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At least 199 records · Page 11

Protoplast fusion in Bacillus species produces frequent, unbiased, genome-wide homologous recombination

Abstract In eukaryotes, fine-scale maps of meiotic recombination events have greatly advanced our understanding of the factors that affect genomic variation patterns and evolution of traits. However, in bacteria that lack natural systems for sexual reproduction, unbiased characterization of recombination landscapes has remained challenging due to variable rates of genetic exchange and influence of natural selection. Here, to overcome these limitations and to gain a genome-wide view on recombination, we crossed Bacillus strains with different genetic distances using protoplast fusion. The offspring displayed complex inheritance patterns with one of the parents consistently contributing the major part of the chromosome backbone and multiple unselected fragments originating from the second parent. Our results demonstrate that this bias was in part due to the action of restriction–modification systems, whereas genome features like GC content and local nucleotide identity did not affect distribution of recombination events around the chromosome. Furthermore, we found that recombination occurred uniformly across the genome without concentration into hotspots. Notably, our results show that species-level genetic distance did not affect genome-wide recombination. This study provides a new insight into the dynamics of recombination in bacteria and a platform for studying recombination patterns in diverse bacterial species.

59 BASIC BIOLOGICAL SCIENCES↗

Quantum biological insights into CRISPR-Cas9 sgRNA efficiency from explainable-AI driven feature engineering

Abstract CRISPR-Cas9 tools have transformed genetic manipulation capabilities in the laboratory. Empirical rules-of-thumb have been developed for only a narrow range of model organisms, and mechanistic underpinnings for sgRNA efficiency remain poorly understood. This work establishes a novel feature set and new public resource, produced with quantum chemical tensors, for interpreting and predicting sgRNA efficiency. Feature engineering for sgRNA efficiency is performed using an explainable-artificial intelligence model: iterative Random Forest (iRF). By encoding quantitative attributes of position-specific sequences for Escherichia coli sgRNAs, we identify important traits for sgRNA design in bacterial species. Additionally, we show that expanding positional encoding to quantum descriptors of base-pair, dimer, trimer, and tetramer sequences captures intricate interactions in local and neighboring nucleotides of the target DNA. These features highlight variation in CRISPR-Cas9 sgRNA dynamics between E. coli and H. sapiens genomes. These novel encodings of sgRNAs enhance our understanding of the elaborate quantum biological processes involved in CRISPR-Cas9 machinery.

59 BASIC BIOLOGICAL SCIENCES↗

Multilayered regulation by RNA thermometers enables precise control of Cas9 expression in E. coli

Cas9-based genome editing technologies can rapidly generate mutations to probe a diverse array of mutant genotypes. However, aberrant Cas9 nuclease translation and activity can occur despite the use of inducible promoters to control expression, leading to extensive cell death. This background killing caused by promoter leakiness severely limits the application of Cas9 for generating mutant libraries because of the potential for population skew. We demonstrate the utility of temperature sensitive RNA elements as a layer of post-transcriptional regulation to reduce the impact of promoter leak. We observe significant temperature-dependent increases in cell survival when certain RNA thermometers (RNATs) are placed upstream of the cas9 coding sequence. We also show that the most highly repressing RNAT, hsp17rep, significantly reduces population skew with a library of characterized guide RNAs in Escherichia coli. This strategy should be applicable to all bacterial Cas9-based methods and technologies.

Kammerdiener, Elise K. [Oak Ridge National Laborat↗

Empirical evaluation of all unique Cas9 protospacers in E. coli reveal widespread functionality and rules for gRNA design

The Cas9 nuclease has become central to modern methods and technologies in synthetic biology, largely due to the ease with which it can be targeted to specific DNA loci via guide RNAs (gRNAs). Reports vary widely on the actual specificity of this targeting, with some studies observing 60% of gRNAs possessing no activity against the genome, yet an assumption persists within the E. coli community that inactive gRNAs are rare. To resolve these contradictions, we evaluated the activity of 463 000 unique gRNAs in the E. coli K12 MG1655 genome. We show that the overwhelming majority (at least 93%) of unique gRNAs are functional while only 0.3% are nonfunctional. These nonfunctional gRNAs exhibit strong spacer self-interaction, which can either be excluded using a simple design rule or “repaired” during library design. Finally, this work provides the greater microbial synthetic biology community both a set of nearly half a million empirically evaluated E. coli gRNAs as well as a thoroughly evaluated experimental procedure, complete with appropriate controls for Cas9 activity, for conducting Cas9 assays in E. coli specifically and bacteria more generally. Lastly, we have produced a webapp to allow users to easily browse and extract gRNA sequences from the E. coli genome, which can be accessed at https://grna.ornl.gov.

