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At least 91 records · Page 5

Quantifying Membrane Structure and Dynamics during Bioproduct Production in Zymomonas mobilis by Molecular Simulation

The conversion of lignocellulosic biomass into biofuels and bioproducts by microbial biorefineries is central to a sustainable chemical industry. Zymomonas mobilis is one such biorefinery chassis and is resistant to ethanol stress, leading to its use in biomass conversion to biofuels and bioproducts. However, Z. mobilis growth is often inhibited by organic acids, aldehydes, alcohols, ketones, and amides found in biomass hydrolysate. The resulting slow growth inhibits production and as a result drives up the price for the resulting products. One hypothesis is that these molecules interact with or disrupt the bacterial membrane, triggering stress responses and hindering growth. To test this hypothesis at the molecular level, we employ all-atom molecular dynamics (MD) simulations to investigate lignocellulose-derived small molecules and their impact on a biologically relevant Z. mobilis membrane model. Simulations were conducted across a range of inhibitor concentrations from 0 to 2.5 mol %, analyzing key membrane properties such as area per lipid (APL), membrane thickness, lipid-order parameter (−S CH ), lateral diffusion coefficient (D xy ), and permeability coefficient (Pm). From simulation, we observed altered membrane structure and dynamics at these modest small molecule concentrations commonly found in hydrolysates. Generally, the membranes become thinner, with a higher area per lipid and lower-order parameter as the small molecule concentration increases. These trends are stronger for more hydrophobic molecules with greater hydrophobic bulk, as isobutanol, propanol, and propanoic acid showed greater membrane perturbations as the concentration increased compared to other small molecules. Tracking small molecule distributions directly in our equilibrium simulations allows us to determine concentration-dependent free energy profiles for these molecules. While the trends are noisy, generally the barriers to crossing the membrane decrease as the concentration increases, indicating that the membranes become leakier as small molecule concentrations rise. Comparing between native Z. mobilis membranes with hopanoids and membranes sharing the same phospholipid composition but without hopanoids, hopanoids stabilize and order the membrane for smaller molecules to maintain membrane structure but appear insufficient for larger hydrophobic molecules like isobutanol. These findings provide a mechanistic understanding of how small molecules found in biomass degradation streams interact with the Z. mobilis membrane, offering valuable insights for future strain engineering efforts to optimize biofuel and bioproduct synthesis from biomass feedstocks by highlighting limits to small molecule tolerance. This knowledge can guide the modification of membrane composition to develop more robust microbes, thereby improving microbial survival and yields in industrial contexts.

Singh, Nitin Kumar [Michigan State Univ., East Lan↗

Carbon cycling across ecosystem succession in a north temperate forest: Controls and management implications

Despite decades of progress, much remains unknown about successional trajectories of carbon (C) cycling in north temperate forests. Drivers and mechanisms of these changes, including the role of different types of disturbances, are particularly elusive. To address this gap, we synthesized decades of data from experimental chronosequences and long-term monitoring at a well-studied, regionally representative field site in northern Michigan, USA. Our study provides a comprehensive assessment of changes in above- and belowground ecosystem components over two centuries of succession, links temporal dynamics in C pools and fluxes with underlying drivers, and offers several conceptual insights to the field of forest ecology. Our first advance shows how temporal dynamics in some ecosystem components are consistent across severe disturbances that reset succession and partial disturbances that slightly modify it: both of these disturbance types increase soil N availability, alter fungal community composition, and alter growth and competitive interactions between short-lived pioneer and longer-lived tree taxa. Further, these changes in turn affect soil C stocks, respiratory emissions, and other belowground processes. Second, we show that some other ecosystem components have effects on C cycling that are not consistent over the course of succession. For example, canopy structure does not influence C uptake early in succession but becomes important as stands develop, and the importance of individual structural properties changes over the course of two centuries of stand development. Third, we show that in recent decades, climate change is masking or overriding the influence of community composition on C uptake, while respiratory emissions are sensitive to both climatic and compositional change. In synthesis, we emphasize that time is not a driver of C cycling; it is a dimension within which ecosystem drivers such as canopy structure, tree and microbial community composition change. Changes in those drivers, not in forest age, are what control forest C trajectories, and those changes can happen quickly or slowly, through natural processes or deliberate intervention. Stemming from this view and a whole-ecosystem perspective on forest succession, we offer management applications from this work and assess its broader relevance to understanding long-term change in other north temperate forest ecosystems.

