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All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA). Final report

The goal of the Stahl lab is to use a combination of isotopic, genomic, and activity-based analyses in the field, of selected field isolates, and defined synthetic communities to identify sources and sinks of different nitrogen species in the field, focusing on the FRC at the ORNL. The most general research objective is to characterize variants in the pathway for denitrification among field isolates and synthetic microbial assemblies as a foundation for predicting the biotic and abiotic controls of nitrogen and carbon transformations in the field. Specifically, we have focused on characterizing environmental controls of nitrous oxide emissions, an important greenhouse gas and ozone depleting molecule. Studies have mapped the flux of nitrous oxide and controlling genetic elements within the FRC subsurface, indicating the importance of different enzyme variants in nitrous oxide production and consumption in controlling surface emissions. We have developed methods to quantify and catalog the isotopic signatures imparted on nitrous oxide by different nitrous oxide reductase variants in isolated organisms and model synthetic communities. We have developed methods to quantify and catalog the nitrous oxide affinity of field relevant isolates and their biomass yield when respiring nitrous oxide on different carbon substrates. We continue to expand the collection of field-relevant microbial isolates within ENIGMA by focused enrichment and isolation from field material. These objectives have been facilitated by the development of new technologies, including: 1) the development of a multiplexed automated optical density measurement system for quantifying growth kinetics of microorganisms dependent on gaseous substrates in monoculture or gas exchange in synthetic communities and 2) the development of a microcalorimetric analysis format to quantify the impact of environmental stress on microbial maintenance energy. Since field relevant processes are mediated by assemblies of organisms interacting with each other and their local environment, we have also collaborated with other ENIGMA investigators on the assembly and characterization of synthetic communities. These studies have evaluated the role of organisms lacking a complete pathway for denitrification in the control of field processes, measuring process rates of assemblies of organisms that individually lack the full denitrification pathway but mediate the complete denitrification when growing together. These efforts have so far resulted in the publication of 18 peer reviewed papers ranging from ecological and physiological drivers of pathway optimization to field-scale process characterization.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Choreographing root architecture and rhizosphere interactions through synthetic biology

Abstract Climate change is driving extreme changes to the environment, posing substantial threats to global food security and bioenergy. Given the direct role of plant roots in mediating plant-environment interactions, engineering the form and function of root systems and their associated microbiota may mitigate these effects. Synthetic genetic circuits have enabled sophisticated control of gene expression in microbial systems for years and a surge of advances has heralded the extension of this approach to multicellular plant species. Targeting these tools to affect root structure, exudation, and microbe activity on root surfaces provide multiple strategies for the advancement of climate-ready crops.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Acoustic force spectroscopy reveals subtle differences in cellulose unbinding behavior of carbohydrate-binding modules

Protein adsorption to solid carbohydrate interfaces is critical to many biological processes, particularly in biomass deconstruction. To engineer more-efficient enzymes for biomass deconstruction into sugars, it is necessary to characterize the complex protein–carbohydrate interfacial interactions. A carbohydrate-binding module (CBM) is often associated with microbial surface-tethered cellulosomes or secreted cellulase enzymes to enhance substrate accessibility. However, it is not well known how CBMs recognize, bind, and dissociate from polysaccharides to facilitate efficient cellulolytic activity, due to the lack of mechanistic understanding and a suitable toolkit to study CBM–substrate interactions. Our work outlines a general approach to study the unbinding behavior of CBMs from polysaccharide surfaces using a highly multiplexed single-molecule force spectroscopy assay. Here, we apply acoustic force spectroscopy (AFS) to probe a Clostridium thermocellum cellulosomal scaffoldin protein (CBM3a) and measure its dissociation from nanocellulose surfaces at physiologically relevant, low force loading rates. An automated microfluidic setup and method for uniform deposition of insoluble polysaccharides on the AFS chip surfaces are demonstrated. The rupture forces of wild-type CBM3a, and its Y67A mutant, unbinding from nanocellulose surfaces suggests distinct multimodal CBM binding conformations, with structural mechanisms further explored using molecular dynamics simulations. Applying classical dynamic force spectroscopy theory, the single-molecule unbinding rate at zero force is extrapolated and found to agree with bulk equilibrium unbinding rates estimated independently using quartz crystal microbalance with dissipation monitoring. However, our results also highlight critical limitations of applying classical theory to explain the highly multivalent binding interactions for cellulose–CBM bond rupture forces exceeding 15 pN.