Kammerdiener, Elise K. [Oak Ridge National Laborat↗

Abiotic Stress Reorganizes Rhizosphere and Endosphere Network Structure of Sorghum bicolor

Sorghum bicolor is a promising bioenergy feedstock with high biomass production and unusual tolerance for stresses, such as water and nutrient limitation. Although the membership of the sorghum microbiome in response to stress has been explored, relatively little is known about how microbe–microbe networks change under water- or nutrient-limited conditions. This is important because network changes can indicate impacts on the functionality and stability of microbial communities. We performed network-based analysis on the core bacterial and archaeal community of an agronomically promising high biomass bioenergy genotype, Grassl, grown under nitrogen and water stress. Stress caused relatively minor changes in bacterial abundances within soil, rhizosphere, and endosphere communities but led to significant changes in bacterial network structure and modularity. We found a complete reorganization of network roles in all plant compartments, as well as an increase in the modularity and proportion of positive associations, which potentially could represent coexistence and cooperation in the sorghum bacterial/archaeal community under stress. Although stressors are often believed to be destabilizing, we found stressed networks were as or more stable than non-stressed networks, likely due to their redundancy and compartmentalization. Together, these findings support the idea that both sorghum and its bacterial/archaeal community can be resilient to future environmental stressors.

09 BIOMASS FUELS↗

Regional Differences in the Structure of Juglans nigra Phytobiome Reflect Geographical Differences in Thousand Cankers Disease Severity

Thousand cankers disease threatens Juglans nigra (Eastern Black Walnut) in urban and natural landscapes. Incidence and severity of thousand cankers disease is higher in the host’s introduced range in the western United States. We hypothesized that these differences are driven partly by geographical variation in the host phytobiome due to its roles in host stress tolerance, nutrient acquisition, and defense. To evaluate the role of the phytobiome in mediating thousand cankers disease, we characterized the J. nigra phytobiome of diseased and healthy trees in portions of its native (Indiana and Tennessee) and introduced (Washington) ranges. Grafted clones present in each state and open-pollinated populations were sampled. DNA was extracted from soil and branch (caulosphere) tissues and internal transcribed spacer and 16s regions were sequenced for characterization of fungal and bacterial communities. We found that microbial communities in the caulosphere and soil differ between native and introduced ranges of J. nigra and harbor different mutualistic and pathogenic microorganisms. Additionally, caulosphere microbial communities were more species rich and diverse in the native range of J. nigra, suggesting greater levels of functional redundancy and multifunctionality in the native-range phytobiome compared with the introduced range. We also found higher network complexity in the caulosphere of trees in the introduced range and evidence for two alternative stable community states associated with diseased and healthy trees. Our results provide support for the hypothesis that geographical variation in thousand cankers disease incidence and severity is partially driven by differences in the phytobiome of J. nigra in its introduced and native ranges.

59 BASIC BIOLOGICAL SCIENCES↗

Chronic drought differentially alters the belowground microbiome of drought tolerant and drought susceptible genotypes of Populus trichocarpa

