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At least 19 records

Deep Learning Prediction of Interspecies Interactions from Self-organized Spatiotemporal Patterns of Co-evolving Organisms

Microorganisms colonizing natural habits such as soils co-evolve to form specific spatial patterns through interspecies interactions. These self-organized patterns are a key ecological phenotype, which provides critical information on their interaction mechanisms. However, conventional network inference techniques that analyze species population data in bulk have yet to be extended to account for such spatial heterogeneity. Here we proposed supervised deep learning as a new network inference tool for predicting interspecies interactions from spatiotemporal patterns of microbial evolution. Due to lack of biological imaging data that can be used for training deep learning networks, we used in silico data generated from high-fidelity agent-based models to determine model structure and parameters. Even though networks were trained under simple configurations where interaction coefficients are assumed to be spatially invariant, we demonstrated that the resulting model can be utilized to successfully predict spatial variation of interactions in more complex domains (i.e., configured with a context-dependent mixture of interaction coefficients) as well as in simple domains without further training. In the further test against real biological data obtained through imaging experiments of a binary consortium (Pseudomonas fluorescens and a mutant of Escherichia coli), our model also predicted the dramatic shifts in interactions of the two organisms across different environmental contexts. Through various successful demonstrations in this work, the combined use of the agent-based model and machine learning algorithm provides a means to use new type of data - microscopic images - for extracting microbial interactions, therefore presenting itself as a useful tool for the analysis of more complex microbial community interactions.

Lee, Joon-Yong↗

The transparent soil microcosm: a window into the spatial distribution and dynamics of carbon utilization and microbial interspecies interactions (Final Report)

This document contains the summary and final report for award DE-SC0013887 (The transparent soil microcosm: a window into the spatial distribution and dynamics of carbon utilization and microbial interspecies interactions), including the overall description of the accomplishments obtained during this work, the manuscripts published with its support, and the list of personnel funded to conduct this research.

59 BASIC BIOLOGICAL SCIENCES↗

Synergistic epistasis enhances the co-operativity of mutualistic interspecies interactions

Abstract Early evolution of mutualism is characterized by big and predictable adaptive changes, including the specialization of interacting partners, such as through deleterious mutations in genes not required for metabolic cross-feeding. We sought to investigate whether these early mutations improve cooperativity by manifesting in synergistic epistasis between genomes of the mutually interacting species. Specifically, we have characterized evolutionary trajectories of syntrophic interactions of Desulfovibrio vulgaris (Dv) with Methanococcus maripaludis (Mm) by longitudinally monitoring mutations accumulated over 1000 generations of nine independently evolved communities with analysis of the genotypic structure of one community down to the single-cell level. We discovered extensive parallelism across communities despite considerable variance in their evolutionary trajectories and the perseverance within many evolution lines of a rare lineage of Dv that retained sulfate-respiration (SR+) capability, which is not required for metabolic cross-feeding. An in-depth investigation revealed that synergistic epistasis across pairings of Dv and Mm genotypes had enhanced cooperativity within SR− and SR+ assemblages, enabling their coexistence within the same community. Thus, our findings demonstrate that cooperativity of a mutualism can improve through synergistic epistasis between genomes of the interacting species, enabling the coexistence of mutualistic assemblages of generalists and their specialized variants.

59 BASIC BIOLOGICAL SCIENCES↗

Probing interspecies metabolic interactions within a synthetic binary microbiome using genome-scale modeling