09 BIOMASS FUELS↗
DOE OSTI2022

Edaphic controls on genome size and GC content of bacteria in soil microbial communities

Nutrient limitation has been shown to reduce bacterial genome size and influence nucleotide composition; however, much of this work has been conducted in marine systems and the factors which shape soil bacterial genomic traits remain largely unknown. Here, for this work, we determined average genome size, GC content, codon usage, and amino acid content from 398 soil metagenomes across a broad geographic range and used machine-learning to determine the environmental parameters that most strongly explain the distribution of these traits. We found that genomic trait averages were most related to pH, which we suggest is primarily due to the correlation of pH with several environmental parameters, particularly soil carbon content. Low pH soils had higher carbon to nitrogen ratios (C:N) and tended to have communities with lower GC content and larger genomes, potentially a response to increased physiological stress and a requirement for metabolic diversity. Conversely, communities in high pH and low soil C:N had smaller genomes and higher GC content—indicating potential resource driven selection against AT base pairs, which have a higher C:N than GC base pairs. Similarly, we found that nutrient conservation also applied to amino acid stoichiometry, where bacteria in soils with low C:N ratios tended to code for amino acids with lower C:N. Together, these relationships point towards fundamental mechanisms that underpin genome size, and nucleotide and amino acid selection in soil bacteria.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Gut microbiome dynamics and associations with mortality in critically ill patients

Abstract Background Critical illness and care within the intensive care unit (ICU) leads to profound changes in the composition of the gut microbiome. The impact of such changes on the patients and their subsequent disease course remains uncertain. We hypothesized that specific changes in the gut microbiome would be more harmful than others, leading to increased mortality in critically ill patients. Methods This was a prospective cohort study of critically ill adults in the ICU. We obtained rectal swabs from 52 patients and assessed the composition the gut microbiome using 16 S rRNA gene sequencing. We followed patients throughout their ICU course and evaluated their mortality rate at 28 days following admission to the ICU. We used selbal, a machine learning method, to identify the balance of microbial taxa most closely associated with 28-day mortality. Results We found that a proportional ratio of four taxa could be used to distinguish patients with a higher risk of mortality from patients with a lower risk of mortality (p = .02). We named this binarized ratio our microbiome mortality index (MMI). Patients with a high MMI had a higher 28-day mortality compared to those with a low MMI (hazard ratio, 2.2, 95% confidence interval 1.1–4.3), and remained significant after adjustment for other ICU mortality predictors, including the presence of the acute respiratory distress syndrome (ARDS) and the Acute Physiology and Chronic Health Evaluation (APACHE II) score (hazard ratio, 2.5, 95% confidence interval 1.4–4.7). High mortality was driven by taxa from theAnaerococcus(genus) andEnterobacteriaceae(family), while lower mortality was driven byParasutterellaandCampylobacter(genera). Conclusions Dysbiosis in the gut of critically ill patients is an independent risk factor for increased mortality at 28 days after adjustment for clinically significant confounders. Gut dysbiosis may represent a potential therapeutic target for future ICU interventions.

Gastroenterology & Hepatology↗
DOE OSTI2020

Microbial carbon limitation: The need for integrating microorganisms into our understanding of ecosystem carbon cycling

Abstract Numerous studies have demonstrated that fertilization with nutrients such as nitrogen, phosphorus, and potassium increases plant productivity in both natural and managed ecosystems, demonstrating that primary productivity is nutrient limited in most terrestrial ecosystems. In contrast, it has been demonstrated that heterotrophic microbial communities in soil are primarily limited by organic carbon or energy. While this concept of contrasting limitations, that is, microbial carbon and plant nutrient limitation, is based on strong evidence that we review in this paper, it is often ignored in discussions of ecosystem response to global environment changes. The plant‐centric perspective has equated plant nutrient limitations with those of whole ecosystems, thereby ignoring the important role of the heterotrophs responsible for soil decomposition in driving ecosystem carbon storage. To truly integrate carbon and nutrient cycles in ecosystem science, we must account for the fact that while plant productivity may be nutrient limited, the secondary productivity by heterotrophic communities is inherently carbon limited. Ecosystem carbon cycling integrates the independent physiological responses of its individual components, as well as tightly coupled exchanges between autotrophs and heterotrophs. To the extent that the interacting autotrophic and heterotrophic processes are controlled by organisms that are limited by nutrient versus carbon accessibility, respectively, we propose that ecosystems by definition cannot be ‘limited’ by nutrients or carbon alone. Here, we outline how models aimed at predicting non‐steady state ecosystem responses over time can benefit from dissecting ecosystems into the organismal components and their inherent limitations to better represent plant–microbe interactions in coupled carbon and nutrient models.

