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Single-cell stable isotope probing in microbial ecology

Abstract Environmental and host-associated microbiomes are typically diverse assemblages of organisms performing myriad activities and engaging in a network of interactions that play out in spatially structured contexts. As the sum of these activities and interactions give rise to overall microbiome function, with important consequences for environmental processes and human health, elucidating specific microbial activities within complex communities is a pressing challenge. Single-cell stable isotope probing (SC-SIP) encompasses multiple techniques that typically utilize Raman microspectroscopy or nanoscale secondary ion mass spectrometry (NanoSIMS) to enable spatially resolved tracking of isotope tracers in cells, cellular components, and metabolites. SC-SIP techniques are uniquely suited for illuminating single-cell activities in microbial communities and for testing hypotheses about cellular functions generated for example from meta-omics datasets. Here, we illustrate the insights enabled by SC-SIP techniques by reviewing selected applications in microbiology and offer a perspective on their potential for future research.

Alcolombri, Uria (ORCID:0000000335615091)↗

GROWdb US River Systems - Samples

GROW Overview We developed the Genome Resolved Open Watersheds database (GROWdb), which aims to increase genomic sampling and understanding of global river microbiomes. An emphasis of GROWdb is to create a publicly available and ever-expanding microbial genome database that is focused on rivers while being interoperable with databases from other ecosystems. GROWdb is based on a network-of-networks approach to move beyond a small collection of well-studied rivers, towards a spatially distributed, global network of systematic observations. GROWdb represents the first microbial, river-focused resource parsed at various scales from genes to MAGs to community level including expression and potential based measurements that will be of interest to microbiologists, ecologists, geochemists, hydrologists, and modelers. Dataset Acknowledgement GROWdb contains data from various research campaigns, please acknowledge the following data generators, as appropriate: WHONDRS derived genomes or samples - include this statement in your acknowledgements: “This study used data from the Worldwide Hydrobiogeochemistry Observation Network for Dynamic River Systems (WHONDRS) under the River Corridor Science Focus Area (SFA) at the Pacific Northwest National Laboratory (PNNL) that was generated at the U.S. Department of Energy (DOE) Joint Genome Institute User Facility. PNNL is operated by Battelle Memorial Institute for the U.S. DOE under Contract No. DE-AC05-76RL01830. The SFA is supported by the U.S. DOE, Office of Biological and Environmental Research (BER), Environmental System Science (ESS) Program.” Total Samples loaded onto this Narrative: 178 Note: Not all GROW samples may be loaded into KBase Data Availability The data underlying GROWdb are accessible across various platforms to ensure all levels of data structure are widely available. First, all reads and MAGs are publicly hosted on National Center for Biotechnology (NCBI) under Bioproject PRJNA946291. Second, all data related data presented here including MAG annotations, extended data tables, phylogenetic tree files, antibiotic resistance gene database files, and MAG abundance tables are available in Zenodo (link). Beyond the flat database files listed above, our aim for GROWdb was to maximize data use by making the data available in searchable and interactive platforms including the National Microbiome Data Collaborative (NMDC) data portal, the Department of Energy’s Systems Biology Knowledgebase (KBase), and a GROW specific user interface released here, GROWdb Explorer. Each platform provides different ways to interact with GROWdb: NMDC GROWdb formed a pilot project for the NMDC. Specifically, individual GROWdb datasets (metagenomes, metatranscriptomes, etc) are easily accessible and searchable through the NMDC data portal, where they are systematically connected to each other and to a rich suite of sample information and standard analysis results, following Findable, Accessible, Interoperable, and Reusable (FAIR) data practices. KBase GROWdb is publicly available within KBase, including samples (this Narrative), MAGs, and corresponding genome scale metabolic models. Access within KBase allows for immediate access and reuse of data, including comparison to private data using KBase’s 500+ analysis tools. Other linked narratives in KBase: GROW Metagenome Assembled Genomes (MAGs) GROW Metabolic Models GROWdb Explorer GROWdb data is also explorable through a graphical user interface built through the Colorado State University Geospatial Centroid (https://geocentroid.shinyapps.io/GROWdatabase/), allowing users to search and graph microbial and spatial data simultaneously. In summary, this microbial genome resource represents the first publicly available genome collection from rivers and offers data that can be leveraged across microbiome studies. GROWdb is an expanding repository to incorporate and unify global river multi-omic data for the future.