Metabolic interactions within a microbial community play a key role in determining the structure, function, and composition of the community. However, due to the complexity and intractability of natural microbiomes, limited knowledge is available on interspecies interactions within a community. In this work, using a binary synthetic microbiome, a methanotroph-photoautotroph (M-P) coculture, as the model system, we examined different genome-scale metabolic modeling (GEM) approaches to gain a better understanding of the metabolic interactions within the coculture, how they contribute to the enhanced growth observed in the coculture, and how they evolve over time. Using batch growth data of the model M-P coculture, we compared three GEM approaches for microbial communities. Two of the methods are existing approaches: SteadyCom, a steady state GEM, and dynamic flux balance analysis (DFBA) Lab, a dynamic GEM. We also proposed an improved dynamic GEM approach, DynamiCom, for the M-P coculture. SteadyCom can predict the metabolic interactions within the coculture but not their dynamic evolutions; DFBA Lab can predict the dynamics of the coculture but cannot identify interspecies interactions. DynamiCom was able to identify the cross-fed metabolite within the coculture, as well as predict the evolution of the interspecies interactions over time. A new dynamic GEM approach, DynamiCom, was developed for a model M-P coculture. Constrained by the predictions from a validated kinetic model, DynamiCom consistently predicted the top metabolites being exchanged in the M-P coculture, as well as the establishment of the mutualistic N-exchange between the methanotroph and cyanobacteria. The interspecies interactions and their dynamic evolution predicted by DynamiCom are supported by ample evidence in the literature on methanotroph, cyanobacteria, and other cyanobacteria-heterotroph cocultures.

59 BASIC BIOLOGICAL SCIENCES↗

Species-specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures

ABSTRACT The development of synthetic microbial consortia in recent years has revealed that complex interspecies interactions, notably the exchange of cytoplasmic material, exist even among organisms that originate from different ecological niches. Although morphogenetic characteristics, viable RNA and protein dyes, and fluorescent reporter proteins have played an essential role in exploring such interactions, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to further investigate interactions in synthetic or semisynthetic consortia. Despite its maturity, several challenges exist in using rRNA-FISH as a tool to quantify individual species population dynamics and interspecies interactions using high-throughput instrumentation such as flow cytometry. In this work, we resolve such challenges and apply rRNA-FISH to double and triple co-cultures of Clostridium acetobutylicum, Clostridium ljungdahlii, and Clostridium kluyveri . In pursuing our goal to capture each organism’s population dynamics, we demonstrate dynamic rRNA, and thus ribosome, exchange between the three species leading to the formation of hybrid cells. We also characterize the localization patterns of the translation machinery in the three species, identifying distinct, dynamic localization patterns among them. Our data also support the use of rRNA-FISH to assess the culture’s health and expansion potential, and, here again, our data find surprising differences among the three species examined. Taken together, our study argues for rRNA-FISH as a valuable and accessible tool for quantitative exploration of interspecies interactions, especially in organisms which cannot be genetically engineered or in consortia where selective pressures to maintain recombinant species cannot be used. IMPORTANCE Though dyes and fluorescent reporter proteins have played an essential role in identifying microbial species in co-cultures, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to quantitatively probe complex interactions between organisms in synthetic consortia. Despite its maturity, several challenges existed before rRNA-FISH could be used to study Clostridium co-cultures of interest. First, species-specific probes for Clostridium acetobutylicum and Clostridium ljungdahlii had not been developed. Second, “state-of-the-art” labeling protocols were tedious and often resulted in sample loss. Third, it was unclear if FISH was compatible with existing fluorescent reporter proteins. We resolved these key challenges and applied the technique to co-cultures of C. acetobutylicum , C. ljungdahlii , and Clostridium kluyveri . We demonstrate that rRNA-FISH is capable of identifying rRNA/ribosome exchange between the three organisms and characterized rRNA localization patterns in each. In combination with flow cytometry, rRNA-FISH can capture sub-population dynamics in co-cultures.

Hill, John D.↗

A dual-species Rydberg array

Large-scale Rydberg atom arrays are used for highly coherent analogue quantum simulations and for digital quantum computations. However, advanced quantum protocols, such as quantum error correction, require midcircuit qubit operations, including the replenishment, reset and read-out of a subset of qubits. A compelling strategy for unlocking these capabilities is a dual-species architecture in which a second atomic species is controlled independently and entangled with the first through Rydberg interactions. Here, we realize a dual-species Rydberg array consisting of rubidium and caesium atoms and explore regimes of interactions and dynamics not accessible in single-species architectures. We achieve enhanced interspecies interactions by electrically tuning the Rydberg states close to a Förster resonance. In this regime, we demonstrate an interspecies Rydberg blockade and implement a quantum state transfer from one species to another. We then generate a Bell state between Rb and Cs hyperfine qubits through an interspecies controlled-phase gate. Finally, we combine interspecies entanglement with a native midcircuit read-out to achieve quantum non-demolition measurements.