Soong, Jennifer L.↗
DOE OSTI2022

Anaerobic Degradation of Alkanes by Marine Archaea

Alkanes are saturated apolar hydrocarbons that range from their simplest form, methane, to high-molecular-weight compounds. Although alkanes were once considered biologically recalcitrant under anaerobic conditions, microbiological investigations have now identified several microbial taxa that can anaerobically degrade alkanes. Here we review recent discoveries in the anaerobic oxidation of alkanes with a specific focus on archaea that use specific methyl coenzyme M reductases to activate their substrates. Our understanding of the diversity of uncultured alkane-oxidizing archaea has expanded through the use of environmental metagenomics and enrichment cultures of syntrophic methane-, ethane-, propane-, and butane-oxidizing marine archaea with sulfate-reducing bacteria. A recently cultured group of archaea directly couples long-chain alkane degradation with methane formation, expanding the range of substrates used for methanogenesis. This article summarizes the rapidly growing knowledge of the diversity, physiology, and habitat distribution of alkane-degrading archaea.

Microbiology↗
DOE OSTI2026

Hypophosphite Is a Naturally Occurring Selective Inhibitor of Syntrophic Methanogenesis

Microbial methanogenesis is a major contributor to global warming, and methane fluxes represent a loss of energy and electrons from industrial ecosystems. The chemical space of methane control strategies is still underexplored. Most known methanogenesis inhibitors target methanogenic archaeal enzymes. However, interference with the exchange of syntrophic electron carriers (H 2 or formate) in methanogenic systems presents an additional target for methane control. Here, we show that hypophosphite (H 2 PO 2 − ), an inorganic formate analogue, is a potent and selective inhibitor of syntrophic methanogenesis versus primary fermentation in rice field sediments and cattle rumens. Hypophosphite is also generally recognized as safe and relatively nontoxic to plants and animals. Genetic screens and physiological assays in the model methanogen Methanococcus maripaludis S2 implicate formate metabolism as the target of hypophosphite inhibition. Currently, there is no known biological pathway for anaerobic hypophosphite oxidation, and hypophosphite is stable in anoxic sediments for weeks to months. Given its widespread natural occurrence, we propose that hypophosphite may modulate the carbon cycle in natural environments. Taken together, our results suggest that hypophosphite could be used as a safe, inexpensive strategy for methane control in syntrophic methanogenic ecosystems.

chemical inhibitor↗
DOE OSTI2023

Influence of sulfide on diazotrophic growth of the methanogen Methanococcus maripaludis and its implications for the origin of nitrogenase

Abstract Methanogens inhabit euxinic (sulfide-rich) or ferruginous (iron-rich) environments that promote the precipitation of transition metals as metal sulfides, such as pyrite, reducing metal or sulfur availability. Such environments have been common throughout Earth’s history raising the question as to how anaerobes obtain(ed) these elements for the synthesis of enzyme cofactors. Here, we show a methanogen can synthesize molybdenum nitrogenase metallocofactors from pyrite as the source of iron and sulfur, enabling nitrogen fixation. Pyrite-grown, nitrogen-fixing cells grow faster and require 25-fold less molybdenum than cells grown under euxinic conditions. Growth yields are 3 to 8 times higher in cultures grown under ferruginous relative to euxinic conditions. Physiological, transcriptomic, and geochemical data indicate these observations are due to sulfide-promoted metal limitation, in particular molybdenum. These findings suggest that molybdenum nitrogenase may have originated in a ferruginous environment that titrated sulfide to form pyrite, facilitating the availability of sufficient iron, sulfur, and molybdenum for cofactor biosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Chapter 14: Dynamic Flux Analysis: An Experimental Approach of Fluxomics