54 ENVIRONMENTAL SCIENCES↗

Utilization of on-site resources for Regenerative Life Support Systems at a lunar outpost

Regenerative life support systems (RLSS) will be required to regenerate air, water, and wastes, and to produce food for human consumption during long-duration stays on the moon. It may be possible to supplement some of the materials needed for RLSS from resources on the moon. Natural materials at the lunar surface may be used for a variety of lunar RLSS needs, including (i) soils or solid-support substrates for plant growth, (ii) sources for extraction of essential, plant-growth nutrients, (iii) substrates for microbial populations in the degradation of wastes, (iv) sources of O2 and H, which may be used to manufacture water, (v) feed stock materials for the synthesis of useful minerals (e.g., molecular sieves), and (vi) shielding materials surrounding the outpost structure to protect humans, plants, and microorganisms from harmful radiation.

Ming, D. W.↗

Cyanobacteria as cell factories for the photosynthetic production of sucrose

Biofuels and other biologically manufactured sustainable goods are growing in popularity and demand. Carbohydrate feedstocks required for industrial fermentation processes have traditionally been supplied by plant biomass, but the large quantities required to produce replacement commodity products may prevent the long-term feasibility of this approach without alternative strategies to produce sugar feedstocks. Cyanobacteria are under consideration as potential candidates for sustainable production of carbohydrate feedstocks, with potentially lower land and water requirements relative to plants. Several cyanobacterial strains have been genetically engineered to export significant quantities of sugars, especially sucrose. Sucrose is not only naturally synthesized and accumulated by cyanobacteria as a compatible solute to tolerate high salt environments, but also an easily fermentable disaccharide used by many heterotrophic bacteria as a carbon source. In this review, we provide a comprehensive summary of the current knowledge of the endogenous cyanobacterial sucrose synthesis and degradation pathways. We also summarize genetic modifications that have been found to increase sucrose production and secretion. Finally, we consider the current state of synthetic microbial consortia that rely on sugar-secreting cyanobacterial strains, which are co-cultivated alongside heterotrophic microbes able to directly convert the sugars into higher-value compounds (e.g., polyhydroxybutyrates, 3-hydroxypropionic acid, or dyes) in a single-pot reaction. We summarize recent advances reported in such cyanobacteria/heterotroph co-cultivation strategies and provide a perspective on future developments that are likely required to realize their bioindustrial potential.

59 BASIC BIOLOGICAL SCIENCES↗

BIOGEOCHEMICAL STUDIES OF PHOTOSYNTHETIC MICROBIAL MATS AND THEIR BIOTA

Photosynthetic microbial mats offer an opportunity to define holistic functionality at the millimeter scale. At the same time. their biogeochemistry contributes to environmental processes on a planetary scale. These mats are possibly direct descendents of the most ancient biological communities; communities in which oxygenic photosynthesis might have been invented. Mats provide one of the best natural systems to study how microbial populations associate to control dynamic biogeochemical gradients. These are self- sustaining, complete ecosystems in which light energy absorbed over a dial (24 hour) cycle drives the synthesis of spatially-organized, diverse biomass. Tightly-coupled microorganisms in the mat have specialized metabolisms that catalyze transformations of carbon, nitrogen, sulfur, and a host of other elements.

DesMarais, David↗

Production of fusel lactates via biocatalysis

Microbial enzymes are used for esterification of biomass-derived substrates for production of industrially valuable esters. E. coli was used as an efficient platform for recombinant synthesis of fusel lactates such as the green solvent ethyl lactate.