atomic and molecular interactions with photons↗

Ir(hkl) Surface Electrochemistry in a Nonadsorbing Acidic Medium

The fundamental properties of electrochemical materials depend on the multiple and often complex interactions between electrode surface sites and electrolyte species at the electrochemical interface. Despite Iridium use in electrolyzer systems, much of its surface electrochemistry remains underexplored. This study investigates the surface electrochemistry of Ir(111), Ir(100), and Ir(110) surfaces in acidic media. Using cyclic voltammetry and CO charge displacement experiments, we establish the charge states and adsorbate coverages as a function of the electrode potential, revealing the presence of hydrogen and hydroxyl co-adsorption at low potentials on (111), and almost no coverage of H ad on (110) facet. In situ Shell Isolated Nanoparticle Enhanced Raman Spectroscopy experiments provide direct evidence of the formation of key adsorbate species, such as hydrogen, hydroxyl, and oxygen, but most importantly, their interactions with interfacial water, confirmed by Density Functional Theory calculations. Our findings highlight the role of co-adsorption and interspecies interactions, with microkinetic adsorption voltammetry simulations corroborating the influence of lateral interactions on adsorption dynamics, particularly for Ir(100) where the OHad formation occurs as a sharp adsorption/desorption current. Our results underscores the importance of interfacial water and hydrogen bonding networks in shaping the electrochemical behavior on Ir surfaces, refining our baseline understanding of the Ir surface electrochemistry necessary for the development of advanced Ir-based electrochemical materials.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Metabolic Interactions between Brachypodium and Pseudomonas fluorescens under Controlled Iron-Limited Conditions

Plant iron (Fe) nutritional status has a significant impact on rhizosphere microbial communities. While this effect has been attributed to alterations in the composition of root exudates, the underlying mechanisms are not known. Here, we investigated the effect of Fe deficiency on the interactions between the grass Brachypodium distachyon and the common soil bacteria Pseudomonas fluorescens SBW25. Increased phytosiderophore production was the most significant response of Brachypodium root exudation to Fe deficiency. We observed upregulated expression of phytosiderophore biosynthesis genes but lower accumulated exudate concentrations in the presence of Pseudomonas, indicating that the bacteria degrade these phytosiderophores. Pseudomonas did not produce endogenous siderophores under the low Fe conditions. Rather, the bacterial transcriptome revealed upregulation of four genes in response to Fe deficiency that contain motifs associated with cellular uptake of small molecules. Collectively, these results suggest that rhizosphere Pseudomonas fluorescens may take up phytosiderophores produced by grasses in response to Fe deficiency, and we propose target genes that may be involved. Our findings provide insight into the molecular basis for the exchange of C, N, and Fe between plants and bacteria under Fe deficient conditions. These interactions may contribute to differences in the rhizosphere microbial community structure across soil pH regimes.

Boiteau, Rene M.↗

WA-IsoC_MSC1.1.0

The soil microbiome is central to the cycling of carbon and other nutrients and to the promotion of plant growth. Despite its importance, analysis of the soil microbiome is difficult due to its sheer complexity, with thousands of interacting species. Here, we reduced this complexity by developing model soil microbial consortia that are simpler and more amenable to experimental analysis but still represent important microbial functions of the native soil ecosystem. Samples were collected from an arid grassland soil and microbial communities (consisting mainly of bacterial species) were enriched on agar plates containing chitin as the main carbon source. Chitin was chosen because it is an abundant carbon and nitrogen polymer in soil that often requires the coordinated action of several microorganisms for complete metabolic degradation. Several soil consortia were derived that had tractable richness (30-50 OTUs) with diverse phyla representative of the native soil, including Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria and Verrucomicrobia. The resulting consortia can be stored as glycerol or lyophilized stocks at -80° C and revived while retaining community composition, greatly increasing their use as tools for the research community at large. One of the consortia that was particularly stable was chosen as a model soil consortium (MSC-1) for further analysis. MSC-1 species interactions were studied using both pairwise co-cultivation in liquid media and during growth in soil under several perturbations. Co-abundance analyses highlighted interspecies interactions and helped to define keystone species, including Mycobacterium, Rhodococcus, and Rhizobiales taxa. These experiments demonstrate the success of an approach based on naturally enriching a community of interacting species that can be stored, revived, and shared. The knowledge gained from querying these communities and their interactions will enable better understanding of the soil microbiome and the roles these interactions play in this environment.

