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CeMbio - The Caenorhabditis elegans Microbiome Resource

The study of microbiomes by sequencing has revealed a plethora of correlations between microbial community composition and various life-history characteristics of the corresponding host species. However, inferring causation from correlation is often hampered by the sheer compositional complexity of microbiomes, even in simple organisms. Synthetic communities offer an effective approach to infer cause-effect relationships in host-microbiome systems. Yet the available communities suffer from several drawbacks, such as artificial (thus non-natural) choice of microbes, microbe-host mismatch (e.g., human microbes in gnotobiotic mice), or hosts lacking genetic tractability. Here we introduce CeMbio, a simplified natural Caenorhabditis elegans microbiota derived from our previous meta-analysis of the natural microbiome of this nematode. The CeMbio resource is amenable to all strengths of the C. elegans model system, strains included are readily culturable, they all colonize the worm gut individually, and comprise a robust community that distinctly affects nematode life-history. Several tools have additionally been developed for the CeMbio strains, including diagnostic PCR primers, completely sequenced genomes, and metabolic network models. With CeMbio, we provide a versatile resource and toolbox for the in-depth dissection of naturally relevant host-microbiome interactions in C. elegans.

59 BASIC BIOLOGICAL SCIENCES↗

Bioactive exometabolites drive maintenance competition in simple bacterial communities

During prolonged resource limitation, bacterial cells can persist in metabolically active states of non-growth. These maintenance periods, such as those experienced in stationary phase, can include upregulation of secondary metabolism and release of exometabolites into the local environment. As resource limitation is common in many environmental microbial habitats, we hypothesized that neighboring bacterial populations employ exometabolites to compete or cooperate during maintenance and that these exometabolite-facilitated interactions can drive community outcomes. Here, we evaluated the consequences of exometabolite interactions over the stationary phase among three environmental strains: Burkholderia thailandensis E264, Chromobacterium subtsugae ATCC 31532, and Pseudomonas syringae pv. tomato DC3000. We assembled them into synthetic communities that only permitted chemical interactions. We compared the responses (transcripts) and outputs (exometabolites) of each member with and without neighbors. We found that transcriptional dynamics were changed with different neighbors and that some of these changes were coordinated between members. The dominant competitor B. thailandensis consistently upregulated biosynthetic gene clusters to produce bioactive exometabolites for both exploitative and interference competition. These results demonstrate that competition strategies during maintenance can contribute to community-level outcomes. It also suggests that the traditional concept of defining competitiveness by growth outcomes may be narrow and that maintenance competition could be an additional or alternative measure.

59 BASIC BIOLOGICAL SCIENCES↗

Impact of inoculation practices on microbiota assembly and community stability in a fabricated ecosystem

Studying plant-microbe-soil interactions is challenging due to their high complexity and variability in natural ecosystems. While fabricated ecosystems provide opportunities to recapitulate aspects of these systems in reduced complexity and controlled environments, inoculation can be a significant source of variation. To tackle this, we evaluated how different bacteria inoculation practices and plant harvesting time points affect the reproducibility of a microbial synthetic community (SynCom) in association with the model grass Brachypodium distachyon. We tested three microbial inoculation practices: seed inoculation, transplant inoculation, and seedling inoculation; and two harvesting points: early (14-day-old plants) and late (21 days post-inoculation). We grew our plants and bacterial strains in sterile devices (EcoFABs) and characterized the microbial community from root, rhizosphere, and sand using 16S ribosomal RNA gene sequencing. The results showed that inoculation practices significantly affected the rhizosphere microbial community only when harvesting at an early time point but not at the late stage. As the SynCom showed a persistent association with B. distachyon at 21 days post-inoculation regardless of inoculation practices, we assessed the reproducibility of each inoculation method and found that transplant inoculation showed the highest reproducibility. Moreover, plant biomass was not adversely affected by transplant inoculation treatment. We concluded that bacteria inoculation while transplanting coupled with a later harvesting time point gives the most reproducible microbial community in the EcoFAB-B. distachyon-SynCom fabricated ecosystem and recommend this method as a standardized protocol for use with fabricated ecosystem experimental systems.

59 BASIC BIOLOGICAL SCIENCES↗

Cultivating the Bacterial Microbiota of Populus Roots

Microbial communities play an integral role in the health and survival of their plant hosts. Many studies have identified key members in these communities and led to the use of synthetic communities for elucidating their function; however, these studies are limited by the available cultured bacterial representatives.

