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At least 1,603 records · Page 89

Fostering Peat Moss Feedbacks to Accelerate Peatland Restoration

Extensive knowledge exists on plant-species traits and functions, but we understand less about how population- or community-level emergent traits influence ecosystem functioning. This knowledge gap is important for ecosystems like peatlands, arid drylands, salt marshes, seagrass meadows and mangroves, where emergent traits of plant communities can create plant-environment feedbacks that amplify or dampen ecosystem processes. Recent insights from restoration ecology suggest that these feedbacks can critically influence restoration success. Despite growing recognition of emergent trait-driven feedbacks in other ecosystems, they remain underexplored in peatland restoration, world’s most carbon-dense ecosystem. Here, we review emergent self-amplifying and self-dampening feedbacks with net positive effects for peat moss-dominated systems. We show how these feedbacks can promote key physical, chemical, and biological processes that enhance peat moss growth, increase water retention, and reduce microbial decomposition of organic matter. Understanding and fostering these feedbacks offers a promising framework to accelerate peatland restoration across diverse degradation states.

Sphagnum↗

Simple measurements in a complex system: soil community responses to nitrogen amendment in a Pinus taeda forest

Abstract Microorganisms regulate the decomposition of soil organic matter and the flow of plant‐available nutrients. In a temperate pine forest in North Carolina, USA , where nitrogen (N) had been experimentally added for eight years, we combined DNA ‐based measures of fungal and bacterial biomass and composition with measures of seven extracellular enzymes (ecoenzymes). These measures were then correlated with soil chemistry. Our goals were to evaluate the relative sensitivity and repeatability of multiple structural and functional measures of community organization to long‐term N deposition. Measurements were conducted at three litter/soil depths, corresponding to the litter, Oa, and mineral A horizons (max depth 10 cm) with ten spatial replicates per experimental plot. Soil chemistry differed significantly with soil depth and N amendment. Total biomass (soil DNA ), as well as fungal and bacterial biomass (measured by qPCR ), was greatest in the litter horizon and declined significantly with depth. Ecoenzyme activity patterns also changed with soil depth, transitioning from high levels of C, N, and P metabolizing activities in the litter horizon to increased oxidative activities at the lower depth. Under N amendment, soil pH decreased and nitrate concentrations increased in all three soil horizons. Correspondingly, the estimated microbial C use efficiency decreased in N‐amended soils at all depths, despite differences in microbial biomass, community composition, and soil chemistry. Overall, bacterial composition was most responsive to nitrogen amendment, but the taxonomic context of the response varied with soil horizons in conjunction with shifts in soil chemistry and enzyme activities. While a single measure that would incorporate all of the C, N, and P metabolizing activities did not emerge, many measures correlated with each other, and/or with depth and/or N amendment.

54 ENVIRONMENTAL SCIENCES↗

Gas generation and intramolecular isotope study in laboratory pyrolysis of the Springfield coal from the Illinois Basin

Position-specific (PS) isotopes of propane have been proposed as a potential geochemical tool to decipher various geological processes (e.g., thermal cracking, biodegradation, H exchange) in natural reservoirs. The limited studies have been conducted on the PS isotopes of propane from the pyrolysis gases from marine shales, and natural gases sourced from lacustrine and marine kerogens, but little is known on gases produced from the humic kerogen. Here, this study investigated the PS δ 13 C of propane in the closed-system pyrolysis of the Springfield coal, Illinois Basin, Indiana, at 310 to 470 °C (Easy %Ro: 0.76 to 3.07). The C kinetic isotope effect (KIE) of CH 4 produced in both this study and previous low-temperature pyrolysis of the same coal indicates the cleavage of C—O bonds is the main generation pathway at the early kerogen cracking stage, followed by the breakdown of alkyl groups. At the wet-gas cracking stage, C 3 H 8 production from thermally stable compounds has a significant influence on the bulk and position-specific C KIE in the pyrolysis of marine Woodford kerogen and Springfield coal. According to the PS δ 13 C of propane, the central site is likely more enriched in 13 C and the δ 13 C of the terminal site is relatively heterogeneous within the propyl group attached to different functional groups of the gas-prone kerogen. Our findings based on the pyrolysis experiments and natural gas samples indicate thermal cracking and biodegradation appear to alter the δ 13 C cen values more significantly than the δ 13 C ter values of propane. The larger magnitude of ΔC c-t in the kMC simulations (Peterson et al., 2018) compared with those from the marine shale and coal possibly implies the non-random distributions of 13 C of propane precursors in the kerogens. As a new dimension of intramolecular isotopic information of propane, the PS δ 13 C values can contribute to fingerprinting the gas origins and identifying the various geological processes (e.g., kerogen cracking, wet-gas cracking, microbial activities) in sedimentary basins.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