59 BASIC BIOLOGICAL SCIENCES↗

Protein N -Glycans in Healthy and Sclerotic Glomeruli in Diabetic Kidney Disease

Diabetes is expected to directly affect renal glycosylation; yet to date, there has not been a comprehensive evaluation of alterations in N-glycan composition in the glomeruli of patients with diabetic kidney disease (DKD). Here, we used untargeted mass spectrometry imaging to identify N-glycan structures in healthy and sclerotic glomeruli in formalin-fixed paraffin-embedded sections from needle biopsies of five patients with DKD and three healthy kidney samples. Regional proteomics was performed on glomeruli from additional biopsies from the same patients to compare the abundances of enzymes involved in glycosylation. Secondary analysis of single-nucleus RNA sequencing (snRNAseq) data were used to inform on transcript levels of glycosylation machinery in different cell types and states. We detected 120 N-glycans, and among them, we identified 12 of these protein post-translated modifications that were significantly increased in glomeruli. All glomeruli-specific N-glycans contained an N-acetyllactosamine epitope. Five N-glycan structures were highly discriminant between sclerotic and healthy glomeruli. Sclerotic glomeruli had an additional set of glycans lacking fucose linked to their core, and they did not show tetra-antennary structures that were common in healthy glomeruli. Orthogonal omics analyses revealed lower protein abundance and lower gene expression involved in synthesizing fucosylated and branched N-glycans in sclerotic podocytes. In snRNAseq and regional proteomics analyses, we observed that genes and/or proteins involved in sialylation and N-acetyllactosamine synthesis were also downregulated in DKD glomeruli, but this alteration remained undetectable by our spatial N-glycomics assay. Integrative spatial glycomics, proteomics, and transcriptomics revealed protein N-glycosylation characteristic of sclerotic glomeruli in DKD.

60 APPLIED LIFE SCIENCES↗

A multimodal and integrated approach to interrogate human kidney biopsies with rigor and reproducibility: guidelines from the Kidney Precision Medicine Project

Comprehensive and spatially mapped molecular atlases of organs at a cellular level are a critical resource to gain insights into pathogenic mechanisms and personalized therapies for diseases. The Kidney Precision Medicine Project (KPMP) is an endeavor to generate three-dimensional (3-D) molecular atlases of healthy and diseased kidney biopsies by using multiple state-of-the-art omics and imaging technologies across several institutions. Obtaining rigorous and reproducible results from disparate methods and at different sites to interrogate biomolecules at a single-cell level or in 3-D space is a significant challenge that can be a futile exercise if not well controlled. Here we describe a “follow the tissue” pipeline for generating a reliable and authentic single-cell/region 3-D molecular atlas of human adult kidney. Our approach emphasizes quality assurance, quality control, validation, and harmonization across different omics and imaging technologies from sample procurement, processing, storage, shipping to data generation, analysis, and sharing. We established benchmarks for quality control, rigor, reproducibility, and feasibility across multiple technologies through a pilot experiment using common source tissue that was processed and analyzed at different institutions and different technologies. A peer review system was established to critically review quality control measures and the reproducibility of data generated by each technology before their being approved to interrogate clinical biopsy specimens. The process established economizes the use of valuable biopsy tissue for multiomics and imaging analysis with stringent quality control to ensure rigor and reproducibility of results and serves as a model for precision medicine projects across laboratories, institutions and consortia.

59 BASIC BIOLOGICAL SCIENCES↗

Documenting the function of non-cultivated microorganisms in terrestrial ecosystems