16S rRNA gene sequencing↗

Exploring the roles of microbes in facilitating plant adaptation to climate change

Plants benefit from their close association with soil microbes which assist in their response to abiotic and biotic stressors. Yet much of what we know about plant stress responses is based on studies where the microbial partners were uncontrolled and unknown. Under climate change, the soil microbial community will also be sensitive to and respond to abiotic and biotic stressors. Thus, facilitating plant adaptation to climate change will require a systems-based approach that accounts for the multi-dimensional nature of plant–microbe–environment interactions. In this perspective, we highlight some of the key factors influencing plant–microbe interactions under stress as well as new tools to facilitate the controlled study of their molecular complexity, such as fabricated ecosystems and synthetic communities. When paired with genomic and biochemical methods, these tools provide researchers with more precision, reproducibility, and manipulability for exploring plant–microbe–environment interactions under a changing climate.

59 BASIC BIOLOGICAL SCIENCES↗

Identifying Hot Spots and Hot Moments of Metabolic Activity in Salt Marsh Sediments through BONCAT-FISH Microscale Mapping (Final Technical Report)

Understanding the biogeography and timing of microbial metabolic activity is a key priority for microbial ecologists. With such knowledge, the cumulative biogeochemical contributions of microbial communities become more predictable, and our ability to both understand the effects of environmental change on microbial activity and build synthetic communities with desired functions will increase substantially. In this project, we advanced this broad, ambitious goal in two substantial ways: we developed a multiplexed Fluorescence In Situ Hybridization (FISH) approach that links microbial identity with metabolic function, and we established a novel Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) technique that can resolve the timing of anabolic activity, providing new resolution of when microbial constituents are growing. We deployed both of these techniques in multiple environmental settings to demonstrate their versatility. At the Little Sippewissett Salt Marsh, we combined a novel dual-BONCAT technique with fluorescence activated cell sorting to determine which population of cells was metabolically active during the day and which was active during the night. We found that Methylobacterium was active during daylight hours, potentially feeding on carbon-rich molecules released from plant roots. Sulfur-cycling microbes dominate the population active during the dark night-time hours. Overall, the work conducted under the auspices of this project developed two promising new techniques for identifying the “hot spots” and “hot moments” of microbial activity in complex communities with taxonomic and functional resolution. We focused largely on a salt marsh sediment context, but are confident that microbial ecologists seeking to understand the microbial role in biogeochemical cycles in a wide range of settings will find our newly developed techniques useful in future work.

54 ENVIRONMENTAL SCIENCES↗

MicroFisher: Fungal taxonomic classification for metatranscriptomic and metagenomic data using multiple short hypervariable markers

AbstractProfiling the taxonomic and functional composition of microbes using metagenomic (MG) and metatranscriptomic (MT) sequencing is advancing our understanding of microbial functions. However, the sensitivity and accuracy of microbial classification using genome– or core protein-based approaches, especially the classification of eukaryotic organisms, is limited by the availability of genomes and the resolution of sequence databases. To address this, we propose the MicroFisher, a novel approach that applies multiple hypervariable marker genes to profile fungal communities from MGs and MTs. This approach utilizes the hypervariable regions of ITS and large subunit (LSU) rRNA genes for fungal identification with high sensitivity and resolution. Simultaneously, we propose a computational pipeline (MicroFisher) to optimize and integrate the results from classifications using multiple hypervariable markers. To test the performance of our method, we applied MicroFisher to the synthetic community profiling and found high performance in fungal prediction and abundance estimation. In addition, we also used MGs from forest soil and MTs of root eukaryotic microbes to test our method and the results showed that MicroFisher provided more accurate profiling of environmental microbiomes compared to other classification tools. Overall, MicroFisher serves as a novel pipeline for classification of fungal communities from MGs and MTs.

Wang, Haihua↗

Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA) (Final Scientific/Technical Report, Subcontract Award No. 6953691)