A generalized computational framework to streamline thermodynamics and kinetics analysis of metabolic pathways

Metabolic engineering is a critical biotechnological approach in addressing global energy and environment challenges. Most engineering efforts, however, consist of laborious and inefficient trial-and-error of target pathways, due in part to the lack of methodologies that can comprehensively assess pathway properties in thermodynamics and kinetics. Metabolic engineering can benefit from computational tools that evaluate feasibility, expense and stability of non-natural metabolic pathways. Such tools can also help us understand natural pathways and their regulation at systems level. Here we introduce a computational toolbox, PathParser, which, for the first time, integrates multiple important functions for pathway analysis including thermodynamics analysis, kinetics-based protein cost optimization and robustness analysis. Specifically, PathParser enables optimization of the driving force of a pathway by minimizing the Gibbs free energy of least thermodynamically favorable reaction. In addition, based on reaction thermodynamics and enzyme kinetics, it can compute the minimal enzyme protein cost that supports metabolic flux, and evaluate pathway stability and flux in response to enzyme concentration perturbations. In a demo analysis of the Calvin-Benson-Bassham cycle and photorespiration pathway in the model cyanobacterium Synechocystis PCC 6803, the computation results are corroborated by experimental proteomics data as well as metabolic engineering outcomes. This toolbox may have broad application in metabolic engineering and systems biology in other microbial systems.

09 BIOMASS FUELS↗

Biosensor and optogenetics for systems biology of yeast branched-chain alcohol production and tolerance

In this project we combined synthetic biology, systems biology, protein engineering and metabolic engineering to study, control, and improve the production of branched chain alcohols (BCAs), a class of advanced biofuels preferred by the DOE, in the yeast Saccharomyces cerevisiae. This involved the development and application of optogenetic systems as a new modality of dynamic control of native and engineered metabolic pathways, using light as inducible or repressible agent. The optogenetic systems include gene circuits for light control of gene expression, as well as light-assembled synthetic organelles and photo-switchable protein binders to control metabolic and protein function with light at the protein level. In addition, we developed the first genetically encoded biosensor for BCA production in yeast, which we used to design high throughput assays to identify highly productive strains, pathways, and enzymes. We also showed that this biosensor can be functionally co-expressed with optogenetic circuits in the same strain, raising the possibility of establishing, for the first time, computer-interfaced closed-loop controls of engineered metabolic pathways. The yeast gene deletion library was also utilized to conduct the first systems-level study on BCA toxicity in yeast, which uncovered key fundamental principles of yeast sensitivity and tolerance to these alcohols, allowing us to design highly tolerant strains with increased BCA production. This project, thus comprises the development of several new technologies, which we integrated to make new discoveries on the dynamics of BCA production and their mechanisms of cellular toxicity and tolerance, as well as to establish new paradigms to engineer and control metabolic pathways and microbial fermentations with light, for the production of BCAs and other products of interest to the DOE.