Populus trichocarpa is an ecologically important tree species and economically important biofeedstock. Belowground, P. trichocarpa interacts with diverse microorganisms in the rhizosphere and root endosphere. These plant-microbial interactions can bolster a variety of plant processes, ranging from pathogen suppression to drought tolerance, yet we know little about the impact of chronic drought stress on P. trichocarpa’s belowground microbiomes. To investigate the interactive effect of chronic drought on belowground microbial communities across genetically different P. trichocarpa hosts, we assessed archaeal/bacterial and fungal communities within the root endosphere, rhizosphere, and surrounding bulk soil of selected genotypes in a long-term drought experiment in Boardman, OR, USA. We sequenced the 16S rRNA and ITS2 gene region on samples collected from 16 distinct P. trichocarpa genotypes in plots with full or reduced irrigation. Eight of these genotypes have been previously identified as drought tolerant while the other eight genotypes were drought susceptible. While reduced irrigation influenced the composition of every archaeal/bacterial microbiome compartment, fungal communities were only affected in the rhizosphere and bulk soil compartments. Drought-tolerant bacteria, such as Actinobacteria, were differentially abundant in reduced irrigation across all belowground microbiomes. Host drought-tolerance influenced plant-associated microbiome compartments but had little impact on the bulk soil compartment. Drought-tolerant trees were enriched for potential growth-promoting microorganisms in the root endosphere and rhizosphere, including Sphingomonas bacteria and ectomycorrhizal fungi. Overall, associations of growth-promoting microbes in drought resistant P. trichocarpa genotypes can be leveraged to improve biofeedstock productivity in regions prone to periodic drought.

59 BASIC BIOLOGICAL SCIENCES↗

Fluid-phase helium: Shock-compression experiments, quantum molecular dynamics simulations, and development of an equation of state

Helium (He) plays a critical role in numerous areas ranging from the study of celestial objects like brown dwarfs and gas giants to modern-day technologies like nuclear energy and rocket propulsion. For many of these applications, it is essential to have a reliable equation of state (EOS) for He that yields an accurate representation of its thermodynamic behavior. To help constrain and develop such EOS models, we have performed a series of shock-compression experiments on cryogenic liquid He to pressures exceeding 100 GPa using a magnetically accelerated flyer plate on Sandia National Laboratories' Z-machine. We have also performed quantum molecular dynamics simulations that are consistent with our shock measurements. None of the previously available EOSs agree with our experimental and simulation results, motivating the development of a fluid-phase He EOS that we present in this study. Here, we show that our EOS yields good agreement with published data that span temperatures and pressures encountered across a diverse array of applications.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

Aqueous harvesting of 88 Zr at a radioactive-ion-beam facility for cross-section measurements

Isotope harvesting is a method of collecting the long-lived radioisotopes that build up during the operation of ion-beam facilities in a way that is useful for subsequent research. As a demonstration of this method for the collection of a group IV metal at a fragmentation facility, the high-energy Zr 88 secondary beam produced from a 140-MeV/u Mo 92 primary beam at the National Superconducting Cyclotron Laboratory (NSCL) was stopped in a water target. The setup aimed to mimic the aqueous beam dump that will be implemented at the Facility for Rare Isotope Beams (FRIB). The collected Zr 88 and accompanying Y 88 decay daughter were radiochemically extracted from the solution and made into target samples suitable for neutron-capture cross-section measurements. These samples were then irradiated at two reactor facilities, and the Zr 88 average thermal-neutron-capture cross section ( σ T ) and resonance integral ( I ) were determined to be σ T = ( 8.04 ± 0.63 ) × 10 5 b and I = ( 2.53 ± 0.28 ) × 10 6 b. The σ T value agrees well with previous results and I , determined for the first time here, was found to be the largest measured resonance integral by two orders of magnitude. The Y 88 thermal-neutron-capture cross section was determined to be less than 1.8 × 10 4 b. This work demonstrates the steps needed to make cross-section measurements with samples produced via aqueous isotope harvesting.