The terrestrial biosphere contains a large fraction of global carbon and nearly 70% of the organic carbon in these systems is found in soils. Global changes in atmospheric CO 2 , temperature, precipitation, and ecosystem N inputs, are expected to impact primary production and carbon inputs to soils, but it remains difficult to predict the response of soil processes to anthropogenic change. Models that predict soil C-cycling as a function of ecosystem properties do not explain well variation in soil processes. Our difficulty in predicting the response of soil processes to environmental change suggests a need for a greater understanding of the biotic mechanisms that govern the soil C-cycle. Changes in microbial community structure and function have been proposed to impact soil C-cycling. However, our ability to predict the impacts of these changes on terrestrial ecosystems is constrained by our limited understanding of mechanisms that drive microbial processes in soil systems. We have applied a newly developed approach of Microbial Food Web Mapping to chart the carbon assimilation dynamics of microbial taxa in soils. This method made it possible to track different classes of 13C-labeled substrates into the DNA of nearly every member of the soil community simultaneously over time. Microbial Food Web Mapping was used to identify and characterize microbial taxa that fill key roles in the soil carbon cycle. We mapped the assimilation of carbon into thousands of discrete soil microorganisms as a function of soil carbon content and pH. In so doing we characterized assimilatory carbon metabolism, an essential component of soil C-cycling, for a vast array of uncultivated soil microorganisms. Further, we characterized the spatial and temporal dynamics of these microorganisms across a series of sites representing variation in soil characteristics. This project revealed fundamental aspects of soil C-cycling and provided ecological and metabolic insights on diverse uncultivated soil organisms that are widespread and play major roles in the global C-cycle. The genetic capacity of microbial communities can be studied through ‘omic approaches but it remains difficult to make direct links between the genetic capacity of microorganisms and their function in the soil C-cycle. Microbial Food Web Mapping makes it possible to link gene sequences to soil C-cycle processes as they occur in soil. This approach allows us to characterize the activity of non-cultivated microorganisms in a range of terrestrial systems. This data will be used to build a base of information about the role of non-cultivated organisms in critical C-cycle processes in terrestrial ecosystems and will provide insight on the manner in which soil communities metabolize soil organic matter. The results generated by this project will improve our ability to examine the impacts of management decisions, soil history, and environmental change on the behavior of microbial communities in terrestrial ecosystems, revealing the ecological mechanisms by which microbes regulate both C mineralization and carbon retention in soils, and improving our ability to predict changes in terrestrial ecosystem processes in the face of accelerating global change.

54 ENVIRONMENTAL SCIENCES↗

DOE new players Carbon cycle (2016-2021)

The overarching scientific goals of our multidisciplinary grant was to further expand understanding about the key microorganisms (players), metabolic strategies (processes), and interspecies relationships (interactions) involved in the formation and oxidation of methane in the environment. This research applied novel environmental metagenomics, transcriptomics, and proteomic techniques, state-of-the-art analytical imaging, stable isotope geochemistry, and reaction-transport modeling to address these goals and develop an ‘ecosystems level’ understanding of the factors which regulate microbial methane cycling in anoxic sedimentary ecosystems. For decades, it was believed that the obligate step in methanogenesis catalyzed by methyl coenzyme M reductase (Mcr) was limited to a specific branch of the archaeal Domain, formerly known as the Euryarchaeota. Less than a decade ago co-I Tyson’s team published a surprising metagenomic-based discovery of divergent Mcr genes in a novel uncultured phylum (Bathyarchaeota), catalyzing a major shift in thinking about the diversity of microorganisms that encode the potential for methane (or higher alkane) metabolism in anoxic environments (Evans et al., 2015). In our work here, we further expand on the groups of archaea harboring the genomic potential for methane or hydrocarbon metabolism using environmental metagenomics and new gene targeted bioinformatics techniques. We additionally advanced understanding about the terminal electron acceptors and metabolic potential supporting the anaerobic oxidation of methane (AOM) in terrestrial ecosystems, specifically focused on new lineages of ANME archaea capable of respiring manganese oxides with methane presumably using large extracellular multi-heme cytochrome complexes. New details about specific syntrophic mechanisms underlying the exchange of electrons during sulfate-coupled methane oxidation between ANME-2 archaea and their sulfate-reducing bacterial partners were also elucidated as part of this funded project. Through a series of experimental ‘omics and single cell stable isotope probing studies with incubated environmental sediment samples and a cultured model electrogenic microorganism combined with model-based predictions. Combined, this work provides strong support for the hypothesis of direct interspecies electron transfer (DIET) is the dominant syntrophic mechanism controlling the anaerobic oxidation of methane with sulfate over other proposed mechanisms and additionally illustrates important spatial constraints and the underlying physico-chemical factors influencing AOM syntrophic consortia structure for DIET and extracellular metal respiration. This collaborative multi-institutional project successfully advanced several of our milestone goals including the identification of new microbial players containing methyl coenzyme M reductases hypothesized to be central to methane or hydrocarbon cycling in anoxic environments and enhancing fundamental knowledge about the role extracellular electron transfer plays in the ecophysiology of methanotrophic archaea respiring metal oxides and in the physical and metabolic structuring of syntrophic interactions in methane-rich sedimentary ecosystems.