2-metyl-1-butanol↗

Recent Advances in Copper Intercalators as Anticancer Agents

Copper is a part of various enzymes and helps them to function properly. It can be effectively used to produce promising anticancer drugs and presently, many studies are being pursued worldwide on the development of copper-based complexes as potential anticancer drugs. Herein, we briefly discuss the importance of reactive oxygen species in biological applications and copper(II) complexes as anticancer drugs. The anti-angiogenic properties of mono-nuclear copper(II) complexes have been demonstrated by in vivo chick embryo angiogenesis analysis. The plausible mechanism behind anticancer activity of these complexes is by the formation of excessive intracellular Reactive Oxygen Species (ROS). ROS is a composite term used for oxygen derivative non-radicals and free radicals of highly reactive components, that enhances the killing response of immune cells to microbial invasion. Previous reports have shown that ROS plays an important role as a messenger in cell cycling and normal cell signal transduction. .

60 APPLIED LIFE SCIENCES↗

Kinetic, metabolic, and statistical analytics: addressing metabolic transport limitations among organelles and microbial communities

Microbial organisms engage in a variety of metabolic interactions. A crucial part of these interactions is the exchange of molecules between different organelles, cells, and the environment. The main forces mediating this metabolic exchange are transporters. This transport can be difficult to measure experimentally because several transport mechanisms remain opaque. However, theoretical calculations about the inputs and outputs of cells via metabolic exchanges have enabled the successful inference of the workings of intra-organismal and inter-organismal systems. Kinetic, metabolic, and statistical modeling approaches in combination with omics data are enhancing our knowledge and understanding about metabolic exchange and mass resource allocation. Furthermore, this model-driven analytics approach can guide effective experimental design and yield new insights into biological function and control.

59 BASIC BIOLOGICAL SCIENCES↗

Rapid Evaluation of Amine-Functionalized Solvents for Biomass Deconstruction Using High-Throughput Screening and One-Pot Enzymatic Saccharification

Efficient and sustainable pretreatment of lignocellulosic biomass is critical for biofuel and biochemical production, yet its optimization is often hindered by slow, labor-intensive experimental methods. Here, we report the first demonstration of a custom-built, miniaturized, high-throughput screening platform integrated with one-pot enzymatic saccharification, enabling parallel evaluation of solvent type, feedstock, and temperature with minimal material use and high reproducibility. As a proof-of-concept, the HTX platform was used to screen five amine-functionalized solvents, including isopropanolamine, butylamine, N-methylbutylamine, ethanolamine, and ethanolamine acetate across three bioenergy crops (sorghum, poplar, and switchgrass) and pretreatment temperatures ranging from 80 to 140 °C. Vacuum drying successfully removed more than 99% of the solvents from the pretreated biomass, eliminating the need for water washing prior to saccharification. Isopropanolamine and N-methylbutylamine yielded the highest glucose (70–80%) and xylose (58–67%) release, with trends reflecting feedstock recalcitrance. The produced hydrolysates supported robust growth of an engineered strain of the yeast Rhodosporidium toruloides, confirming biocompatibility. This high-throughput platform provides a scalable, feedstock-agnostic framework for rapid pretreatment screening, accelerating solvent–feedstock pairing and process optimization. Its ability to integrate pretreatment, solvent removal, saccharification, and microbial conversion in a miniaturized format offers significant advantages for cost-competitive biorefinery development.

Biomass↗

MURMoT: Design and Application of Microbial Uranium Reduction Monitoring Tools

The overarching project goal of the MURMoT project was the design of tools to elucidate the presence, abundance, dynamics, spatial distribution, and activity of metal- and radionuclide-transforming bacteria. To accomplish these objectives, an integrated approach that combined nucleic acid-based tools, proteomic workflows, uranium isotope measurements, and U(IV) speciation and structure analyses using the Advanced Photon Source (APS) at Argonne National Laboratory was developed.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

Development of a plant carbon–nitrogen interface coupling framework in a coupled biophysical-ecosystem–biogeochemical model (SSiB5/TRIFFID/DayCent-SOM v1.0)