54 ENVIRONMENTAL SCIENCES↗

Unconventional Charge Transport in MgCr 2 O 4 and Implications for Battery Intercalation Hosts

Ion transport in solid-state cathode materials prescribes a fundamental limit to the rates batteries can operate; therefore, an accurate understanding of ion transport is a critical missing piece to enable new battery technologies, such as magnesium batteries. Based on our conventional understanding of lithium-ion materials, MgCr 2 O 4 is a promising magnesium-ion cathode material given its high capacity, high voltage against an Mg anode, and acceptable computed diffusion barriers. Electrochemical examinations of MgCr 2 O 4 , however, reveal significant energetic limitations. Motivated by these disparate observations; herein, we examine long-range ion transport by electrically polarizing dense pellets of MgCr 2 O 4 . Our conventional understanding of ion transport in battery cathode materials, e.g., Nernst-Einstein conduction, cannot explain the measured response since it neglects frictional interactions between mobile species and their nonideal free energies. In this work, we propose an extended theory that incorporates these interactions and reduces to the Nernst-Einstein conduction under dilute conditions. This theory describes the measured response, and we report the first study of long-range ion transport behavior in MgCr 2 O 4 . We conclusively show that the Mg chemical diffusivity is comparable to lithium-ion electrode materials, whereas the total conductivity is rate-limiting. Given these differences, energy storage in MgCr 2 O 4 is limited by particle-scale voltage drops, unlike lithium-ion particles that are limited by concentration gradients. Future materials design efforts should consider the interspecies interactions described in this extended theory, particularly with respect to multivalent-ion systems and their resultant effects on continuum transport properties.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Transcriptomic Network Analysis of Cyanobacterial-Methylotroph Interactions in Coculture and Axenic Conditions

A previous study demonstrated the potential for Cyanobacterial-Methylotroph cocultures to facilitate biogas processing as well as to be used in other biotechnological applications. To advance this technology, we investigated potential interactions between Cyanobacterium stanieri HL-69 (HL69) and Methylotuvimicrobium alkaliphilum 20Z (20Z) by inferring and analyzing gene co-expression networks under co-culture and axenic conditions. Five different co-expression networks were examined. These networks were inferred using gene expression profiles for 20Z axenic condition, HL-69 axenic, HL-69, 20Z coculture, HL-69 coculture, and cross-species HL-69-20Z coculture. Through the analysis of node (gene) betweenness and node normalized degree values in all five network cases, we compared adjustments in gene expression between growth conditions (axenic vs co-culture) as well as identify biological functions relevant to interspecies interactions. This analysis was done to distinguish between gene interactions within an organism and gene interactions between two organisms. Moreover, for all five cases we investigated two different network cutoff levels of 3,000 and 10,000. By shedding light on inter- and intra- species interactions, we hope to gain a better understanding of how these two organisms interact. This research will allow the investigation of further biotechnological applications of coculture systems and optimization of such applications for biotechnological purposes.

59 BASIC BIOLOGICAL SCIENCES↗

Harnessing photoautotroph-methanotroph interactions for biogas conversion to fuels and chemicals using binary consortia (Project Final Technical Report)