03 NATURAL GAS↗

Single-Cell Metabolomics with Rapid Determination of Chemical Formulas from Isotopic Fine Structures

Metabolomic measurements can provide functional readouts of cellular states and phenotypes. Here, we present a protocol for single-cell metabolomics that permits direct untargeted detection of a broad number of metabolites under ambient conditions, without the need for sample processing, and with high confidence in the discovery and identification of the molecular formulas for detected metabolites. This protocol describes combining fiber-based laser ablation electrospray ionization (f-LAESI) with a 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer (21T-FTICR-MS) to obtain high confidence molecular formula information about detected metabolites. The f-LAESI source utilizes mid-infrared laser ablation through a sharp optical fiber tip, affording direct ambient analysis of cells without the need for sample processing. Using the 21T-FTICR-MS as a mass analyzer enabled measurement of the isotopic fine structure (IFS) for numerous metabolites simultaneously from single cells, and the IFSs were in turn computationally processed to rapidly determine the corresponding elemental compositions. This metabolomics technique complements other single cell omics measurement methods, helping to resolve complex molecular interactions that take place within cells unattainable from single cell transcriptomic and proteomics methods.

mass spectrometry, FTICR, FTMS, ultrahigh mass res↗

Accounting for hydrological and microbial processes on greenhouse gas budgets from river systems (Final Report)

River systems have a disproportionately large impact on carbon and nitrogen cycling on this planet. For instance, despite their much smaller size in area, it is estimated that global carbon dioxide emission from river systems exceed the emission from lakes and reservoirs, and are on par with the estimated oceanic and land carbon sinks. Closing carbon and nitrogen budgets in river systems requires knowledge of end member greenhouse gas (GHG) products (e.g. carbon dioxide, nitrous oxide, methane), which typically have not been measured in river systems. Moreover, while the production of these gasses is thought to be microbially derived, little is known about microbial activities in sediments of large rivers, despite their potentially high influence on biogeochemical budgets. Typically, when we think of rivers we visualize the channels that carve through the earth, but this only represents a small part of the hydrologic exchange that a river participates in. Rivers are more than conduits; they are dynamically exchanging water and materials with surrounding terrestrial and subsurface environments. In fact, 96% of the carbon dioxide produced within river systems occurs in hyporheic zones, or sediment regions beneath and alongside a river where groundwater and river water mix. In fact, it is widely recognized that microbial processes in hyporheic zones, also known as the river’s liver, strongly influence the fate of carbon, nutrients, and contaminants transported through watersheds. Despite this general knowledge, observational studies linking nitrogen and carbon gas production to microbial activity, and studies of overall gas emissions, are limited across river systems, but especially absent from larger rivers. To identify microbial processes responsible for GHG emissions from large rivers, and to quantify their environmental drivers and dependencies, we selected the Columbia River as a representative field site. This is the largest river west of the North American Continental Divide, and produces a drainage and discharge roughly double of the Nile in Egypt. This river is also an important source for agricultural irrigation and generates more hydroelectric power than any other North American river. Dams manage the release of water in the river, with these changes in river discharge patterns strongly impacting microbial activities in hyporheic zones. Specifically, we target a region down-river from the Priest Rapids Dam (WA) associated with the PNNL SFA, where water discharge from the dam is highly controlled, resulting in 1.5 m fluctuations in river stage daily. This dynamic river stage creates an ideal research opportunity for investigating hydrological perturbations in GHG production, uptake, and emission from large river systems. In this exploratory 1-year project, we conducted data-driven field research to provide observations needed to support predictions about GHG flux from river ecosystems. Using porewater dialysis and surface flux measurements, we observed where and when GHGs were produced and is the emission impacted by river stage. Using meta-omics tools, we determined what microbial processes control production and consumption of GHG in these systems. Finally, we provided key observations to parameterize models that could predict the effect of environmental conditions on GHG emission in the Columbia River near shore environment, at the scale that the modeled processes operate. Closing knowledge gaps in the linkages between river stage, microbial processes, and GHG production and emission, as well as the spatial distribution and ecosystem dependencies of these processes, is necessary to better parameterize land surface models. We provide some of the first, detailed GHG measurements paired microbial activity from large river systems. Our findings have direct relevance to many other large rivers with flows altered by hydroelectric energy, reservoirs, snowmelt, tides, or agriculture.