Plant and microbial nitrogen (N) dynamics and N availability regulate the photosynthetic capacity and capture, allocation, and turnover of carbon (C) in terrestrial ecosystems. Studies have shown that a wide divergence in representations of N dynamics in land surface models leads to large uncertainties in the biogeochemical cycle of terrestrial ecosystems and then in climate simulations as well as the projections of future trajectories. In this study, a plant C–N interface coupling framework is developed and implemented in a coupled biophysical-ecosystem–biogeochemical model (SSiB5/TRIFFID/DayCent-SOM v1.0). The main concept and structure of this plant C–N framework and its coupling strategy are presented in this study. This framework takes more plant N-related processes into account. The dynamic ratio (CNR) for each plant functional type (PFT) is introduced to consider plant resistance and adaptation to N availability to better evaluate the plant response to N limitation. Furthermore, when available N is less than plant N demand, plant growth is restricted by a lower maximum carboxylation capacity of RuBisCO (V c,max ), reducing gross primary productivity (GPP). In addition, a module for plant respiration rates is introduced by adjusting the respiration with different rates for different plant components at the same N concentration. Since insufficient N can potentially give rise to lags in plant phenology, the phenological scheme is also adjusted in response to N availability. All these considerations ensure a more comprehensive incorporation of N regulations to plant growth and C cycling. This new approach has been tested systematically to assess the effects of this coupling framework and N limitation on the terrestrial carbon cycle. Long-term measurements from flux tower sites with different PFTs and global satellite-derived products are employed as references to assess these effects. The results show a general improvement with the new plant C–N coupling framework, with more consistent emergent properties, such as GPP and leaf area index (LAI), compared to the observations. The main improvements occur in tropical Africa and boreal regions, accompanied by a decrease in the bias in global GPP and LAI by 16.3 % and 27.1 %, respectively.

54 ENVIRONMENTAL SCIENCES↗

Scripting a new dialogue between diazotrophs and crops

Diazotrophs are bacteria and archaea that can reduce atmospheric dinitrogen (N 2 ) into ammonium. Plant–diazotroph interactions have been explored for over a century as a nitrogen (N) source for crops to improve agricultural productivity and sustainability. This scientific quest has generated much information about the molecular mechanisms underlying the function, assembly, and regulation of nitrogenase, ammonium assimilation, and plant–diazotroph interactions. This review presents various approaches to manipulating N fixation activity, ammonium release by diazotrophs, and plant–diazotroph interactions. Here, we discuss the research avenues explored in this area, propose potential future routes, emphasizing engineering at the metabolic level via biorthogonal signaling, and conclude by highlighting the importance of biocontrol measures and public acceptance.

59 BASIC BIOLOGICAL SCIENCES↗

IMPACTS OF INTERACTING ELEVATED ATMOSPHERIC CO2 AND O3 ON THE STRUCTURE AND FUNCTIONING OF A NORTHERN FOREST ECOSYSTEM: OPERATING AND DECOMMISSIONING THE ASPEN FACE PROJECT