Industrial, municipal, and agricultural waste streams containing stranded organic carbon represent a significant and underutilized feedstock to produce fuels and chemicals. With anaerobic digestion deployed at large scales to capture organic waste streams, over 6 million tons of biogas are available today. However, the utilization of biogas represents a significant challenge due to its low pressure and presence of contaminants such as H 2 S, ammonia, and volatile organic carbon compounds. To tap into this immense potential, effective biotechnologies that co-utilize both CO 2 and CH 4 are needed. Recent studies demonstrated that, in nature, microbial communities have developed a highly efficient way to recover energy and capture carbon from both CH 4 and CO 2 through metabolic coupling of methane oxidation to oxygenic photosynthesis. Using two synthetic methanotroph – photoautotroph (M-P) co-cultures that exhibit stable growth under a broad range of cultivation conditions, in this project we proposed to harness the interspecies interactions within these cocultures for biogas conversion to fuels and chemicals. To facilitate this overarching objective, we aim to develop experimental and computational tools to gain qualitative and quantitative understandings on the interactions and dynamics of the coculture at both systems and molecular levels, and to validate our findings through experiments and mutant development. The fundamental understanding on the interactions and dynamics of the photoautotroph-methanotroph will lay the foundation for the design and optimization of synthetic binary consortia for production of fuels and chemicals from biogas. We expect the knowledge gained from this project may be generally applicable to other cross-feeding binary consortium, and the tools developed can be adapted to study the interactions and dynamics of other multi-organism platforms.

09 BIOMASS FUELS↗

CarbStor: Development, Analysis and Modification of Carbon Storing Model Soil Communities

Soil microbial communities carry out a number of key processes including plant growth promotion, bioremediation and cycling of nutrients. Carbon cycling is among the most important of these nutrients that are metabolized and processed by the soil microbial community. Many of the carbon inputs are converted to alternative organic forms of carbon that can be used by plants or act as biomass for microbial growth. However, inorganic forms of carbon can also be produced by soil microbial communities including calcium carbonate (CaCO 3 ). Production of calcium carbonate is beneficial for the ecosystem in several ways: it can stabilize soils and improve soil health, especially denser soils with high clay content, it can act as a method of bioremediation, it can serve as an alternative carbon source for plants and it can be a way to store carbon in soil in a stable, inorganic manner for the long term. While the chemistry surrounding individual species carrying out this process is well known what is lacking is an understanding of how species interact in a community to drive carbonate production. As all microbial species in soil exist in a community setting gaining this knowledge is critical to our predicting and controlling this microbial phenotype to greatly improve soil health. The CarbStor project is focused on developing, analyzing and modifying defined microbial soil consortia that express phenotypes at both the species and community level to convert carbon into recalcitrant stable sources such as precipitated carbonate or microbial necromass. To take full advantage of the soil community for this process we will need to fill several key knowledge gaps (KG), three of which are the focus of CarbStor. KG1: Whether and to what degree microbial communities can be developed that produce precipitated carbon via microbial metabolism. KG2: What interspecies interactions drive the individual member phenotypes in defined communities that lead to carbon precipitation. KG3: How can these interactions be modified to enhance carbon sequestration beyond what native communities are capable of. We hypothesize that in a carbon sequestering community only a subset of species will express phenotypes related to carbon storage processes. We also hypothesize that these phenotypes are expressed as a result of interactions with other species in the community that are not involved in carbon storage processes and that these interactions can be harnessed to enhance community carbon sequestration.

54 ENVIRONMENTAL SCIENCES↗

Learning from nature’s plant engineers: Hijacking metabolism and development beyond genetics

Plant metabolism underpins the food, fiber, and fuel that support our economy, driving strong interest in new strategies to rewire plant metabolism for emerging applications. While most synthetic biology efforts are reliant on genetic engineering, plants can be manipulated in many other ways that remain comparatively underexplored. Across nature, diverse organisms, including bacteria, fungi, and insects, have evolved sophisticated mechanisms to exploit plant metabolic richness, reshaping it for purposes that span from basic nutrition to the construction of complex, novel structures for shelters. These interspecies interactions and non-model systems represent unique manners in which plants can be reprogrammed or hijacked by other organisms, offering inspiration for novel approaches to engineering plant metabolism. By better understanding the basis of how organisms induce these remarkable transformations in plants, we can expand the conceptual boundaries of synthetic biology and reveal alternative routes to manipulating plants for the production of a diverse array of valuable compounds and materials. Deeper insight into these mechanisms will yield novel blueprints for rethinking the scope and breadth in which we can redesign plant metabolism across many applications.

