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At least 937 records · Page 52
All sourcesNASA NTRSDOE OSTI
NASA NTRS2005

Intraspecific differences in bacterial responses to modelled reduced gravity

AIMS: Bacteria are important residents of water systems, including those of space stations which feature specific environmental conditions, such as lowered effects of gravity. The purpose of this study was to compare responses with modelled reduced gravity of space station, water system bacterial isolates with other isolates of the same species. METHODS AND RESULTS: Bacterial isolates, Stenotrophomonas paucimobilis and Acinetobacter radioresistens, originally recovered from the water supply aboard the International Space Station (ISS) were grown in nutrient broth under modelled reduced gravity. Their growth was compared with type strains S. paucimobilis ATCC 10829 and A. radioresistens ATCC 49000. Acinetobacter radioresistens ATCC 49000 and the two ISS isolates showed similar growth profiles under modelled reduced gravity compared with normal gravity, whereas S. paucimobilis ATCC 10829 was negatively affected by modelled reduced gravity. CONCLUSIONS: These results suggest that microgravity might have selected for bacteria that were able to thrive under this unusual condition. These responses, coupled with impacts of other features (such as radiation resistance and ability to persist under very oligotrophic conditions), may contribute to the success of these water system bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Water quality is a significant factor in many environments including the ISS. Efforts to remove microbial contaminants are likely to be complicated by the features of these bacteria which allow them to persist under the extreme conditions of the systems.

Non-NASA Center↗
DOE OSTI2026

Tendencies of Soil Microbial NO Emissions During HI‐SCALE as Predicted by a Nitrification/Denitrification Scheme

Many atmospheric chemical processes, including the formation of secondary organic aerosol (SOA), are strongly modulated by the reactions of NO and NO 2 (NO x ). Though NO x is controlled by anthropogenic emissions near urban areas, in rural areas soil microbes can be a significant contribution to NO emission globally. The relative rates of emissions of different nitrogen-containing species (e.g., NO, N 2 O, HONO, and N 2 ) are strong functions of soil properties such as temperature, moisture content, pH, and soil carbon and nitrogen pools. However, typical large-scale biogeochemical models either express these emissions simplistically, or not at all. Here we investigate the potential impact of soil NO emissions on atmospheric chemistry and SOA formation over regional and monthly time scales, specifically the 2016 spring and summer Intensive Observational Periods (IOPs) of the Holistic Interactions of Shallow Clouds, Aerosols and Land Ecosystems (HI-SCALE) field campaign. We implement the soil NO nitrification/denitrification parameterization of Rasool et al. (2019) into the Weather Research and Forecasting model coupled with Chemistry (WRF-Chem), supplemented by a 1-km soil moisture analysis. We then simulate both IOPs over the U.S. Great Plains, evaluating against ground stations and flight data. We show that soil NO emissions can account for a large fraction of total NO x and locally increase O 3 concentrations by up to 25%, while alleviating negative biases of gases and aerosols toward observations. Soil moisture and temperature changes between IOP1 and IOP2 lead to overall differences in emissions, but with large regional variability due to heterogeneous surface characteristics.

Atmoshpheric Chemistry↗
DOE OSTI2026

Multi-omic characterization of a soil microbial consortium reveals critical role of succinate and glutamate metabolism during calcium carbonate precipitation