Two of the most important and pervasive greenhouse gases driving global change and impacting forests in the U.S. and around the world are atmospheric CO2 and tropospheric O3. As the only free air, large-scale manipulative experiment studying the interaction of elevated CO2 and O3 on forests, the Aspen FACE experiment was uniquely designed to address the long-term ecosystem level impacts of these two greenhouse gases on aspen-birch-maple forests, which dominate the richly forested Lake States region. The project was established in 1997 to address the overarching scientific question: “What are the effects of elevated [CO2] and [O3], alone and in combination, on the structure and functioning of northern hardwood forest ecosystems?” From 1998 through the middle of the 2009 growing season, we examined the interacting effects of elevated CO2 and O3 on ecosystem processes in an aggrading northern forest ecosystem to compare the responses of early-successional, rapid-growing shade intolerant trembling aspen and paper birch to those of a late successional, slower growing shade tolerant sugar maple. Fumigations with elevated CO2 (560 ppm during daylight hours) and O3 (approximately 1.5 x ambient) were conducted during the growing season from 1998 to 2008, and in 2009 through harvest date. Response variables quantified during the experiment included growth, competitive interactions and stand dynamics, physiological processes, plant nutrient status and uptake, tissue biochemistry, litter quality and decomposition rates, hydrology, soil respiration, microbial community composition and respiration, VOC production, treatment-pest interactions, and treatment-phenology interactions. In 2009, we conducted a detailed harvest of the site. The harvest included detailed sampling of a subset of trees by component (leaves and buds, fine branches, coarse branches and stem, coarse roots, fine roots) and excavation of soil to a depth of 1 m. Throughout the experiment, aspen and birch photosynthesis increased with elevated CO2 and tended to decrease with elevated O3, compared to the control. In contrast to aspen and birch, maple photosynthesis was not enhanced by elevated CO2. Elevated O3 did not cause significant reductions in maximum photosynthesis in birch or maple. In addition, photosynthesis in ozone sensitive clones was affected to a much greater degree than that in ozone tolerant aspen clones. Treatment effects on photosynthesis contributed to CO2 stimulation of aboveground and belowground growth that was species and genotype dependent, with birch and aspen being most responsive and maple being least responsive. The positive effects of elevated CO2 on net primary productivity NPP were sustained through the end of the experiment, but negative effects of elevated O3 on NPP had dissipated during the final three years of treatments. The declining response to O3 over time resulted from the compensatory growth of O3-tolerant genotypes and species as the growth of O3-sensitive individuals declined over time. Cumulative NPP over the entire experiment was 39% greater under elevated CO2 and 10% lower under elevated O3. Enhanced NPP under elevated CO2 was sustained by greater root exploration of soil for growth-limiting N, as well as more rapid rates of litter decomposition and microbial N release during decay. Results from Aspen FACE clearly indicate that plants growing under elevated carbon dioxide, regardless of community type or ozone level, obtained significantly greater amounts of soil N. These results indicate that greater plant growth under elevated carbon dioxide has not led to “progressive N limitation”. If similar forests growing throughout northeastern North America respond in the same manner, then enhanced forest NPP under elevated CO2 may be sustained for a longer duration than previously thought, and the negative effect of elevated O3 may be diminished by compensatory growth of O3-tolerant plants as they begin to dominate forest communities. By the end of the experiment, elevated CO2 increased ecosystem C content by 11%, whereas elevated O3 decreased ecosystem C content by 9%. Total ecosystem C content in the interaction treatment (elevated CO2 and O3) did not significantly differ from that of the control. Total ecosystem C content responded similarly to the treatments across the three forest communities. The treatment effects on ecosystem C content resulted from differences in tree biomass, particularly woody tissues (branches, stem, and coarse roots), and lower C content in the near-surface mineral soil. During its duration, the Aspen FACE project involved collaboration between scientists from 9 countries, and over the course of the experiment there were over 120 Aspen FACE scientific users. These scientists helped produce 75 publications during the most recent funding period (2008-2014) and 207 peer-reviewed publications (169 in refereed journals) since the beginning of the project.