The overarching objective the Baliga lab is to develop a mechanistic understanding of the field relevant metabolic processes underlying community partitioning of respiration pathways and physiological state shifts. In order to dissect how environmental perturbations influence microbial regulatory networks, we have constructed data-driven models that capture the dynamic interplay between abiotic and biotic factors during laboratory simulations. Specifically, we have developed multiple computational models (i.e., cMonkey2, EGRIN 2.0, and multiple genome-scale metabolic models) connecting environmental influence to genome-encoded regulatory programs. In addition, we have generated new tools such as Live Anaerobic Cell Sorting (LAnCS) and Fluidized Bed Reactors (FBRs) to investigate the dynamics of community partitioning of metabolic processes supporting sulfate or nitrate respiration, which are two critical activities observed from the field site at Oak Ridge National Labs (ORNL). This framework of connecting field phenomena with laboratory simulations and computational modeling has created the Environmental Simulations and Modelling (EnvSim) campaign within ENIGMA. The campaign, which spans multiple laboratories across ENIGMA and is led by the Baliga group, has established synthetic communities (SynComs) to discover, characterize, and dissect key microbial processes relevant to field observations, such as emissions of the greenhouse gas nitrous oxide (N 2 O). For example, the EnvSim campaign so far has deduced four potential mechanisms that may account for the N 2 O emissions at the FRC and are currently being investigated by labs across ENIGMA. Denitrification may be driven by complete denitrifiers, however, their NosZ enzymes, which catalyze the final step in denitrification by converting N 2 O to N 2 , may be sensitive at a lower pH. Additionally, the denitrification process could be partitioned among organisms and some may have pH-sensitive NosZ genes. Another factor contributing to variable N 2 O emissions relates to the metal co-factors involved in the denitrification pathway. For instance, excess Cu, Al, Mn, U, Ni, Co, Cu, and/or Cd may have inhibitory effects on multiple enzymatic steps during denitrification, while the enzymatic production of nitrite, the precursor of N 2 O, can be limited by the essential metal Mo. Lastly, abiotic production of N 2 O may occur as a result of chemodenitrification, in which metals like Fe, Mn, and some organic compounds can drive redox reactions that convert nitrogen cycle intermediates to N 2 O under the right conditions. Current investigations and analyses have generated multiple transcriptomic profiles aiding the refinement of new metabolic and gene regulatory network model for our denitrifying SynCom. In sum, the previous funding cycle has generated 33 peer-reviewed publications spanning predictive network biology to co-operativity of mutualistic interspecies interactions of evolved communities. Our integration of multiple omics datasets and the development of new network modelling tools has revealed important insights that can explain field observed phenomenon and generated new hypotheses to be investigated in the field.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Microfabrication of a Chamber for High-Resolution, In Situ Imaging of the Whole Root for Plant–Microbe Interactions

Fabricated ecosystems (EcoFABs) offer an innovative approach to in situ examination of microbial establishment patterns around plant roots using nondestructive, high-resolution microscopy. Previously high-resolution imaging was challenging because the roots were not constrained to a fixed distance from the objective. Here, we describe a new ‘Imaging EcoFAB’ and the use of this device to image the entire root system of growing Brachypodium distachyon at high resolutions (20×, 40×) over a 3-week period. The device is capable of investigating root–microbe interactions of multimember communities. We examined nine strains of Pseudomonas simiae with different fluorescent constructs to B. distachyon and individual cells on root hairs were visible. Succession in the rhizosphere using two different strains of P. simiae was examined, where the second addition was shown to be able to establish in the root tissue. The device was suitable for imaging with different solid media at high magnification, allowing for the imaging of fungal establishment in the rhizosphere. Overall, the Imaging EcoFAB could improve our ability to investigate the spatiotemporal dynamics of the rhizosphere, including studies of fluorescently-tagged, multimember, synthetic communities.

59 BASIC BIOLOGICAL SCIENCES↗

Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format

Plate-based proteomic sample preparation offers a solution to the large sample throughput demands in the biotechnology field where hundreds or thousands of engineered microbes are constructed for testing is routine. Meanwhile, sample preparation methods that work efficiently on broader microbial groups are desirable for new applications of proteomics in other fields, such as microbial communities. Here, we detail a step-by-step protocol that consists of cell lysis in an alkaline chemical buffer (NaOH/SDS) followed by protein precipitation with high-ionic strength acetone in 96-well format. The protocol works for a broad range of microbes ( e . g ., Gram-negative bacteria, Gram-positive bacteria, non-filamentous fungi) and the resulting proteins are ready for tryptic digestion for bottom-up quantitative proteomic analysis without the need for desalting column cleanup. The yield of protein using this protocol increases linearly with respect to the amount of starting biomass from 0.5–2.0 OD*mL of cells. By using a bench-top automated liquid dispenser, a cost-effective and environmentally-friendly option to eliminating pipette tips and reducing reagent waste, the protocol takes approximately 30 minutes to extract protein from 96 samples. Tests on mock mixtures showed expected results that the biomass composition structure is in close agreement with the experimental design. Lastly, we applied the protocol for the composition analysis of a synthetic community of environmental isolates grown on two different media. This protocol has been developed to facilitate rapid, low-variance sample preparation of hundreds of samples and allow flexibility for future protocol development.