Microbially induced calcium carbonate precipitation (MICP) holds potential for use in soil stabilization and carbon sequestration, with the overall efficiency of the process being a major determinant for use in many environmental and civil engineering applications. While the biogeochemical pathways and enzymes driving MICP are known, the microbial metabolic networks and community dynamics underlying such precipitation remain poorly characterized. To address this gap, we developed a four-member consortium of soil bacteria (Curtobacterium flaccumfaciens, Rhodococcus qingshengii, Microbacterium sp., and Bacillus toyonensis), termed carbon storing consortium - A (CSC-A), that is capable of MICP. Prior work shows that MICP production is higher in CSC-A compared to the sum of carbonate produced by each member, suggesting carbonate production is driven by consortium dynamics. To that end we used a multi-omic integration approach of genomics, transcriptomics, and metabolomics to investigate potential inter-species interactions that may influence the MICP phenotype. Genomic life history characterizations identified evidence of niche specialization by B. toyonensis and Microbacterium, while metatranscriptomic analysis suggests R. qingshengii is a keystone species during growth in urea. By comparing individual species’ metabolomes to the metabolic profile of a shared well of precipitated metabolites, we identified over 200 metabolites predicted to be produced or consumed by CSC-A members. Integrating both data types to search the KEGG reactome highlighted a network centered around glutamine metabolism and branched chain amino acid biosynthesis under regulation during CSC-A growth in urea. Succinate metabolism was also a major node in this network and laboratory assays confirmed that increasing the amount of succinate in the growth medium leads to increased carbonate precipitation by CSC-A, a critical confirmation of our modeling approach. By isolating and identifying the interconnected metabolic components underlying MICP in CSC-A, we identified keystone taxa, metabolites, and pathways important for future optimization of the application of this consortia to carbonate precipitation.

carbon storing consortium - A (CSC-A)↗
NASA NTRS2007

Spaceflight Alters Bacterial Gene Expression and Virulence and Reveals Role for Global Regulator Hfq

A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the spaceflight environment has never been accomplished due to significant technological and logistical hurdles. Moreover, the effects of spaceflight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared to identical ground control cultures. Global microarray and proteomic analyses revealed 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground based microgravity culture model. Spaceflight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during spaceflight missions and provide novel therapeutic options on Earth.

Wilson, J. W.↗
DOE OSTI2022

Data for Reshaping the 2-Pyrone Synthase Active Site for Chemoselective Biosynthesis of Polyketides

Engineering enzymes with novel reactivity and applying them in metabolic pathways to produce valuable products are quite challenging due to the intrinsic complexity of metabolic networks and the need for high in vivo catalytic efficiency. Triacetic acid lactone (TAL), naturally generated by 2-pyrone synthase (2PS), is a platform molecule that can be produced via microbial fermentation and further converted into value-added products. However, these conversions require extra synthetic steps under harsh conditions. We herein report a biocatalytic system for direct generation of TAL derivatives under mild conditions with controlled chemoselectivity by rationally engineering the 2PS active site and then rewiring the biocatalytic pathway in the metabolic network of E. coli to produce high-value products, such as kavalactone precursors, with yields up to 17 mg/L culture. Computer modeling indicates sterics and hydrogen-bond interactions play key roles in tuning the selectivity, efficiency, and yield.

Conversion↗
DOE OSTI2020

A Microbe Associated with Sleep Revealed by a Novel Systems Genetic Analysis of the Microbiome in Collaborative Cross Mice

The microbiome influences health and disease through complex networks of host genetics, genomics, microbes, and environment. Identifying the mechanisms of these interactions has remained challenging. Systems genetics in laboratory mice (Mus musculus) enables data-driven discovery of biological network components and mechanisms of host–microbial interactions underlying disease phenotypes. To examine the interplay among the whole host genome, transcriptome, and microbiome, we mapped QTL and correlated the abundance of cecal messenger RNA, luminal microflora, physiology, and behavior in a highly diverse Collaborative Cross breeding population. One such relationship, regulated by a variant on chromosome 7, was the association of Odoribacter (Bacteroidales) abundance and sleep phenotypes. In a test of this association in the BKS.Cg-Dock7 m +/+ Lepr db /J mouse model of obesity and diabetes, known to have abnormal sleep and colonization by Odoribacter, treatment with antibiotics altered sleep in a genotype-dependent fashion. The many other relationships extracted from this study can be used to interrogate other diseases, microbes, and mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Arctic Permafrost Thawing Enhances Sulfide Oxidation