54 ENVIRONMENTAL SCIENCES↗

Elucidation of Aromatic Catabolic Pathways in White-Rot Fungi

This project aims to investigate the hypothesis that white-rot fungi can simultaneously depolymerize lignin extracellularly and catabolize depolymerization products intracellularly as carbon and energy sources. Evaluating this hypothesis will provide deeper understanding of the role of white-rot fungi in facilitating carbon sequestration in Nature. Additionally, identifying the most promising fungal strains for lignin turnover and catabolism will catalyze future efforts in genetic tool development to enable metabolic engineering in white-rot fungi for lignin bioconversion to bioproducts. Lignin is the second most abundant plant-based biopolymer on Earth and represents up to 40% of the energy density of lignocellulosic biomass. Even though lignin is a massive natural carbon and energy reservoir, only a small group of basidiomycete fungi, namely white-rot fungi (WRF), have evolved the ability to efficiently depolymerize and mineralize lignin to CO2 and H2O. Considerable research efforts have been undertaken to understand how WRF depolymerize lignin but the biochemical reactions that convert lignin into CO2 have been largely neglected. In fact, it is unclear if WRF intracellularly catabolize lignin-derived aromatic compounds to utilize them as a carbon and energy source, or rather if lignin is depolymerized and mineralized extracellularly merely to facilitate access to cellulose and hemicellulose for use as a primary carbon source. To date, we have employed 13C-isotope labeling, systems biology approaches, and in vitro enzyme assays to definitively demonstrate that two WRF, Trametes versicolor and Gelatoporia (Ceriporiopsis) subvermispora, funnel carbon from lignin-derived aromatic compounds into central carbon metabolism via intracellular catabolic pathways [1]. Specifically, 13C-isotopic labeling approaches showed that these WRF utilize poplar-derived aromatic compounds (e.g. 4-hydroxybenzoic acid (4-HBA)) as a carbon source. In silico genome analysis led us to hypothesize a complete catabolic pathway for 4-HBA and identify multiple homologous sequences for enzymes with putative oxidative decarboxylase, hydroxylase, and ring-opening dioxygenase activities, which are among the main biochemical reactions acting on aromatic compounds. Spatial and differential proteomic and metabolomic analyses supported the proposed catabolic pathways and showed alternative catabolic steps in T. versicolor that were not present in G. subvermispora. Based on the in silico, proteomics, and transcriptomics results, we down-selected enzymes for further in vitro characterization, and we have assigned a function to six fungal enzymes (including oxidative decarboxylases, hydroxylases, and ring-opening dioxygenases). Interestingly, even though we selected homologous enzyme pairs from both WRF with similar -omics trends, in a few cases only one of the studied fungi showed activity for the proposed substrate. Based on all the observations from this study [1], we hypothesized that 4-HBA preferentially undergoes oxidative decarboxylation to hydroquinone and subsequent hydroxylation to 1,2,4-benzenetriol in G. subvermispora before ring cleavage, whereas 4-HBA would preferentially undergo hydroxylation to protocatechuate and further oxidative decarboxylation to 1,2,4-benzenetriol in T. versicolor. Examining additional decarboxylases and hydroxylases as well as enzymes from other protein families that can perform the same or similar oxidative reactions, such as cytochromes P450 with aromatic hydroxylation activity, will also be key for elucidating enzyme preferences for specific substrates. Overall, this work forms the foundation of a new research area based on lignin catabolism by WRF, which could be further exploited to convert the undervalued biopolymer lignin into value-added compounds.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗

KS4A-Omics1.0_FspDS682

Soil fungi facilitate the translocation of inorganic nutrients from soil minerals to other microorganisms and plants. This ability is particularly advantageous in impoverished soils, because fungal mycelial networks can bridge otherwise spatially disconnected and inaccessible nutrient hotspots. However, the molecular mechanisms underlying fungal mineral weathering and transport through soil remains poorly understood. Here, we addressed this knowledge gap by directly visualizing nutrient acquisition and transport through fungal hyphae in a mineral doped soil micromodel using a multimodal imaging approach. Here, we observed how a representative of common saprotrophic soil fungi, Fusarium sp. DS 682, exhibited a mechanosensory response (thigmotropism) around obstacles and through pore spaces (~12 μm) in the presence of minerals.This study establishes the significance of fungal biology and nutrient translocation mechanisms in maintaining fungal growth under water and nutrient limitations in a soil-like microenvironment, using a high-throughput multi-omic analysis approach. Data package KS4A-Omics.1.0_FspDS682 (Publication: Fungal Mineral Weathering Mechanisms Revealed Through Direct Molecular Visualization) contents reported here are the first version (1.0) and contain pre- and post-processed data using high throughput data capture technologies for multi-omic analysis and integration, this data package contains raw and post-processed experimental data for X-Ray Absorption Near Edge Structure Spectroscopy (XANES/XRF), Optical Microscopy, Proteomics, Scanning Electron Microscope (SEM), Time-of-Flight Secondary Ion Mass Spectroscopy (ToF-SIMS), X-Ray Diffraction Spectroscopy (XRD) files, and X- Ray Photoelectron Spectroscopy (XPS) using EMSL capabilities. This data package DOI contains a comprehensive collection of high-throughput multi-omics data and process metadata catalog. Support files include additional data download contents “Read Me” with dataset descriptor information and data source method application ontologies (see data dictionary section). Reported data download content is structured for compliance with reported guidelines provided by community standard initiatives and publisher stakeholder policies supporting FAIR data principles.

47 OTHER INSTRUMENTATION↗

Soil gas probes for monitoring trace gas messengers of microbial activity

Abstract Soil microbes vigorously produce and consume gases that reflect active soil biogeochemical processes. Soil gas measurements are therefore a powerful tool to monitor microbial activity. Yet, the majority of soil gases lack non-disruptive subsurface measurement methods at spatiotemporal scales relevant to microbial processes and soil structure. To address this need, we developed a soil gas sampling system that uses novel diffusive soil probes and sample transfer approaches for high-resolution sampling from discrete subsurface regions. Probe sampling requires transferring soil gas samples to above-ground gas analyzers where concentrations and isotopologues are measured. Obtaining representative soil gas samples has historically required balancing disruption to soil gas composition with measurement frequency and analyzer volume demand. These considerations have limited attempts to quantify trace gas spatial concentration gradients and heterogeneity at scales relevant to the soil microbiome. Here, we describe our new flexible diffusive probe sampling system integrated with a modified, reduced volume trace gas analyzer and demonstrate its application for subsurface monitoring of biogeochemical cycling of nitrous oxide (N 2 O) and its site-specific isotopologues, methane, carbon dioxide, and nitric oxide in controlled soil columns. The sampling system observed reproducible responses of soil gas concentrations to manipulations of soil nutrients and redox state, providing a new window into the microbial response to these key environmental forcings. Using site-specific N 2 O isotopologues as indicators of microbial processes, we constrain the dynamics of in situ microbial activity. Unlocking trace gas messengers of microbial activity will complement -omics approaches, challenge subsurface models, and improve understanding of soil heterogeneity to disentangle interactive processes in the subsurface biome.

04 OIL SHALES AND TAR SANDS↗

Comparative Genomics of Nitrogen Cycling Pathways in Bacteria and Archaea

Despite the explosion of metagenomic sequencing data, using -omics data to indicate environmental biogeochemistry remains a challenge. One or a few genes (referred to as marker genes) in a metabolic pathway of interest in meta-omic data are normally used to represent the prevalence of a biogeochemical reaction. This method often fails to demonstrate a consistent relationship between gene abundance and an ecosystem process rate. One reason this may occur is if a marker gene is not a good representative of a complete pathway. Here, we map the presence of 11 nitrogen (N)-cycling pathways in over 6000 complete bacterial and archaeal genomes using the Integrated Microbial Genomes database. Incomplete N-cycling pathways occurred in 39% of surveyed archaeal and bacterial species revealing a weakness in current marker-gene analyses. Moreover, we found that most organisms have limited ability to utilize inorganic N in multiple oxidation states. This suggests that inter-organism exchange of inorganic N compounds is common, highlighting the importance of both community composition and spatial structure in determining the extent of recycling versus loss in an ecosystem.