54 ENVIRONMENTAL SCIENCES↗

Conotoxin Prediction: New Features to Increase Prediction Accuracy

Conotoxins are toxic, disulfide-bond-rich peptides from cone snail venom that target a wide range of receptors and ion channels with multiple pathophysiological effects. Conotoxins have extraordinary potential for medical therapeutics that include cancer, microbial infections, epilepsy, autoimmune diseases, neurological conditions, and cardiovascular disorders. Despite the potential for these compounds in novel therapeutic treatment development, the process of identifying and characterizing the toxicities of conotoxins is difficult, costly, and time-consuming. This challenge requires a series of diverse, complex, and labor-intensive biological, toxicological, and analytical techniques for effective characterization. While recent attempts, using machine learning based solely on primary amino acid sequences to predict biological toxins (e.g., conotoxins and animal venoms), have improved toxin identification, these methods are limited due to peptide conformational flexibility and the high frequency of cysteines present in toxin sequences. This results in an enumerable set of disulfide-bridged foldamers with different conformations of the same primary amino acid sequence that affect function and toxicity levels. Consequently, a given peptide may be toxic when its cysteine residues form a particular disulfide-bond pattern, while alternative bonding patterns (isoforms) or its reduced form (free cysteines with no disulfide bridges) may have little or no toxicological effects. Similarly, the same disulfide-bond pattern may be possible for other peptide sequences and result in different conformations that all exhibit varying toxicities to the same receptor or to different receptors. We present here new features, when combined with primary sequence features to train machine learning algorithms to predict conotoxins, that significantly increase prediction accuracy.

collisional cross section↗

Microbiomes Have the Power to Help or Hinder Your Health

They are everywhere: countless microorganisms that inhabit our world—even in your skin, mouth, gut and other parts of your body. Called microbiomes, these communities play a fundamental role in our ecosystem and our bodies, influencing everything from climate to human health. And scientists at PNNL are studying microbiomes to better understand how they influence our daily lives. Scientist Janet Jansson studies complex microbiomes in soil and the human intestine to understand changes in the composition or function of microbes. In the soil, these microbes are associated with carbon cycling and degrading pollutants, as well as plant health. In the intestine, they are responsible for digestion of our food and protection from pathogens. When they go awry, they can be associated with numerous inflammatory bowel diseases, such as Crohn’s. Understanding the factors underlying these microbiome changes will ultimately help researchers develop solutions to problems encountered within our world and our bodies.

54 ENVIRONMENTAL SCIENCES↗

A Structural Basis for Inhibition of the Complement Initiator Protease C1r by Lyme Disease Spirochetes

Abstract Complement evasion is a hallmark of extracellular microbial pathogens such as Borrelia burgdorferi, the causative agent of Lyme disease. Lyme disease spirochetes express nearly a dozen outer surface lipoproteins that bind complement components and interfere with their native activities. Among these, BBK32 is unique in its selective inhibition of the classical pathway. BBK32 blocks activation of this pathway by selectively binding and inhibiting the C1r serine protease of the first component of complement, C1. To understand the structural basis for BBK32-mediated C1r inhibition, we performed crystallography and size-exclusion chromatography–coupled small angle X-ray scattering experiments, which revealed a molecular model of BBK32-C in complex with activated human C1r. Structure-guided site-directed mutagenesis was combined with surface plasmon resonance binding experiments and assays of complement function to validate the predicted molecular interface. Analysis of the structures shows that BBK32 inhibits activated forms of C1r by occluding substrate interaction subsites (i.e., S1 and S1’) and reveals a surprising role for C1r B loop–interacting residues for full inhibitory activity of BBK32. The studies reported in this article provide for the first time (to our knowledge) a structural basis for classical pathway–specific inhibition by a human pathogen.

Garrigues, Ryan J. (ORCID:0000000259233250)↗

A meta-analysis of global fungal distribution reveals climate-driven patterns

The evolutionary and environmental factors that shape fungal biogeography are incompletely understood. Here, we assemble a large dataset consisting of previously generated mycobiome data linked to specific geographical locations across the world. We use this dataset to describe the distribution of fungal taxa and to look for correlations with different environmental factors such as climate, soil and vegetation variables. Our meta-study identifies climate as an important driver of different aspects of fungal biogeography, including the global distribution of common fungi as well as the composition and diversity of fungal communities. In our analysis, fungal diversity is concentrated at high latitudes, in contrast with the opposite pattern previously shown for plants and other organisms. Mycorrhizal fungi appear to have narrower climatic tolerances than pathogenic fungi. We speculate that climate change could affect ecosystem functioning because of the narrow climatic tolerances of key fungal taxa.