Permafrost degradation is altering biogeochemical processes throughout the Arctic. Thaw-induced changes in organic matter transformations and mineral weathering reactions are impacting fluxes of inorganic carbon (IC) and alkalinity (ALK) in Arctic rivers. However, the net impact of these changing fluxes on the concentration of carbon dioxide in the atmosphere (pCO 2 ) is relatively unconstrained. Resolving this uncertainty is important as thaw-driven changes in the fluxes of IC and ALK could produce feedbacks in the global carbon cycle. Enhanced production of sulfuric acid through sulfide oxidation is particularly poorly quantified despite its potential to remove ALK from the ocean-atmosphere system and increase pCO 2 , producing a positive feedback leading to more warming and permafrost degradation. In this work, we quantified weathering in the Koyukuk River, a major tributary of the Yukon River draining discontinuous permafrost in central Alaska, based on water and sediment samples collected near the village of Huslia in summer 2018. Using measurements of major ion abundances and sulfate (SO 4 2- ) sulfur ( 34 S/ 32 S) and oxygen ( 18 O/ 16 O) isotope ratios, we employed the MEANDIR inversion model to quantify the relative importance of a suite of weathering processes and their net impact on pCO 2 . Calculations found that approximately 80% of SO 4 2- in mainstem samples derived from sulfide oxidation with the remainder from evaporite dissolution. Moreover, 34 S/ 32 S ratios, 13 C/ 12 C ratios of dissolved IC, and sulfur X-ray absorption spectra of mainstem, secondary channel, and floodplain pore fluid and sediment samples revealed modest degrees of microbial sulfate reduction within the floodplain. Weathering fluxes of ALK and IC result in lower values of pCO 2 over timescales shorter than carbonate compensation (~10 4 yr) and, for mainstem samples, higher values of pCO 2 over timescales longer than carbonate compensation but shorter than the residence time of marine (~10 7 yr). Furthermore, the absolute concentrations of and Mg 2+ in the Koyukuk River, as well as the ratios of SO 4 2- and Mg 2+ to other dissolved weathering products, have increased over the past 50 years. Through analogy to similar trends in the Yukon River, we interpret these changes as reflecting enhanced sulfide oxidation due to ongoing exposure of previously frozen sediment and changes in the contributions of shallow and deep flow paths to the active channel. Overall, these findings confirm that sulfide oxidation is a substantial outcome of permafrost degradation and that the sulfur cycle responds to permafrost thaw with a timescale-dependent feedback on warming.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

Robust Rapid Cellular Metabolite Sensing Using Benchtop NMR and SABRE-Hyperpolarized [1- 13 C]Pyruvate

Hyperpolarized NMR has emerged as a powerful analytical technique to significantly enhance targeted NMR signals, improving the sensitivity for investigations of unique chemical and biological dynamics. Here, we demonstrate the use of a hyperpolarization strategy based on Signal Amplification By Reversible Exchange (SABRE) to generate highly reproducible doses of a hyperpolarized [1- 13 C]pyruvate probe for benchtop characterization of yeast metabolism. This method allows rapid, scalable, and benchtop preparation of biocompatible hyperpolarized solutions suitable for live-cell experiments. We show that this production can be dove-tailed into a modular, compact workflow to characterize real-time metabolism in cell cultures, using Saccharomyces cerevisiae (Baker’s yeast) as a model organism. With high temporal resolution, we show that this method can resolve the conversion of hyperpolarized [1- 13 C]pyruvate into oxidative decarboxylation products CO 2 and bicarbonate. This conversion exhibits sustained and detectable metabolic activity for over 300 s after introduction of the agent to the cells. We model the metabolite kinetics to show decarboxylation activity and derive estimates of the pH over time from the CO 2 and bicarbonate (carbonic acid buffer system) equilibrium to probe changes in the cellular environment during active metabolism. These results highlight the utility of benchtop SABRE-hyperpolarized [1- 13 C]pyruvate as a scalable, specific probe for metabolic phenotyping of living cells using compact, low-cost instrumentation well-suited for future high-throughput applications across microbial engineering, drug response profiling, and dynamic metabolic screening.