59 BASIC BIOLOGICAL SCIENCES↗

Metaoptics Enabled Multifunctional Imaging

Despite the fact that most microorganisms occupy two- and three-dimensional space in heterogeneous arrangements and in proximity to other microorganisms of different species, much of our current knowledge about their metabolic processes is derived from homogenizing, then analyzing, the chemical content of cultures containing a single microbial species. This project addressed this fundamental dichotomy by developing a set of optical imaging principles combining multiple new technologies. The imaging strategies developed in this project combine metaoptics structures with active in situ nanoscale control of the chemical environment and applied them to a microbial system, Myxococcus xanthus, with particular relevance to the DoE mission. The combination of metaoptical architectures and nanoscale control over the molecular environment enables: (a) precise control over the electromagnetic (EM) field at length scales smaller than the wavelength of light; (b) control of the interaction of the EM field with critical molecular systems in DOE relevant microbes; (c) control over the chemical environment – especially the presence and quantity of reactive oxygen species (ROS) that can affect redox homeostasis; and (d) the ability to ask new kinds of questions not accessible to ‘omics’ approaches or standard methods of biological imaging. These capabilities are applicable to detailed studies of metabolic pathways in microbes and to lignocellulosic biomass deconstruction. To accomplish these objectives, we pursued two over-arching technical goals: (1) the development of new metaoptics-enabled approaches to imaging and spectroscopic characterization; and (2) the development of tools to control the chemical environment of a microbial sample with nanometer-scale precision. Goal 1 was addressed through the design, fabrication, and characterization of new metasurfaces capable of super-resolution imaging through extreme confinement of the optical field. Goal 2 was addressed by controlling the redox potential on the nanoscale in microbial communities and characterizing their effect on intrinsic bacterial fluorophores which act as molecular sentinels and through characterization of soluble factors secreted by Myxococcus xanthus by confocal Raman imaging. The optical imaging/sensing approaches developed here make it possible to use these powerful new imaging and sensing modalities in metabolic studies by making it possible to visualize and track the spatial and temporal expression patterns of natural or engineered pathways in microorganisms.

09 BIOMASS FUELS↗

Development and refinement of an in situ ‘molecular microscope’ utilizing ultrahigh resolution mass spectrometry