Sarkiss, Anthony E. [University of California, Ber↗

Analysis of biofilm assembly by large area automated AFM

Biofilms are complex microbial communities critical in medical, industrial, and environmental contexts. Understanding their assembly, structure, genetic regulation, interspecies interactions, and environmental responses is key to developing effective control and mitigation strategies. While atomic force microscopy (AFM) offers critically important high-resolution insights on structural and functional properties at the cellular and even sub-cellular level, its limited scan range and labor-intensive nature restricts the ability to link these smaller scale features to the functional macroscale organization of the films. We begin to address this limitation by introducing an automated large area AFM approach capable of capturing high-resolution images over millimeter-scale areas, aided by machine learning for seamless image stitching, cell detection, and classification. Large area AFM is shown to provide a very detailed view of spatial heterogeneity and cellular morphology during the early stages of biofilm formation which were previously obscured. Using this approach, we examined the organization of Pantoea sp. YR343 on PFOTS-treated glass surfaces. Our findings reveal a preferred cellular orientation among surface-attached cells, forming a distinctive honeycomb pattern. Detailed mapping of flagella interactions suggests that flagellar coordination plays a role in biofilm assembly beyond initial attachment. Additionally, we use large-area AFM to characterize surface modifications on silicon substrates, observing a significant reduction in bacterial density. This highlights the potential of this method for studying surface modifications to better understand and control bacterial adhesion and biofilm formation.

59 BASIC BIOLOGICAL SCIENCES↗

Defining the Expression, Production, and Signaling Roles of Specialized Metabolites during Bacillus subtilis Differentiation

Bacterial specialized (or secondary) metabolites are structurally diverse molecules that mediate intra- and interspecies interactions by altering growth and cellular physiology and differentiation. Bacillus subtilis, a Gram-positive model bacterium commonly used to study biofilm formation and sporulation, has the capacity to produce more than 10 specialized metabolites. Some of these B. subtilis specialized metabolites have been investigated for their role in facilitating cellular differentiation, but only rarely has the behavior of multiple metabolites been simultaneously investigated. In this study, we explored the interconnectivity of differentiation (biofilm and sporulation) and specialized metabolites in B. subtilis. Specifically, we interrogated how development influences specialized metabolites and vice versa. Using the sporulation-inducing medium DSM, we found that the majority of the specialized metabolites examined are expressed and produced during biofilm formation and sporulation. Additionally, we found that six of these metabolites (surfactin, ComX, bacillibactin, bacilysin, subtilosin A, and plipastatin) are necessary signaling molecules for proper progression of B. subtilis differentiation. This study further supports the growing body of work demonstrating that specialized metabolites have essential physiological functions as cell-cell communication signals in bacteria.

59 BASIC BIOLOGICAL SCIENCES↗

BONCAT-Live for isolation and cultivation of active environmental bacteria

In diverse environments, microbes drive a myriad of processes, from geochemical and nutrient cycling to interspecies interactions, including associations with plants and animals. Their physiological state is dynamic and impacted by abiotic and biotic conditions, responding to environmental fluctuations by changes in cellular metabolism, according to their genetic potential. Molecular, cellular, and genomic approaches can identify and measure microbial responses and adaptation to environmental changes in native communities. However, isolating individual microbial cells that respond to specific changes for cultivation has been difficult. To address this, we implemented a novel bacterial isolation approach (BONCAT-Live) by integrating bio-orthogonal non-canonical amino acid tagging (BONCAT) in diverse native communities, with isolation and cultivation of cells responding to specific stimuli, at different time scales. In frozen Arctic permafrost samples, we identified and isolated dormant bacteria that become active after thawing under native or nutrient-enriched conditions. From the Populus tree rhizosphere, we isolated strains that thrive under high concentrations of root exudates that act as defense compounds and nutrients. In the human microbiome, we identified and isolated bacteria that rapidly proliferated when exposed to metabolites provided by the host or other co-occurring microbes. Further characterization of isolated bacterial strains will provide opportunities for in-depth determination of how these microbes adapt to changes in their environments, individually and as part of model communities.

Analytical Methods↗