fungi↗
NASA NTRSDate not supplied

A Lunar Ground Truth of Microbes that are Integral to Sustaining Biological Life Support Systems

A lunar outpost such as Gateway or a Mars transit vehicle will use a semi-closed to closed loop biological life support system (BLSS) adapted for microgravity and high levels of radiation. A moon habitat will likely exist under partial gravity conditions and sustained levels of high radiation as a semi-closed loop system able to get resources from Earth via Gateway. Additionally, the moon will act as a closed-loop testbed for Mars habitat operations and will employ various elements of in situ resource utilization (ISRU). Perhaps the longest running BLSS collective study to date is that of the European MELiSSA (Micro Ecological Life Support System Alternative) project, a circular life support system, established to gain knowledge on regenerative systems aimed at the highest degree of autonomy to produce food, water and oxygen from mission wastes. This setup has evaluated the use of discrete microbial compartments and a higher plant compartment to carry out the necessary life support functions to support crew. Such compartments utilize thermophilic anaerobes to break down human and inedible plant wastes, photoheterotrophic bacteria that can further metabolize volatile fatty acids, nitrifying bacteria that can convert ammonium to plant and microalgae available nitrates, and photoautotrophic bacteria and higher plants which will convert carbon dioxide to oxygen, purify water, and provide food for human consumption. This being the most advanced BLSS model example to date -aside from the Yuegong-1, Chinese Lunar Palace- with the most defined compartment composition, and the assumption that other BLSSs will build upon such models, it becomes relevant to study the effects of the lunar environment upon the microbes that are integral to a BLSS. In ramping up to advanced stage BLSS systems, relatively simple experiments can be conducted on Artemis missions using continuous culturing and sampling of the model microbes, subsequently subjected to DNA sequencing for mutational analysis monitoring and chemical analysis to assess the sustained ability to carry out their hallmark biochemical processes efficiently. A suite of microbes should be assessed, prioritizing those with the duel capacity to be utilized in an BLSS and the ability to biochemically facilitate ISRU goals, such as the ability to transform the biogeochemistry of Moon or Martian regolith into materials that can support crop growth or extract elements of industrial significance such as aluminum or iron.

Biological Life Support Systems↗
NASA NTRSDate not supplied

Constraining Venus Cloud Habitability: Earth's Aerobiosphere as an Analogue Environment

The potential habitability of Venus's cloud and haze layers has been debated for decades, with interest fueled by observations including disequilibria in atmospheric chemistry, strongly UV-absorbing particles, and (most recently) a controversial phosphine detection. Venus cloud temperature and pressure are clement by Earth standards; however, the calculated high acid activity and low water activity may exceed the limits of Earth biochemistry, based on observed life in extreme environments. A significant challenge in understanding Venus's habitability is the lack of an appropriate terrestrial analogue environment. Life has not been found in hydrothermal systems approaching Venus aerosol acidities, but these systems also contain high levels of other harsh solutes. Earth's stratospheric sulfate layer is a partial match in terms of acidity, desiccation, size, and isolation from the surface, but the few samples returned from these altitudes have yielded only sparse, inactive cells. Earth's tropospheric cloud droplets have an active microbial presence, but are larger and far more water-rich than Venus aerosols; tropospheric times aloft are also similar to microbial generation times, making Earth's aerobiosphere dependent on continuous surface replenishment. Direct detection of potential biosignatures from a single transect is challenging; Earth's cloud microbiota yields 102 - 108 viable cells per mL, equivalent to one per 103 - 109 particles, with highly dynamic and heterogeneous distribution. The most important in situ measurement for Venus cloud habitability is detailed aerosol composition: confirmation of acid and water activity, presence of potentially bioavailable nutrients and energy, and potential presence of organic matter. Modeling Venus cloud aerosol residence time in comparison to periodic influxes of water and photochemical energy, and thus the ability of an ecosystem to maintain itself over time, would also address a significant habitability constraint particular to aerobiospheres.