54 ENVIRONMENTAL SCIENCES↗

Signatures of Mollicutes-related endobacteria in publicly available Mucoromycota genomes

ABSTRACT Mucoromycota fungi and their Mollicutes-related endobacteria (MRE) are an ideal system for studying bacterial–fungal interactions and evolution due to the long-term and intimate nature of their interactions. However, methods for detecting MRE face specific challenges due to the poor representation of MRE in sequencing databases coupled with the high sequence divergence of their genomes, making traditional similarity searches unreliable. This has precluded estimations on the diversity of MRE associated with Mucoromycota. To determine the prevalence of previously undetected MRE in fungal genome sequences, we scanned 389 Mucoromycota genome assemblies available from the National Center for Biotechnology Information for the presence of MRE sequences using publicly available tools to map contigs from fungal assemblies to publicly available MRE genomes. We demonstrate a higher diversity of MRE genomes than previously described in Mucoromycota and a lack of cophylogeny between MRE and the majority of their fungal hosts. This supports the late invasion hypothesis regarding MRE acquisition across most of the examined fungal families. In contrast with other Mucoromycota lineages, MRE from the Gigasporaceae displayed some degree of cophylogeny with their hosts, which may indicate that horizontal transmission is restricted between members of this family or that transmission is strictly vertical. These results underscore the need for a refined process to capture sequencing data from potential fungal endosymbionts to discern their evolution and transmission. Screens of fungal genomes for MRE can help improve the quality of fungal genome assemblies while identifying new MRE lineages to further test hypotheses on their origin and evolution. IMPORTANCE Mollicutes-related endobacteria (MRE) are obligate intracellular bacteria found within Mucoromycota fungi. Despite their frequent detection, MRE roles in host functioning are still unknown. Comparative genomic investigations can improve our understanding of the impact of MRE on their fungal hosts by identifying similarities and differences in MRE genome evolution. However, MRE genomes have only been assembled from a small fraction of Mucoromycota hosts. Here, we demonstrate that MRE can be present yet undetected in publicly available Mucoromycota genome assemblies. We use these newfound sequences to assess the broader diversity of MRE and their phylogenetic relationships with respect to their hosts. We demonstrate that publicly available tools can be used to extract novel MRE sequences from assembled fungal genomes leading to insights on MRE evolution. This work contributes to a greater understanding of the fungal microbiome, which is crucial to improving knowledge on the dynamics and impacts of fungi in microbial ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗

Proton transfer reactions in donor site mutants of ESR, a retinal protein from Exiguobacterium sibiricum

Light-driven proton transport by microbial retinal proteins such as archaeal bacteriorhodopsin involves carboxylic residues as internal proton donors to the catalytic center which is a retinal Schiff base (SB). The proton donor, Asp96 in bacteriorhodopsin, supplies a proton to the transiently deprotonated Schiff base during the photochemical cycle. Subsequent proton uptake resets the protonated state of the donor. Here this two step process became a distinctive signature of retinal based proton pumps. Similar steps are observed also in many natural variants of bacterial proteorhodopsins and xanthorhodopsins where glutamic acid residues serve as a proton donor. Recently, however, an exception to this rule was found. A retinal protein from Exiguobacterium sibiricum, ESR, contains a Lys residue in place of Asp or Glu, which facilitates proton transfer from the bulk to the SB. Lys96 can be functionally replaced with the more common donor residues, Asp or Glu. Proton transfer to the SB in the mutants containing these replacements (K96E and K96D/A47T) is much faster than in the proteins lacking the proton donor (K96A and similar mutants), and in the case of K96D/A47T, comparable with that in the wild type, indicating that carboxylic residues can replace Lys96 as proton donors in ESR. We show here that there are important differences in the functioning of these residues in ESR from the way Asp96 functions in bacteriorhodopsin. Reprotonation of the SB and proton uptake from the bulk occur almost simultaneously during the M to N transition (as in the wild type ESR at neutral pH), whereas in bacteriorhodopsin these two steps are well separated in time and occur during the M to N and N to O transitions, respectively.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