic printing of bacteria with REX-NIMS imaging will extend the range of analytical capabilities available for characterization of microbial interactions with mass spectrometry.

A standardized quantitative analysis strategy for stable isotope probing metagenomics.

Stable isotope probing (SIP) facilitates culture-independent identification of active microbial populations within complex ecosystems through isotopic enrichment of nucleic acids. Many DNA-SIP studies rely on 16S rRNA gene sequences to identify active taxa, but connecting these sequences to specific bacterial genomes is often challenging. Here, we describe a standardized laboratory and analysis framework to quantify isotopic enrichment on a per-genome basis using shotgun metagenomics instead of 16S rRNA gene sequencing. To develop this framework, we explored various sample processing and analysis approaches using a designed microbiome where the identity of labeled genomes and their level of isotopic enrichment were experimentally controlled. With this ground truth dataset, we empirically assessed the accuracy of different analytical models for identifying active taxa and examined how sequencing depth impacts the detection of isotopically labeled genomes. We also demonstrate that using synthetic DNA internal standards to measure absolute genome abundances in SIP density fractions improves estimates of isotopic enrichment. In addition, our study illustrates the utility of internal standards to reveal anomalies in sample handling that could negatively impact SIP metagenomic analyses if left undetected. Finally, we present SIPmg, an R package to facilitate the estimation of absolute abundances and perform statistical analyses for identifying labeled genomes within SIP metagenomic data. This experimentally validated analysis framework strengthens the foundation of DNA-SIP metagenomics as a tool for accurately measuring the in situ activity of environmental microbial populations and assessing their genomic potential. IMPORTANCE Answering the questions, "who is eating what?" and "who is active?" within complex microbial communities is paramount for our ability to model, predict, and modulate microbiomes for improved human and planetary health. These questions can be pursued using stable isotope probing to track the incorporation of labeled compounds into cellular DNA during microbial growth. However, with traditional stable isotope methods, it is challenging to establish links between an active microorganism's taxonomic identity and genome composition while providing quantitative estimates of the microorganism's isotope incorporation rate. Here, we report an experimental and analytical workflow that lays the foundation for improved detection of metabolically active microorganisms and better quantitative estimates of genome-resolved isotope incorporation, which can be used to further refine ecosystem-scale models for carbon and nutrient fluxes within microbiomes.

Synthase-selected sorting approach identifies a beta-lactone synthase in a nudibranch symbiotic bacterium.

BACKGROUND: Nudibranchs comprise a group of > 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva. RESULTS: We recovered the genome of Candidatus Doriopsillibacter californiensis from the Ca. Tethybacterales order, an uncultured lineage of sponge symbionts not found in nudibranchs previously. It forms part of the core skin microbiome of D. fulva and is nearly absent in its internal organs. We showed that crude extracts of D. fulva contained secondary metabolites that were consistent with the presence of a beta-lactone encoded in Ca. D. californiensis genome. Beta-lactones represent an underexplored group of secondary metabolites with pharmaceutical potential that have not been reported in nudibranchs previously. CONCLUSIONS: Altogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo. Video Abstract.

Thermophilic Dehalococcoidia with unusual traits shed light on an unexpected past.

Although the phylum Chloroflexota is ubiquitous, its biology and evolution are poorly understood due to limited cultivability. Here, we isolated two motile, thermophilic bacteria from hot spring sediments belonging to the genus Tepidiforma and class Dehalococcoidia within the phylum Chloroflexota. A combination of cryo-electron tomography, exometabolomics, and cultivation experiments using stable isotopes of carbon revealed three unusual traits: flagellar motility, a peptidoglycan-containing cell envelope, and heterotrophic activity on aromatics and plant-associated compounds. Outside of this genus, flagellar motility has not been observed in Chloroflexota, and peptidoglycan-containing cell envelopes have not been described in Dehalococcoidia. Although these traits are unusual among cultivated Chloroflexota and Dehalococcoidia, ancestral character state reconstructions showed flagellar motility and peptidoglycan-containing cell envelopes were ancestral within the Dehalococcoidia, and subsequently lost prior to a major adaptive radiation of Dehalococcoidia into marine environments. However, despite the predominantly vertical evolutionary histories of flagellar motility and peptidoglycan biosynthesis, the evolution of enzymes for degradation of aromatics and plant-associated compounds was predominantly horizontal and complex. Together, the presence of these unusual traits in Dehalococcoidia and their evolutionary histories raise new questions about the timing and selective forces driving their successful niche expansion into global oceans.

Acoustic deposition with NIMS as a high-throughput enzyme activity assay.

Mass spectrometry (MS)-based enzyme assay has been shown to be a useful tool for screening enzymatic activities from environmental samples. Recently, reported approaches for high-specificity multiplexed characterization of enzymatic activities allow for providing detailed information on the range of enzymatic products and monitoring multiple enzymatic reactions. However, the throughput has been limited by the slow liquid-liquid handling and manual analysis. This rapid communication demonstrates the integration of acoustic sample deposition with nanostructure initiator mass spectrometry (NIMS) imaging to provide reproducible measurements of multiple enzymatic reactions at a throughput that is tenfold to 100-fold faster than conventional MS-based enzyme assay. It also provides a simple means for the visualization of multiple reactions and reaction pathways.