Venus↗
DOE OSTI2020

The EGS Collab Project: Learnings from Experiment 1

The primary objective of the EGS Collab Project sponsored by DOE is to increase the understanding needed to efficiently implement enhanced geothermal systems (EGS). One goal of the EGS Collab project is to create a collaborative research environment in which to study stimulation of crystalline rock at the 10 meter scale. Key to this effort is the collection of high quality data to allow comparison to numerical coupled process models in an effort to build confidence in the codes and modeling techniques used. In response to this, the EGS Collab team has created an underground test bed at the Sanford Underground Research Facility (SURF) in Lead SD at a depth of approximately 1.5 km to examine hydraulic fracturing (Experiment 1). We are currently designing a second test bed aimed at investigating shear stimulation (Experiment 2). At the Experiment 1 location, we have characterized our host rock using laboratory testing and numerous field-based geophysical and geological techniques, and created a well-instrumented test bed to allow us to carefully monitor stimulation events and flow tests. In addition to the installed geophysical sensors, we have used tracer tests, differences in the ambient microbial communities at flow collection locations, and cold water injection to inform us about dynamic flow pathways. In Experiment 1, we have hydraulically stimulated the host rock in a number times at several locations in one well, creating new fractures that connect to existing fractures between the injection and production boreholes. We have performed long-term ambient and chilled water injection tests as an analog to EGS, and have monitored system changes resulting from these water injections through geophysical monitoring, flow and pressure measurements, tracer tests, and microbiology. Here, we summarize the tests performed, issues identified including poroelastic and thermoelastic effects, Joule-Thomson effects, restarting effects, indications of flow channeling, and the primary learnings from Experiment 1.

Enhanced Geothermal Systems, EGS Collab, stimulati↗
DOE OSTI2024

Formation of a constructed microbial community in a nutrient-rich environment indicates bacterial interspecific competition

ABSTRACT Understanding the organizational principles of microbial communities is essential for interpreting ecosystem stability. Previous studies have investigated the formation of bacterial communities under nutrient-poor conditions or obligate relationships to observe cooperative interactions among different species. How microorganisms form stabilized communities in nutrient-rich environments, without obligate metabolic interdependency for growth, is still not fully disclosed. In this study, three bacterial strains isolated from the Populus deltoides rhizosphere were co-cultured in complex medium, and their growth behavior was tracked. These strains co-exist in mixed culture over serial transfer for multiple growth-dilution cycles. Competition is proposed as an emergent interaction relationship among the three bacteria based on their significantly decreased growth levels. The effects of different initial inoculum ratios, up to three orders of magnitude, on community structure were investigated, and the final compositions of the mixed communities with various starting composition indicate that community structure is not dependent on the initial inoculum ratio. Furthermore, the competitive relationships within the community were not altered by different initial inoculum ratios. The community structure was simulated by generalized Lotka-Volterra and dynamic flux balance analysis to provide mechanistic predictions into emergence of community structure under a nutrient-rich environment. Metaproteomic analyses provide support for the metabolite exchanges predicted by computational modeling and for highly altered physiologies when microbes are grown in co-culture. These findings broaden our understanding of bacterial community dynamics and metabolic diversity in higher-order interactions and could be significant in the management of rhizospheric bacterial communities. IMPORTANCE Bacteria naturally co-exist in multispecies consortia, and the ability to engineer such systems can be useful in biotechnology. Despite this, few studies have been performed to understand how bacteria form a stable community and interact with each other under nutrient-rich conditions. In this study, we investigated the effects of initial inoculum ratios on bacterial community structure using a complex medium and found that the initial inoculum ratio has no significant impact on resultant community structure or on interaction patterns between community members. The microbial population profiles were simulated using computational tools in order to understand intermicrobial relationships and to identify potential metabolic exchanges that occur during stabilization of the bacterial community. Studying microbial community assembly processes is essential for understanding fundamental ecological principles in microbial ecosystems and can be critical in predicting microbial community structure and function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Contributions of anoxic microsites to soil carbon protection across soil textures