Metabolic flux analysis represents an essential perspective to understand cellular physiology and offers quantitative information to guide pathway engineering. A valuable approach for experimental elucidation of metabolic flux is dynamic flux analysis, which estimates the relative or absolute flow rates through a series of metabolic intermediates in a given pathway. It is based on kinetic isotope labeling experiments, liquid chromatography-mass spectrometry (LC-MS), and computational analysis that relate kinetic isotope trajectories of metabolites to pathway activity. Herein, we illustrate the mathematic principles underlying the dynamic flux analysis and mainly focus on describing the experimental procedures for data generation. This protocol is exemplified using cyanobacterial metabolism as an example, for which reliable labeling data for central carbon metabolites can be acquired quantitatively. This protocol is applicable to other microbial systems as well and can be readily adapted to address different metabolic processes.

BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii

Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Protistan community composition and metabolism in the North Pacific Subtropical Gyre: Influences of mesoscale eddies and depth

Abstract Marine protists and their metabolic activities are intricately tied to the cycling of nutrients and the flow of energy through microbial food webs. Physiochemical changes in the environment, such as those that result from mesoscale eddies, may impact protistan communities, but the effects that such changes have on protists are poorly known. A metatranscriptomic study was conducted to investigate how eddies affected protists at adjacent cyclonic and anticyclonic eddy sites in the oligotrophic ocean at four depths from 25 to 250 m. Eddy polarity impacted protists at all depths sampled, although the effects of eddy polarity were secondary to the impact of depth across the depth range. Eddy‐induced vertical shifts in the water column yielded differences in the cyclonic and anticyclonic eddy protistan communities, and these differences were the most pronounced at and just below the deep chlorophyll maximum. An analysis of transcripts associated with protistan nutritional physiology at 150 m revealed that cyclonic eddies may support a more heterotrophic community, while anticyclonic eddies promote a more phototrophic community. The results of this study indicate that eddies alter the metabolism of protists particularly in the lower euphotic zone and may therefore impact carbon export from the euphotic zone.

Microbiology↗
DOE OSTI2021

Anaerobic Microbial Metabolism of Dichloroacetate

Dichloroacetate (DCA) commonly occurs in the environment due to natural production and anthropogenic releases, but its fate under anoxic conditions is uncertain. Mixed culture RM comprising “Candidatus Dichloromethanomonas elyunquensis” strain RM utilizes DCA as an energy source, and the transient formation of formate, H 2 , and carbon monoxide (CO) was observed during growth. Only about half of the DCA was recovered as acetate, suggesting a fermentative catabolic route rather than a reductive dechlorination pathway. Sequencing of 16S rRNA gene amplicons and 16S rRNA gene-targeted quantitative real-time PCR (qPCR) implicated “Candidatus Dichloromethanomonas elyunquensis” strain RM in DCA degradation. An (S)-2-haloacid dehalogenase (HAD) encoded on the genome of strain RM was heterologously expressed, and the purified HAD demonstrated the cofactor-independent stoichiometric conversion of DCA to glyoxylate at a rate of 90 ± 4.6 nkat mg -1 protein. Differential protein expression analysis identified enzymes catalyzing the conversion of DCA to acetyl coenzyme A (acetyl-CoA) via glyoxylate as well as enzymes of the Wood-Ljungdahl pathway. Glyoxylate carboligase, which catalyzes the condensation of two molecules of glyoxylate to form tartronate semialdehyde, was highly abundant in DCA-grown cells. The physiological, biochemical, and proteogenomic data demonstrate the involvement of an HAD and the Wood-Ljungdahl pathway in the anaerobic fermentation of DCA, which has implications for DCA turnover in natural and engineered environments, as well as the metabolism of the cancer drug DCA by gut microbiota.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Bioactive diterpenoids impact the composition of the root-associated microbiome in maize (Zea mays)