59 BASIC BIOLOGICAL SCIENCES↗

Engineering microbial consortia with rationally designed cellular interactions

Synthetic microbial consortia represent a frontier of synthetic biology that promises versatile engineering of cellular functions. They are primarily developed through the design and construction of cellular interactions that coordinate individual dynamics and generate collective behaviors. Here we review recent advances in the engineering of synthetic communities through cellular-interaction programming. We first examine fundamental building blocks for intercellular communication and unidirectional positive and negative interactions. Here, we then recap the assembly of the building blocks for creating bidirectional interactions in two-species ecosystems, which is followed by the discussion of engineering toward complex communities with increasing species numbers, under spatial contexts, and via model-guided design. We conclude by summarizing major challenges and future opportunities of engineered microbial ecosystems.

42 ENGINEERING↗

Benchmarking second and third-generation sequencing platforms for microbial metagenomics

Shotgun metagenomic sequencing is a common approach for studying the taxonomic diversity and metabolic potential of complex microbial communities. Current methods primarily use second generation short read sequencing, yet advances in third generation long read technologies provide opportunities to overcome some of the limitations of short read sequencing. Here, we compared seven platforms, encompassing second generation sequencers (Illumina HiSeq 300, MGI DNBSEQ-G400 and DNBSEQ-T7, ThermoFisher Ion GeneStudio S5 and Ion Proton P1) and third generation sequencers (Oxford Nanopore Technologies MinION R9 and Pacific Biosciences Sequel II). We constructed three uneven synthetic microbial communities composed of up to 87 genomic microbial strains DNAs per mock, spanning 29 bacterial and archaeal phyla, and representing the most complex and diverse synthetic communities used for sequencing technology comparisons. Our results demonstrate that third generation sequencing have advantages over second generation platforms in analyzing complex microbial communities, but require careful sequencing library preparation for optimal quantitative metagenomic analysis. Our sequencing data also provides a valuable resource for testing and benchmarking bioinformatics software for metagenomics.

59 BASIC BIOLOGICAL SCIENCES↗

Cross-Feeding of a Toxic Metabolite in a Synthetic Lignocellulose-Degrading Microbial Community

The recalcitrance of complex organic polymers such as lignocellulose is one of the major obstacles to sustainable energy production from plant biomass, and the generation of toxic intermediates can negatively impact the efficiency of microbial lignocellulose degradation. Here, we describe the development of a model microbial consortium for studying lignocellulose degradation, with the specific goal of mitigating the production of the toxin formaldehyde during the breakdown of methoxylated aromatic compounds. Included are Pseudomonas putida, a lignin degrader; Cellulomonas fimi, a cellulose degrader; and sometimes Yarrowia lipolytica, an oleaginous yeast. Unique to our system is the inclusion of Methylorubrum extorquens, a methylotroph capable of using formaldehyde for growth. We developed a defined minimal “Model Lignocellulose” growth medium for reproducible coculture experiments. We demonstrated that the formaldehyde produced by P. putida growing on vanillic acid can exceed the minimum inhibitory concentration for C. fimi, and, furthermore, that the presence of M. extorquens lowers those concentrations. We also uncovered unexpected ecological dynamics, including resource competition, and interspecies differences in growth requirements and toxin sensitivities. Finally, we introduced the possibility for a mutualistic interaction between C. fimi and M. extorquens through metabolite exchange. This study lays the foundation to enable future work incorporating metabolomic analysis and modeling, genetic engineering, and laboratory evolution, on a model system that is appropriate both for fundamental eco-evolutionary studies and for the optimization of efficiency and yield in microbially-mediated biomass transformation.

Methylorubrum extorquens↗

Science and Solutions for a Planet Under Pressure: Synthetic Research, Analytics, and Community Engagement on Five Priority Theme Areas

Support from the Department of Energy proved instrumental in supporting the work of Global Council for Science and the Environment (GCSE) on synthetic research, analysis, and community engagement, oriented around the themes and discussions from the GCSE Drawdown 2021 Conference — Research to Action: Science and Solutions for a Planet Under Pressure. These efforts helped inform and enhance the societal relevance of the knowledge that has been generated through the conference and, at the same time, advance the scientific insights acquired through synthesis and scientific translation efforts. The work centered on three main objectives: 1) documentation of findings from the completed GCSE 2021, culminating in the publication of a synthesis report; 2) development and vetting of integrated use cases that illustrate synthesis methods and enable scientific outreach, communications, and translational discussions among universities and the broader research community; and 3) strengthening the scientific basis for energy education research by sharing findings in university-educator fora.