Davis, Ryan Wesley↗

Biogeochemical Processes in Microbial Ecosystems

The hierarchical organization of microbial ecosystems determines process rates that shape Earth's environment, create the biomarker sedimentary and atmospheric signatures of life and define the stage upon which major evolutionary events occurred. In order to understand how microorganisms have shaped the global environment of Earth and potentially, other worlds, we must develop an experimental paradigm that links biogeochemical processes with ever-changing temporal and spatial distributions of microbial population, and their metabolic properties. Photosynthetic microbial mats offer an opportunity to define holistic functionality at the millimeter scale. At the same time, their Biogeochemistry contributes to environmental processes on a planetary scale. These mats are possibly direct descendents of the most ancient biological communities; communities in which oxygenic photosynthesis might have been invented. Mats provide one of the best natural systems to study how microbial populations associate to control dynamic biogeochemical gradients. These are self-sustaining, complete ecosystems in which light energy absorbed over a diel (24 hour) cycle drives the synthesis of spatially-organized, diverse biomass. Tightly-coupled microorganisms in the mat have specialized metabolisms that catalyze transformations of carbon, nitrogen. sulfur, and a host of other elements.

DesMarais, David J.↗

"Ames Research Center: Linking our Origins to our Future"

Our research traces a path from interstellar materials to inhabited worlds and beyond. We examine how protoplanetary disks evolve and form terrestrial planets, the evolutionary paths of habitable planets, and how external factors (e.g., orbital eccentricity) and internal factors (atmospheric circulation) affect habitability. We trace, spectroscopically and chemically, the evolution of organic molecules from the interstellar medium onto habitable bodies. We examine how membranes might form under prebiotic planetary conditions. We evolve proteins capable of sustaining early metabolism, such as synthesis of biopolymers and transport of ions across membranes. We estimate the frequency of finding a functional prebiotic protein that formed spontaneously. We characterize the formation of diagnostic microbial biosignatures in rock-hosted ecosystems in ophiolite springs as an analog for subsurface life within our solar system, and photosynthetic microbial mats as biota that could be detected on extrasolar planets. We develop quantitative models that simulate energy relationships, biogeochemical cycling, trace gas exchange, and biodiversity. We examine the effects of climate variability on a vegetation-rich biosphere over intermediate time scales, using South American ecosystems as a model. We address natural transport of life beyond its planet of origin, such as on a meteorite, where survivors must withstand radiation, desiccation, and time in transit. We fly organisms and ecosystems in low Earth orbit to test their resistance to space. The Ames E&PO program disseminates these themes to national- and international-scale audiences through partnerships with the California Academy of Sciences, Yellow stone National Park, New York Hall of Science, and several K-14 educational organizations.

DesMarais, David J.↗

Novel plant–microbe interactions: Rapid evolution of a legume–rhizobium mutualism in restored prairies

Abstract When plants colonize new habitats, the novel interactions they form with new mutualists or enemies can immediately affect plant performance. These novel interactions also may provoke rapid evolutionary responses and can be ideal scenarios for investigating how species interactions influence plant evolution. To explore how mutualists influence the evolution of colonizing plant populations, we capitalized on an experiment in which two former agricultural fields were seeded with identical prairie seed mixes in 2010. Six years later, we compared how populations of the legume Chamaecrista fasciculata from these sites and their original (shared) source population responded to nitrogen‐fixing rhizobia from the restoration sites in a greenhouse reciprocal cross‐inoculation experiment. We found that the two populations differed both from their original source population and from each other in the benefits they derive from rhizobia, and that one population has evolved reduced allocation to rhizobia (i.e. forms fewer rhizobium‐housing nodules). Synthesis . Our results suggest that these plant populations have evolved different ways of interacting with rhizobia, potentially in response to differences in rhizobium quality between sites. Our study illustrates how microbial mutualists may shape plant evolution in new environments and highlights how variation in microbial mutualists potentially may select for different evolutionary strategies in plant hosts.