Abstract Plants deploy both primary and species-specific, specialized metabolites to communicate with other organisms and adapt to environmental challenges, including interactions with soil-dwelling microbial communities. However, the role of specialized metabolites in modulating plant-microbiome interactions often remains elusive. In this study, we report that maize ( Zea mays ) diterpenoid metabolites with known antifungal bioactivities also influence rhizosphere bacterial communities. Metabolite profiling showed that dolabralexins, antibiotic diterpenoids that are highly abundant in roots of some maize varieties, can be exuded from the roots. Comparative 16S rRNA gene sequencing determined the bacterial community composition of the maize mutant Zman2 ( anther ear 2 ), which is deficient in dolabralexins and closely related bioactive kauralexin diterpenoids. The Zman2 rhizosphere microbiome differed significantly from the wild-type sibling with the most significant changes observed for Alphaproteobacteria of the order Sphingomonadales. Metabolomics analyses support that these differences are attributed to the diterpenoid deficiency of the Zman2 mutant, rather than other large-scale metabolome alterations. Together, these findings support physiological functions of maize diterpenoids beyond known chemical defenses, including the assembly of the rhizosphere microbiome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Heterospecific Neighbor Plants Impact Root Microbiome Diversity and Molecular Function of Root Fungi

Within the forest community, competition and facilitation between adjacent-growing conspecific and heterospecific plants are mediated by interactions involving common mycorrhizal networks. The ability of plants to alter their neighbor’s microbiome is well documented, but the molecular biology of plant-fungal interactions during competition and facilitation has not been previously examined. We used a common soil-plant bioassay experiment to study molecular plant-microbial interactions among rhizosphere communities associated with Pinus taeda (native host) and Populus trichocarpa (non-native host). Gene expression of interacting fungal and bacterial rhizosphere communities was compared among three plant-pairs: Populus growing with Populus , Populus with Pinus , and Pinus with Pinus . Our results demonstrate that heterospecific plant partners affect the assembly of root microbiomes, including the changes in the structure of host specific community. Comparative metatranscriptomics reveals that several species of ectomycorrhizal fungi (EMF) and saprotrophic fungi exhibit different patterns of functional and regulatory gene expression with these two plant hosts. Heterospecific plants affect the transcriptional expression pattern of EMF host-specialists (e.g., Pinus -associated Suillus spp.) on both plant species, mainly including the genes involved in the transportation of amino acids, carbohydrates, and inorganic ions. Alteration of root microbiome by neighboring plants may help regulate basic plant physiological processes via modulation of molecular functions in the root microbiome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Bioavailable iron titrations reveal oceanic Synechococcus ecotypes optimized for different iron availabilities

Abstract The trace metal iron (Fe) controls the diversity and activity of phytoplankton across the surface oceans, a paradigm established through decades of in situ and mesocosm experimental studies. Despite widespread Fe-limitation within high-nutrient, low chlorophyll (HNLC) waters, significant contributions of the cyanobacterium Synechococcus to the phytoplankton stock can be found. Correlations among differing strains of Synechococcus across different Fe-regimes have suggested the existence of Fe-adapted ecotypes. However, experimental evidence of high- versus low-Fe adapted strains of Synechococcus is lacking, and so we investigated the transcriptional responses of microbial communities inhabiting the HNLC, sub-Antarctic region of the Southern Ocean during the Spring of 2018. Analysis of metatranscriptomes generated from on-deck incubation experiments reflecting a gradient of Fe-availabilities reveal transcriptomic signatures indicative of co-occurring Synechococcus ecotypes adapted to differing Fe-regimes. Functional analyses comparing low-Fe and high-Fe conditions point to various Fe-acquisition mechanisms that may allow persistence of low-Fe adapted Synechococcus under Fe-limitation. Comparison of in situ surface conditions to the Fe-titrations indicate ecological relevance of these mechanisms as well as persistence of both putative ecotypes within this region. This Fe-titration approach, combined with transcriptomics, highlights the short-term responses of the in situ phytoplankton community to Fe-availability that are often overlooked by examining genomic content or bulk physiological responses alone. These findings expand our knowledge about how phytoplankton in HNLC Southern Ocean waters adapt and respond to changing Fe supply.

Gilbert, Naomi E. (ORCID:0000000256915547)↗
DOE OSTI2024

Quantifying microbial roles in environmental iron oxidation via an integrated kinetics, `omics and metabolic modeling study (Final Report)