38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEA↗

Measurement of the 230 Th( p ,2n)Pa229 and 230 Th( p ,3n)Pa228 reaction cross sections from 14.1 to 16.9 MeV

Actinium-225 is of interest for medical isotope production and there is on-going research into methods of producing Ac 225 , either directly or via the decay of its parent isotopes ( Th 229 , Pa 229 , and Ra 225 ). One method that has been suggested is the Th 230 ( p , 2 n ) Pa 229 reaction. However, there is no available cross-section data for this reaction in the literature. Purpose: Measure the Th 230 ( p , 2 n ) and Th 230 ( p , 3 n ) reaction cross sections in the energy range where the ( p , 2 n ) reaction is predicted to peak to determine the feasibility of Ac 225 production via the Th 230 ( p , 2 n ) reaction. Methods: Targets naturally enriched in Th 230 were irradiated at the Center for Accelerator Mass Spectrometry at Lawrence Livermore National Laboratory with energies ranging from 14.1 to 16.9 MeV. Furthermore, chemical processing was used to separate the protactinium activation products, followed by γ -ray spectroscopy to measure the activities of Pa 228 , 229 , 230 , 232 produced in the irradiation. Results: We find that excitation functions are reported for the first time in the literature for the Th 230 ( p , 2 n ) and Th 230 ( p , 3 n ) reactions in this energy range. The peak measured value of the Th 230 ( p , 2 n ) reaction was found to be 182 ± 12 mb at 14.4 ± 0.1 MeV. The Th 232 ( p , n ) Pa 232 reaction was used to verify the experimental conditions, the measured values are reported and are comparable to the existing literature values. From the γ -ray spectrometry data, the half-life of Pa 229 was measured as 1.5 ± 0.1 days, which is within the error of the half-life reported in the evaluated nuclear data as well as in the recent measurements, and the half-life of Pa 228 was measured as 19.5 ± 0.4 hours. Conclusions: Overall, the Th 230 ( p , 2 n ) Pa 229 reaction could reasonably be used for Ac 225 isotope production, although significant amounts of relatively isotopically pure Th 230 would be needed for significant production because the low alpha-decay branching ratio of Pa 229 and long half-life of Th 229 inhibit the in-growth of significant amounts of Ac 225 .

38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEA↗

Measurement of the π - -Ar total hadronic cross section at the LArIAT experiment

We present the first measurement of the negative pion total hadronic cross section on argon in a restricted phase space, which we performed at the Liquid Argon In A Testbeam (LArIAT) experiment. All hadronic reaction channels, as well as hadronic elastic interactions with scattering angle greater than 5° are included. The pions have kinetic energies in the range 100–700 MeV and are produced by a beam of charged particles impinging on a solid target at the Fermilab test beam facility. LArIAT employs a 0.24 ton active mass liquid argon time projection chamber (LArTPC) to measure the pion hadronic interactions. For this measurement, LArIAT has developed the “thin slice method,” a new technique to measure cross sections with LArTPCs. While moderately higher, our measurement of the π - -Ar total hadronic cross section is generally in agreement with the geant4 prediction.

72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS↗

A Science Gateway for the Repeatable Analysis of Machine Learning Predicted Gravity Anomalies

In recent years, deep learning has become an increasingly popular alternative for modeling in geoscience applications due to its scalability and efficiency. However, the interpretability, compute, data volume, and hyperparameter tuning requirements of deep learning models make development and monitoring difficult. Furthermore, model explainability and communicating results obtained by these models to users or domain experts is a challenge, as domain experts in geoscience also need to have a deep understanding of how those models function in order to support their scientific works. Here, we describe a science gateway and machine learning pipeline for predicting gravity anomalies from geophysical data. The gateway, built on open-source technologies, provides a holistic view of the pipeline through interactive visualizations aimed at enabling efficient exploratory data analysis. The repeatability, reproducibility, and monitoring capabilities of this overall system allow us to iterate and analyze at scale. Using this pipeline and gateway, we can repeatedly produce accurate high-resolution gravity anomaly datasets. By describing the underlying technologies, implementation, and results, here we provide a foundation for the broader adoption of science gateways into cross-cutting geoscience and machine learning research projects as a means to improve the scientific discovery and collaboration in the geophysics and computational sciences community.