Anoxic microsites, zones of oxygen depletion in otherwise oxic soils, may slow soil C turnover. However, the abundance of anoxic microsites and their contribution to soil C protection is yet undefined. In this study, we determine the contribution of anoxic microsites to soil C protection in soils of three distinct textures (clay loam, loam, sandy loam) across a range of soil moistures. We examined the influence of soil oxygen supply by increasing oxygen content in the incubation atmosphere (oxygen enrichment) and through disaggregation. We attributed increases in CO 2 efflux to the aeration of anoxic microsites. The contribution of anoxic microsites to soil C protection increased with decreasing clay content. Clay loam CO 2 efflux was relatively unaffected by aeration. Moderately moist, loam soils had CO 2 effluxes that did not increase with oxygen enrichment but increased by 375% upon disaggregation. Sandy loam soil CO 2 efflux increased by 50–75% with oxygen enrichment and 250% with disaggregation. Geochemical and microbial data reveal that anoxic microsite abundance also increased with decreasing clay content. The proportion of acid extractable Fe present as Fe(II) increased with decreasing clay content, and methanogens were more abundant in loam and sandy loam soils. Our results suggest that oxygen demand, rather than supply, can regulate anoxic microsite formation and that anoxic protection of soil C can be diminished through physical disruption of soil structure. Our findings further illustrate that anoxic microsites should be included in conceptual models of soil C protection to avoid soil C loss and improve predictions of soil C response to disturbance.

58 GEOSCIENCES↗
DOE OSTI2021

Cytochrome Nanowire XRD

Microbial metabolism plays an important role in the global cycling of carbon, nutrients, and metals. Recent discovery of microorganisms sharing energy using direct electrical connections might represent a prominent strategy by which anaerobic microorganisms interact with the diversity of environments. The purpose of the proposed research is to apply these newly developed principles of electron exchange in microbial communities to the study of biogeochemical cycling in climate and subsurface systems. Previous studies revealed that soil bacteria Geobacter sulfurreducens produce conductive pili nanofilaments that facilitate long-range electron transport to extracellular electron acceptors and other cells. This discovery has already transformed our understanding of the function of microbial communities in diverse environments, making basic contributions to microbial ecology and helping to improve practical applications such as increasing current output of microbial fuel cells and enhancing the conversion of organic waste to methane. Microbial electron exchange via pili may also contribute to methane production in terrestrial environments that are major sources of atmospheric methane. However, the mechanism of electron transport through pili is not well understood. Initial studies have suggested that pili exhibit metallic-like conductivity similar to conducting polymer polyaniline. However, these studies were limited to electrical measurements. X-ray diffraction studies have suggested that aromatic amino acids tyrosine and phenylalanine are closely packed in pili. But these studies used homology modeling to predict the pilus assembly and information about the number of monomers in each pilus assembly unit and their organization are still lacking. The central hypothesis of this proposal is aromatics in pili enable intermolecular electron delocalization due to pi-stacking that can give rise to metallic-like conductivity. By analyzing pili structure using infrared scattering-scanning near field optical microscope (IR s-SNOM) and combining with solution NMR, Focused Ion Beam (FIB and computational capabilities, this work will provide comprehensive understanding of the conduction mechanism from a structure-function perspective. The specific aims are: 1) With infrared scattering scanning near field optical microscope (IR s-SNOM), image the organization and density of pilin monomers and pH-induced conformational changes in a pilus filament. 2) With solution NMR, resolve pH-induced structural changes in the pilin monomer. 3) Using Focused Ion Beam (FIB), draw electrodes on single pili to measure their conductivity. 4) With computational modeling using the NWChem, simulate the effect of environment on the electronic properties of pili as a function of pH. The proposed work will gain the nanoscale insight into the structure of the pili, as the basis for understanding interspecies electrical communications. The improved understanding will be harnessed to accelerate bioenergy production and bioremediation using pili as well as for predictive modeling of carbon cycling in diverse environments to reach the central goals of DOE-BER to develop sustainable energy sources, regulate the contaminants in the subsurface and understand the effects of greenhouse gas emissions on biosphere. The proposed studies will provide design principles for engineering of electrical interactions among these communities by manipulating the amino acid composition of pili. Moreover, these studies will identify the forces that maintain the function of these communities in the face of environmental perturbation. Therefore, these studies will help to address the need "to incorporate process understanding of biogeochemical cycling into a scalable hierarchy of predictive capabilities" specified in EMSL's Science Themes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Enzymatic carbon–fluorine bond cleavage by human gut microbes