Iron oxyhydroxides are extremely reactive components of environmental systems, and therefore exert a strong influence on biogeochemical cycles. These oxyhydroxides strongly adsorb many biologically-relevant elements, including organic carbon and phosphate, as well as a wide range of metals including uranium and actinide species. Thus, the formation mechanism of iron oxyhydroxides are key to understanding both nutrient and contaminant cycling. Microorganisms can catalyze iron oxidation and promote the formation of Fe biominerals and thus are increasingly recognized as important players in biogeochemical cycling. However, it is completely unknown how much of environmental iron oxidation is biologically mediated versus abiotic, and various challenges in studying microbial iron oxidation have hindered accurate incorporation into hydrobiogeochemical models. The overarching goal of our work was to quantify and constrain microbial iron oxidation rates and use ‘omics to gain insight into the controls on this process, while developing tools to enable integration of biotic iron oxidation into hydrobiogeochemical models. Our work focused on the Savannah River Site (SRS) in South Carolina, where extensive microbial iron oxidation has been observed. At Tims Branch, part of the Argonne National Laboratory Wetland Hydrobiogeochemistry Science Focus Area (Argonne SFA), where groundwater discharges into a stream, iron-oxidizing microbial mats form and appear to be a major sink of uranium. In the wetlands that surround Tims Branch, there are wide swaths of iron microbial mats and flocs (mobilized mat). We measured biotic and abiotic iron oxidation rates using mats and water sampled from these sites and found that iron oxidation is primarily carried out by chemolithotrophic microorganisms. The resulting rate constants can be incorporated into models. These mats were characterized by metagenomics and metatranscriptomics, which showed that aerobic chemolithotrophs were the dominant iron-oxidizing bacteria (FeOB), and these included Gallionellaceae and Leptothrix, and possibly Rhodoferax, which is known as an Fe-reducer but may also oxidize Fe(II). This demonstrated that diverse FeOB can coexist and suggests that there are a range of niches and therefore drivers of chemolithotrophic iron oxidation. Analysis of reconstructed genomes strongly suggests that a major factor in diversity is carbon source, as genomes contained varied pathways for autotrophy and heterotrophy. We performed an in-depth analysis of Leptothrix ochracea genomes, since this sheath-former is one of the primary mat builders, yet its physiology remained unresolved. A combination of genomics, transcriptomics, and metabolic modeling suggest that L. ochracea grows mixotrophically using a combination of Fe(II) and organics for energy and both inorganic and organic carbon to create biomass. This contrasts with the largely autotrophic Gallionellaceae (Gallionella, Sideroxydans, and Ferriphaselus) also present in the mats and flocs. Remarkably, multiple FeOB, both Leptothrix and Gallionellaceae, showed activity in response to Fe(II) in live mat incubations. We tracked the gene expression of individual MAGs to Fe(II) and found that various autotrophic and heterotrophic FeOB responded to Fe(II), increasing expression of both carbon fixation and organic utilization genes. The results of the integrated field, kinetics, and omics studies give detailed insight into 1) the taxa that oxidize Fe, and 2) how they connect Fe, C, and N cycles. Towards the goal of connecting omics data to hydrobiogeochemical models, we worked with the KBase team to create a template metabolic model for chemolithotrophic iron oxidation. We initially modeled the well-characterized isolate Gallionellaceae Sideroxydans lithotrophicus, and also applied the model to the mixotroph L. ochracea. In all, we have characterized diverse FeOB in a representative wetland system and solved key problems that enable better incorporation of iron-oxidizing microbes into hydrobiogeochemical models.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Controlling circuitry underlies the growth optimization of Saccharomyces cerevisiae

Microbial growth emerges from coordinated synthesis of various cellular components from limited resources. In Saccharomyces cerevisiae, cyclic AMP (cAMP)-mediated signaling is shown to orchestrate cellular metabolism; however, it remains unclear quantitatively how the controlling circuit drives resource partition and subsequently shapes biomass growth. Here we combined experiment with mathematical modeling to dissect the signaling-mediated growth optimization of S. cerevisiae. We showed that, through cAMP-mediated control, the organism achieves maximal or nearly maximal steady-state growth during the utilization of multiple tested substrates as well as under perturbations impairing glucose uptake. However, the optimal cAMP concentration varies across cases, suggesting that different modes of resource allocation are adopted for varied conditions. Under settings with nutrient alterations, S. cerevisiae tunes its cAMP level to dynamically reprogram itself to realize rapid adaptation. Moreover, to achieve growth maximization, cells employ additional regulatory systems such as the GCN2-mediated amino acid control. This study establishes a systematic understanding of global resource allocation in S. cerevisiae, providing insights into quantitative yeast physiology as well as metabolic strain engineering for biotechnological applications.

59 BASIC BIOLOGICAL SCIENCES↗