Magnoli, Susan M.↗

KBase Narrative - Atacama Trench & Sn Clemente 2018 cruise Metagenomes -Decontamination through Metagenomic Fractionation and analysis of Metabolic Potential-

Deep-sea microbiomes sampled with traditional or pressure-retaining instrumentation A fundamental property of life in the sea is that with increasing depth must come increased adaptation to elevated hydrostatic pressures. This project aims to assess technology for the accurate characterization of microbial biodiversity and biochemistry at greater depths, and validate its use in some of the deepest and least-explored waters in the Ocean: Hadal Trench. Compared to the 0.1 megapascal (MPa) of surface waters, organisms inhabiting at the average depth of the Ocean (3800 m) have to deal with pressures of 38 MPa (5,500 psi). The effects of these high pressure have been most thoroughly investigated in microorganisms, where it has been found to affect membrane integrity and transport, cytoskeletal assembly and both DNA and protein synthesis. We will explore the diversity and activity of microorganisms from 2000 m to >8,000 m depths within the highly productive water column of hadal trenches, comparing seawater samples obtained with and without substantial pressure changes during recovery. A pressure retaining device was used to sample the microbial communities of the watercolumn with reduced decompression during retreival (Peoples et. al., 2019). References: Peoples LM, Norenberg M, Price D, McGoldrick M, Novotny M, Bochdansky A, Bartlett DH. A full-ocean-depth rated modular lander and pressure-retaining sampler capable of collecting hadal-endemic microbes under in situ conditions. Deep Sea Research Part I: Oceanographic Research Papers. 2019 Jan 1;143:50-7.

Plominsky, Alvaro M.↗

KBase Narrative - Atacama Trench & Sn Clemente 2018 cruise Metagenomes -Sequence quality filtration & Assembly-

Deep-sea microbiomes sampled with traditional or pressure-retaining instrumentation A fundamental property of life in the sea is that with increasing depth must come increased adaptation to elevated hydrostatic pressures. This project aims to assess technology for the accurate characterization of microbial biodiversity and biochemistry at greater depths, and validate its use in some of the deepest and least-explored waters in the Ocean: Hadal Trench. Compared to the 0.1 megapascal (MPa) of surface waters, organisms inhabiting at the average depth of the Ocean (3800 m) have to deal with pressures of 38 MPa (5,500 psi). The effects of these high pressure have been most thoroughly investigated in microorganisms, where it has been found to affect membrane integrity and transport, cytoskeletal assembly and both DNA and protein synthesis. We will explore the diversity and activity of microorganisms from 2000 m to >8,000 m depths within the highly productive water column of hadal trenches, comparing seawater samples obtained with and without substantial pressure changes during recovery. A pressure retaining device was used to sample the microbial communities of the watercolumn with reduced decompression during retreival (Peoples et. al., 2019). References: Peoples LM, Norenberg M, Price D, McGoldrick M, Novotny M, Bochdansky A, Bartlett DH. A full-ocean-depth rated modular lander and pressure-retaining sampler capable of collecting hadal-endemic microbes under in situ conditions. Deep Sea Research Part I: Oceanographic Research Papers. 2019 Jan 1;143:50-7.

Plominsky, Alvaro M.↗

Reverse β-oxidation pathways for efficient chemical production

Abstract Microbial production of fuels, chemicals, and materials has the potential to reduce greenhouse gas emissions and contribute to a sustainable bioeconomy. While synthetic biology allows readjusting of native metabolic pathways for the synthesis of desired products, often these native pathways do not support maximum efficiency and are affected by complex regulatory mechanisms. A synthetic or engineered pathway that allows modular synthesis of versatile bioproducts with minimal enzyme requirement and regulation while achieving high carbon and energy efficiency could be an alternative solution to address these issues. The reverse β-oxidation (rBOX) pathways enable iterative non-decarboxylative elongation of carbon molecules of varying chain lengths and functional groups with only four core enzymes and no ATP requirement. Here, we describe recent developments in rBOX pathway engineering to produce alcohols and carboxylic acids with diverse functional groups, along with other commercially important molecules such as polyketides. We discuss the application of rBOX beyond the pathway itself by its interfacing with various carbon-utilization pathways and deployment in different organisms, which allows feedstock diversification from sugars to glycerol, carbon dioxide, methane, and other substrates.