58 GEOSCIENCES↗

Luminescent Solar Concentrator Tandem-on-Silicon with above 700mV Passivated Contact Silicon Bottom Cell

Luminescent solar concentrator (LSC) tandem-on-silicon (Si) provides a route towards achieving higher than 30% overall efficiency which can overcome the theoretical efficiency limit of a single junction Si cell. Here, we present optical coupling and performance of high Voc passivated contact Si bottom cell for LSC tandem-on-Si where the top module consists of highly efficient luminophores and an array of micro InGaP cells embedded in a poly (lauryl methacrylate) waveguide. In this device configuration, InGaP cell area coverage is only ~0.5% of the total LSC area which significantly reduces the high cost III-V material usage. The performance of Si sub-cell is investigated under LSC spectrum and is compared against the measurement done under 1 um thick InGaP filter which mimics the spectrum seen by Si bottom cell in a conventional III-V/Si tandem. V oc of greater than 700 mV has been observed for the passivated contact Si bottom cell in these tandem applications.

14 SOLAR ENERGY↗

Self-Aligned, Selective Area Poly-Si/SiO2 Passivated Contacts for Enhanced Photocurrent in Front/Back Solar Cells

Poly-Si/SiO 2 passivated contact front/back solar cells suffer low Jsc due to parasitic absorption in the front poly-Si layer. We demonstrate a self-aligned, selective area front contact dry-etch technique that retains the as-deposited poly-Si beneath the metal grid lines but thins it elsewhere. Jsc improves by 0.7 mA/cm 2 over our standard 40 nm thick poly-Si. Greater improvements are expected with thicker poly-Si needed for fired metal contacts. Surface passivation is slightly diminished with poly-Si thinning but can be partially restored with rehydrogenation.

14 SOLAR ENERGY↗

Ant handling changes myrmecochore seed coat microbiomes and alters diversity of seed-borne plant pathogenic fungi

1. The putative benefits to seeds in myrmecochory (ant- mediated seed dispersal) are often cast in a reward context. However, microbes have been mostly overlooked as seed mortality agents in myrmecochory, as have potential treatments provided by ant-handling. 2. We investigated the effects of ant handling on the diversity of seed coat fungal communities of three myrmecochorous plant species. 3. Ant-handling altered measures of both alpha and beta diversity of fungal communities. Ant-handled seeds harboured different overall fungal communities and plant pathogen communities than non-ant-handled seeds. The myrmecochore pathogenic fungal community showed high dissimilarity (high pairwise community turnover) between ant-handled and control seeds, while beta diversity measures for ant- handled seeds and seeds with manually removed elaiosomes were less dissimilar. 4. Ant handling may offer an additional benefit to myrmecochorous seeds via the reduction in the seed coat pathogenic community, which may be driven by elaiosome removal or as a by-product of ant cleaning behaviours and chemical secretions.

59 BASIC BIOLOGICAL SCIENCES↗

A dynamic microbial sulfur cycle in a serpentinizing continental ophiolite

Summary Serpentinization is the hydration and oxidation of ultramafic rock, which occurs as oceanic lithosphere is emplaced onto continental margins (ophiolites), and along the seafloor as faulting exposes this mantle‐derived material to circulating hydrothermal fluids. This process leads to distinctive fluid chemistries as molecular hydrogen (H 2 ) and hydroxyl ions (OH − ) are produced and reduced carbon compounds are mobilized. Serpentinizing ophiolites also serve as a vector to transport sulfur compounds from the seafloor onto the continents. We investigated hyperalkaline, sulfur‐rich, brackish groundwater in a serpentinizing continental ophiolite to elucidate the role of sulfur compounds in fuelling in situ microbial activities. Here we illustrate that key sulfur‐cycling taxa, including Dethiobacter , Desulfitispora and ‘ Desulforudis ’, persist throughout this extreme environment. Biologically catalysed redox reactions involving sulfate, sulfide and intermediate sulfur compounds are thermodynamically favourable in the groundwater, which indicates they may be vital to sustaining life in these characteristically oxidant‐ and energy‐limited systems. Furthermore, metagenomic and metatranscriptomic analyses reveal a complex network involving sulfate reduction, sulfide oxidation and thiosulfate reactions. Our findings highlight the importance of the complete inorganic sulfur cycle in serpentinizing fluids and suggest sulfur biogeochemistry provides a key link between terrestrial serpentinizing ecosystems and their submarine heritage.

Sabuda, Mary C.↗