Fluorinated compounds are used for agrochemical, pharmaceutical, and numerous industrial applications, resulting in global contamination. In many molecules, fluorine is incorporated to enhance the half-life and improve bioavailability. Fluorinated compounds enter the human body through food, water, and xenobiotics including pharmaceuticals, exposing gut microbes to these substances. The human gut microbiota is known for its xenobiotic biotransformation capabilities, but it was not previously known whether gut microbial enzymes could break carbon-fluorine bonds, potentially altering the toxicity of these compounds. Here, through the development of a rapid, miniaturized fluoride detection assay for whole-cell screening, we identified active gut microbial defluorinases. We biochemically characterized enzymes from diverse human gut microbial classes including Clostridia, Bacilli, and Coriobacteriia, with the capacity to hydrolyze (di)fluorinated organic acids and a fluorinated amino acid. Whole-protein alanine scanning, molecular dynamics simulations, and chimeric protein design enabled the identification of a disordered C-terminal protein segment involved in defluorination activity. Domain swapping exclusively of the C-terminus conferred defluorination activity to a nondefluorinating dehalogenase. To advance our understanding of the structural and sequence differences between defluorinating and nondefluorinating dehalogenases, we trained machine learning models which identified protein termini as important features. Models trained on 41-amino acid segments from protein C termini alone predicted defluorination activity with 83% accuracy (compared to 95% accuracy based on full-length protein features). This work is relevant for therapeutic interventions and environmental and human health by uncovering specificity-determining signatures of fluorine biochemistry from the gut microbiome.

Probst, Silke I↗
DOE OSTI2022

Optical O 2 Sensors Also Respond to Redox Active Molecules Commonly Secreted by Bacteria

From a metabolic perspective, molecular oxygen (O 2 ) is arguably the most significant constituent of Earth’s atmosphere. Nearly every facet of microbial physiology is sensitive to the presence and concentration of O 2 , which is the most favorable terminal electron acceptor used by organisms and also a dangerously reactive oxidant. As O 2 has such sweeping implications for physiology, researchers have developed diverse approaches to measure O 2 concentrations in natural and laboratory settings. Recent improvements to phosphorescent O 2 sensors piqued our interest due to the promise of optical measurement of spatiotemporal O 2 dynamics. However, we found that our preferred bacterial model, Pseudomonas aeruginosa PA14, secretes more than one molecule that quenches such sen sors, complicating O 2 measurements in PA14 cultures and biofilms. Assaying supernatants from cultures of 9 bacterial species demonstrated that this phenotype is common: all super natants quenched a soluble O 2 probe substantially. Phosphorescent O 2 probes are often embedded in solid support for protection, but an embedded probe called O 2 NS was quenched by most supernatants as well. Measurements using pure compounds indicated that quenching is due to interactions with redox-active small molecules, including phena zines and flavins. Uncharged and weakly polar molecules like pyocyanin were especially potent quenchers of O 2 NS. These findings underscore that optical O 2 measurements made in the presence of bacteria should be carefully controlled to ensure that O 2 , and not bacterial secretions, is measured, and motivate the design of custom O 2 probes for specific organisms to circumvent sensitivity to redox-active metabolites.

59 BASIC BIOLOGICAL SCIENCES↗