59 BASIC BIOLOGICAL SCIENCES↗

Scarcity of fixed carbon transfer in a model microbial phototroph–heterotroph interaction

Although the green alga Chlamydomonas reinhardtii has long served as a reference organism, few studies have interrogated its role as a primary producer in microbial interactions. Here, we quantitatively investigated C. reinhardtii’s capacity to support a heterotrophic microbe using the established coculture system with Mesorhizobium japonicum, a vitamin B 12 -producing α-proteobacterium. Using stable isotope probing and nanoscale secondary ion mass spectrometry (nanoSIMS), we tracked the flow of photosynthetic fixed carbon and consequent bacterial biomass synthesis under continuous and diurnal light with single-cell resolution. We found that more 13 C fixed by the alga was taken up by bacterial cells under continuous light, invalidating the hypothesis that the alga’s fermentative degradation of starch reserves during the night would boost M. japonicum heterotrophy. 15 NH 4 assimilation rates and changes in cell size revealed that M. japonicum cells reduced new biomass synthesis in coculture with the alga but continued to divide—a hallmark of nutrient limitation often referred to as reductive division. Despite this sign of starvation, the bacterium still synthesized vitamin B 12 and supported the growth of a B 12 -dependent C. reinhardtii mutant. Finally, we showed that bacterial proliferation could be supported solely by the algal lysis that occurred in coculture, highlighting the role of necromass in carbon cycling. Collectively, these results reveal the scarcity of fixed carbon in this microbial trophic relationship (particularly under environmentally relevant light regimes), demonstrate B 12 exchange even during bacterial starvation, and underscore the importance of quantitative approaches for assessing metabolic coupling in algal–bacterial interactions.

59 BASIC BIOLOGICAL SCIENCES↗

Drought and plant litter chemistry alter microbial gene expression and metabolite production

Drought represents a significant stress to microorganisms and is known to reduce microbial activity and organic matter decomposition in Mediterranean ecosystems. However, we lack a detailed understanding of the drought stress response of microbial decomposers. Here we present metatranscriptomic and metabolomic data on the physiological response of in situ microbial communities on plant litter to long-term drought in Californian grass and shrub ecosystems. We hypothesised that drought causes greater microbial allocation to stress tolerance relative to growth pathways. In grass litter, communities from the decade-long ambient and reduced precipitation treatments had distinct taxonomic and functional profiles. The most discernable physiological signatures of drought were production or uptake of compatible solutes to maintain cellular osmotic balance, and synthesis of capsular and extracellular polymeric substances as a mechanism to retain water. The results show a clear functional response to drought in grass litter communities with greater allocation to survival relative to growth that could affect decomposition under drought. In contrast, communities on chemically more diverse and complex shrub litter had smaller physiological differences in response to long-term drought but higher investment in resource acquisition traits across precipitation treatments, suggesting that the functional response to drought is constrained by substrate quality. Our findings suggest, for the first time in a field setting, a trade off between microbial drought stress tolerance, resource acquisition and growth traits in plant litter microbial communities.

59 BASIC BIOLOGICAL SCIENCES↗

Benchmarking and Improving Microbial-explicit Soil Biogeochemistry Models

Soils represent the largest terrestrial carbon pool on Earth. Yet emerging theories regarding stabilization of soil organic matter remain poorly represented in global-scale models; thus, underestimating the true uncertainty associated with potential terrestrial carbon cycle – climate feedbacks. With this project we created a testbed to compare and evaluate soil biogeochemical models (W. Wieder at the Univ. of CO, Boulder), data synthesis (R. Phillips, Indiana Univ.) and parameter estimation (S Allison, Univ. CA Irvine) to quantify uncertainty associated with potential soil C changes. This project resulted in 14 Presentations, 14 Publications and helped support the training of three graduate students. A significant accomplishment with this work was the creation of a soil biogeochemical testbed, which forces three soil carbon models (CASA, MIMICS, and CORPSE) with common environmental drivers.