The ability to elucidate complex metabolic behavior of living organisms in situ and with cellular resolution is a bioanalytical grand challenge that is yet to be realized. Mass spectrometry imaging (MSI) using laser ablation electrospray ionization mass spectrometry (LAESI) has recently been demonstrated, and holds promise for direct spatially resolved molecular profiling in a variety of living systems of DOE relevance (e.g., plant leaf, stem, and root tissue). The LAESI MSI approach has revealed cell-to-cell heterogeneity of metabolites within living plant tissue, in a multiplexed fashion and without the need for labeling. Cellular resolution by in situ, ambient pressure MSI methods are far from routine, however, and have inadequate spatial resolution for many biological questions of interest. We propose to develop next-generation LAESI approaches, using advanced fiber optics and transmission-based geometries, in efforts to attain dynamic bioanalysis of living systems at better than 20 µm lateral resolution. In parallel with LAESI innovations, attendant increases in sensitivity and dynamic range of the mass spectrometer system will also be necessary for detection of the limited amount of material being ionized. To respond to this need, a new, high magnetic field (21 Tesla) Fourier Transform Ion Cyclotron Resonance (FTICR) MS at the EMSL/PNNL will be coupled with the newly developed LAESI source. This mass spectrometer, currently being assembled, will be one of only two such systems in the world, and the only one in a DOE laboratory or user facility. Fundamentally, all key figures of merits of FTICR MS performance improve with increased magnetic field strength, providing the ability to achieve unparalleled molecular specificity (near unequivocal molecular constituent identification) at the highest possible mass resolving power and sensitivity. The work proposed here will greatly increase the utility of this FTICR MS instrument, providing unique capabilities to support BER-relevant science. The research plan is comprised of three tasks, two of which are directed by the academic leads (University of Missouri and George Washington University), and one of which will be performed in conjunction with the BER user facility at EMSL/PNNL. In task 1, advancements of the LAESI technology will be achieved, with oversight and leadership of Professor Akos Vertes of George Washington University. In task 2, incorporation of this ion source with EMSL’s new 21T FTICR MS system will be undertaken, supplemented by global omics measurements and calibrated against more conventional MSI platforms also available at EMSL (Drs. Pasa-Tolic, Anderton, and Koppenaal team). In the final task, the resultant innovations and advancements will be tested and validated by addressing important BER relevant science questions on model systems of interest under the direction of Professor Gary Stacey of the University of Missouri. This research follows on key recommendations of the recent BER “Research in Sustainable Bioenergy” report that, for example, stressed that “The design of sustainable biofuel systems requires knowledge about key plant-microbe-environment interactions that provide a range of ecosystem services.” This task will focus on validation of the instrumentation developed through this proposal, advertising this capability to the community through demonstration and, finally, discovering new biology relevant to the BER mission. This final objective will make use of Setaria viridis, a model C4 grass species, as well as switchgrass and sorghum, two promising biofuel crops. Specifically, we will demonstrate applicability to plant-microbe rhizosphere interactions focusing on the biological nitrogen fixation by monitoring metabolic changes in real-time with nutrient, growth condition, and other perturbations.

09 BIOMASS FUELS↗

Members of the Genus Methylobacter Are Inferred To Account for the Majority of Aerobic Methane Oxidation in Oxic Soils from a Freshwater Wetland

ABSTRACT Microbial carbon degradation and methanogenesis in wetland soils generate a large proportion of atmospheric methane, a highly potent greenhouse gas. Despite their potential to mitigate greenhouse gas emissions, knowledge about methane-consuming methanotrophs is often limited to lower-resolution single-gene surveys that fail to capture the taxonomic and metabolic diversity of these microorganisms in soils. Here our objective was to use genome-enabled approaches to investigate methanotroph membership, distribution, and in situ activity across spatial and seasonal gradients in a freshwater wetland near Lake Erie. 16S rRNA gene analyses demonstrated that members of the methanotrophic Methylococcales were dominant, with the dominance largely driven by the relative abundance of four taxa, and enriched in oxic surface soils. Three methanotroph genomes from assembled soil metagenomes were assigned to the genus Methylobacter and represented the most abundant methanotrophs across the wetland. Paired metatranscriptomes confirmed that these Old Woman Creek (OWC) Methylobacter members accounted for nearly all the aerobic methanotrophic activity across two seasons. In addition to having the capacity to couple methane oxidation to aerobic respiration, these new genomes encoded denitrification potential that may sustain energy generation in soils with lower dissolved oxygen concentrations. We further show that Methylobacter members that were closely related to the OWC members were present in many other high-methane-emitting freshwater and soil sites, suggesting that this lineage could participate in methane consumption in analogous ecosystems. This work contributes to the growing body of research suggesting that Methylobacter may represent critical mediators of methane fluxes in freshwater saturated sediments and soils worldwide. IMPORTANCE Here we used soil metagenomics and metatranscriptomics to uncover novel members within the genus Methylobacter . We denote these closely related genomes as members of the lineage OWC Methylobacter . Despite the incredibly high microbial diversity in soils, here we present findings that unexpectedly showed that methane cycling was primarily mediated by a single genus for both methane production (“ Candidatus Methanothrix paradoxum”) and methane consumption (OWC Methylobacter ). Metatranscriptomic analyses revealed that decreased methanotrophic activity rather than increased methanogenic activity possibly contributed to the greater methane emissions that we had previously observed in summer months, findings important for biogeochemical methane models. Although members of this Methylococcales order have been cultivated for decades, multi-omic approaches continue to illuminate the methanotroph phylogenetic and metabolic diversity harbored in terrestrial and marine ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