54 ENVIRONMENTAL SCIENCES↗

CRISPR-based engineering of phages for in situ bacterial base editing

Investigation of microbial gene function is essential to the elucidation of ecological roles and complex genetic interactions that take place in microbial communities. While microbiome studies have increased in prevalence, the lack of viable in situ editing strategies impedes experimental progress, rendering genetic knowledge and manipulation of microbial communities largely inaccessible. Here, we demonstrate the utility of phage-delivered CRISPR-Cas payloads to perform targeted genetic manipulation within a community context, deploying a fabricated ecosystem (EcoFAB) as an analog for the soil microbiome. First, we detail the engineering of two classical phages for community editing using recombination to replace nonessential genes through Cas9-based selection. We show efficient engineering of T7, then demonstrate the expression of antibiotic resistance and fluorescent genes from an engineered λ prophage within an Escherichia coli host. Next, we modify λ to express an APOBEC-1-based cytosine base editor (CBE), which we leverage to perform C-to-T point mutations guided by a modified Cas9 containing only a single active nucleolytic domain (nCas9). We strategically introduce these base substitutions to create premature stop codons in-frame, inactivating both chromosomal ( lacZ ) and plasmid-encoded genes (mCherry and ampicillin resistance) without perturbation of the surrounding genomic regions. Furthermore, using a multigenera synthetic soil community, we employ phage-assisted base editing to induce host-specific phenotypic alterations in a community context both in vitro and within the EcoFAB, observing editing efficiencies from 10 to 28% across the bacterial population. The concurrent use of a synthetic microbial community, soil matrix, and EcoFAB device provides a controlled and reproducible model to more closely approximate in situ editing of the soil microbiome.

59 BASIC BIOLOGICAL SCIENCES↗

Mixed and membrane-separated culturing of synthetic cyanobacteria-yeast consortia reveals metabolic cross-talk mimicking natural cyanolichens

Metabolite exchange mediates crucial interactions in microbial communities, significantly impacting global carbon and nitrogen cycling. Understanding these chemically-mediated interactions is essential for elucidating natural community functions and developing engineered synthetic communities. This study investigated membrane-separated bioreactors (mBRs) as a novel tool to identify transient metabolites and their producers/consumers in mixed microbial communities. We compared three co-culture methods (direct mixed, 2-chamber mBR, and 3-chamber mBR) to grow a synthetic binary community of the cyanobacterium Synechococcus elongatus PCC 7942 and the fungus Rhodotorula toruloides NBRC 0880, as well as axenic S. elongatus. Despite not being natural lichen constituents, these organisms exhibited interactions resembling those in cyanolichens. S. elongatus fixed CO 2 into sugars as the primary shared metabolite, while R. toruloides secreted various biochemicals, predominantly sugar alcohols, mirroring the metabolite exchange observed in natural lichens. The mBR systems successfully captured metabolite gradients and revealed rapidly consumed compounds, including TCA cycle intermediates and amino acids. Our approach demonstrated that the 2-chamber mBR optimally balanced metabolite exchange and growth dynamics. This study provides insights into cross-species metabolic interactions and presents a valuable tool for investigating and engineering synthetic microbial communities with potential applications in biotechnology and environmental science.

59 BASIC BIOLOGICAL SCIENCES↗

Using Methyl Bromide for Interspecies Cell–Cell Signaling and As a Reporter in a Model Soil Consortium

Synthetic microbial communities are emerging as model systems for studying consortia-scale phenotypes. To establish synthetic biology tools for studying these communities in hard-to-image environmental materials, we evaluated whether a single member of a model soil consortium (MSC) can be programmed to report on gene expression without requiring matrix disruption. For these studies, we targeted a five member MSC that includes Dyadobacter fermentans, Ensifer adhaerens, Rhodococcus sp003130705, Streptomyces sp001905665, and Variovorax beijingensis. By coupling the expression of a methyl halide transferase to a constitutive promoter, we show that Variovorax beijingensis can be programmed to synthesize methyl halides that accumulate in the soil headspace at levels that are ≥24-fold higher than all other MSC members across a range of environmentally-relevant hydration conditions. We find that methyl halide production can report on a MSC promoter that is activated by changes in water potential, and we demonstrate that a synthetic gas signal can be read out directly using gas chromatography and indirectly using a soil-derived Methylorubrum that is programmed to produce a visual output in response to methyl halides. Finally, these tools will be useful for future studies that investigate how MSC respond to dynamic hydration conditions, such as drought and flood events induced by climate change, which can alter soil water potential and induce the release of stored carbon.

59 BASIC BIOLOGICAL SCIENCES↗