54 ENVIRONMENTAL SCIENCES↗

Conservation of endo -glucanase 16 (EG16) activity across highly divergent plant lineages

Plant cell walls are highly dynamic structures that are composed predominately of polysaccharides. As such, endogenous carbohydrate active enzymes (CAZymes) are central to the synthesis and subsequent modification of plant cells during morphogenesis. The endo-glucanase 16 (EG16) members constitute a distinct group of plant CAZymes, angiosperm orthologs of which were recently shown to have dual β-glucan/xyloglucan hydrolase activity. Molecular phylogeny indicates that EG16 members comprise a sister clade with a deep evolutionary relationship to the widely studied apoplastic xyloglucan endo-transglycosylases/hydrolases (XTH). A cross-genome survey indicated that EG16 members occur as a single ortholog across species and are widespread in early diverging plants, including the non-vascular bryophytes, for which functional data were previously lacking. Remarkably, enzymological characterization of an EG16 ortholog from the model moss Physcomitrella patens (PpEG16) revealed that EG16 activity and sequence/structure are highly conserved across 500 million years of plant evolution, vis-à-vis orthologs from grapevine and poplar. Ex vivo biomechanical assays demonstrated that the application of EG16 gene products caused abrupt breakage of etiolated hypocotyls rather than slow extension, thereby indicating a mode-of-action distinct from endogenous expansins and microbial endo-glucanases. As a result, the biochemical data presented here will inform future genomic, genetic, and physiological studies of EG16 enzymes.

59 BASIC BIOLOGICAL SCIENCES↗

Diacylglycerol enantiomer selectivity of diacylglycerol acyltransferases highlights metabolic specialization in triacylglycerol synthesis across the tree of life

Triacylglycerols are the major energy storage lipids in plants, animals, and microorganisms, and are predominantly produced by acyl-CoA:diacylglycerol (DAG) acyltransferases (DGATs). Two enantiomers of the DAG substrate, sn -1,2 and sn -2,3, can be produced by different biological mechanisms; however, little is known about which species produce each enantiomer, the selectivity of DGAT isoforms for either enantiomer, or whether DGAT enantiomer selectivity varies across organisms. Here, DAG enantiomer selectivity of DGAT1 and DGAT2 was measured from eight seed plants, two mammals, one oleaginous yeast, and one photosynthetic microalga using enantiomer-specific in vitro DGAT assays. Across most plants, DGAT1 favored sn -1,2-DAG, whereas DGAT2 preferentially utilized sn -2,3-DAG. However, there were several exceptions. Mammalian DGAT1, DGAT2, and microbial DGAT1s efficiently used both DAG enantiomers, while microbial DGAT2s had unique selectivity. The selectivity of several DGATs for combined acyl-CoA and DAG enantiomer molecular species were also evaluated for biotechnical applications. Therefore, DGAT DAG enantiomer selectivity is common yet strongly dependent on lineage and isoform and likely shaped in part by species-specific metabolic context of triacylglycerol synthesis, turnover, and remodeling. This work expands our understanding of DGAT function and establishes a foundation for leveraging enantiomer-selective acyltransferases in metabolic engineering of tailored lipid products.

Arabidopsis thaliana↗

Opportunities to advance the synthesis of ecology and evolution

Despite decades of research on the interactions between ecology and evolution, opportunities still remain to further integrate the two disciplines, especially when considering multispecies systems. Here, we discuss two such opportunities. First, the traditional emphasis on the distinction between evolutionary and ecological processes should be further relaxed as it is particularly unhelpful in the study of microbial communities, where the very notion of species is hard to define. Second, key processes of evolutionary theory such as adaptation should be exported to hierarchical levels higher than populations to make sense of biodiversity dynamics. Together, we argue that broadening our perspective of eco-evolutionary dynamics to be more inclusive of all biodiversity, both phylogenetically and hierarchically, will open up fertile new research directions and help us to address one of the major scientific challenges of our time, that is, to understand and predict changes in biodiversity in the face of rapid environmental change.

54 ENVIRONMENTAL SCIENCES↗