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All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Structures of Fission Yeast Inositol Pyrophosphate Kinase Asp1 in Ligand-Free, Substrate-Bound, and Product-Bound States

Expression of the fission yeast Schizosaccharomyces pombe phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate signaling molecule 1,5-IP 8 . IP 8 dynamics are determined by Asp1, a bifunctional enzyme consisting of an N-terminal kinase domain and a C-terminal pyrophosphatase domain that catalyze IP 8 synthesis and catabolism, respectively. Here, we report structures of the Asp1 kinase domain, crystallized with two protomers in the asymmetric unit, one of which was complexed with ligands (ADPNP, ADP, or ATP; Mg 2+ or Mn 2+ ; IP 6 , 5-IP 7 , or 1,5-IP 8 ) and the other which was ligand-free. The ligand-free enzyme adopts an “open” conformation that allows ingress of substrates and egress of products. ADPNP, ADP, and ATP and associated metal ions occupy a deep phospho-donor pocket in the active site. IP 6 or 5-IP 7 engagement above the nucleotide favors adoption of a “closed” conformation, in which surface protein segments undergo movement and a disordered-to-ordered transition to form an inositol polyphosphate-binding site. In a structure mimetic of the kinase Michaelis complex, the anionic 5-IP 7 phosphates are encaged by an ensemble of nine cationic amino acids: Lys43, Arg223, Lys224, Lys260, Arg274, Arg285, Lys290, Arg293, and Lys341. Alanine mutagenesis of amino acids that contact the adenosine nucleoside of the ATP donor underscored the contributions of Asp258 interaction with the ribose 3'-OH and of Glu248 with adenine-N 6 . Changing Glu248 to Gln elicited a gain of function whereby the kinase became adept at using GTP as phosphate donor. Wild-type Asp1 kinase can utilize N 6 -benzyl-ATP as phosphate donor.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Complete genome of Erwinia spp. str . LJJL01 isolated from waste charcoal

We present the complete genome sequence of Erwinia spp. str. LJJL01, isolated from waste charcoal in Colorado, USA, using Oxford Nanopore sequencing. This sequence provides important insights into this bacterium’s metabolic and catabolic robustness to utilize sugars, acids, and aromatics, highlighting its potential as a bio-industrial strain for various feedstocks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Combinatorial Biosynthesis of Sulfated Benzenediol Lactones with a Phenolic Sulfotransferase from Fusarium graminearum PH-1

Total biosynthesis or whole-cell biocatalytic production of sulfated small molecules relies on the discovery and implementation of appropriate sulfotransferase enzymes. Although fungi are prominent biocatalysts and have been used to sulfate drug-like phenolics, no gene encoding a sulfotransferase enzyme has been functionally characterized from these organisms. Here, we identify a phenolic sulfotransferase, FgSULT1, by genome mining from the plant-pathogenic fungus Fusarium graminearum PH-1. We expressed FgSULT1 in a Saccharomyces cerevisiae chassis to modify a broad range of benzenediol lactones and their nonmacrocyclic congeners, together with an anthraquinone, with the resulting unnatural natural product (uNP) sulfates displaying increased solubility. FgSULT1 shares low similarity with known animal and plant sulfotransferases. Instead, it forms a sulfotransferase family with putative bacterial and fungal enzymes for phase II detoxification of xenobiotics and allelochemicals. Among fungi, putative FgSULT1 homologues are encoded in the genomes of Fusarium spp. and a few other genera in nonsyntenic regions, some of which may be related to catabolic sulfur recycling. Computational structure modeling combined with site-directed mutagenesis revealed that FgSULT1 retains the key catalytic residues and the typical fold of characterized animal and plant sulfotransferases. Our work opens the way for the discovery of hitherto unknown fungal sulfotransferases and provides a synthetic biological and enzymatic platform that can be adapted to produce bioactive sulfates, together with sulfate ester standards and probes for masked mycotoxins, precarcinogenic toxins, and xenobiotics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A Gene Cluster That Encodes Histone Deacetylase Inhibitors Contributes to Bacterial Persistence and Antibiotic Tolerance in Burkholderia thailandensis

Persister cells are genetically identical variants in a bacterial population that have phenotypically modified their physiology to survive environmental stress. In bacterial pathogens, persisters are able to survive antibiotic treatment and reinfect patients in a frustrating cycle of chronic infection. To better define core persistence mechanisms for therapeutics development, we performed transcriptomics analyses of Burkholderia thailandensis populations enriched for persisters via three methods: flow sorting for low proton motive force, meropenem treatment, and culture aging. Although the three persister-enriched populations generally displayed divergent gene expression profiles that reflect the multimechanistic nature of stress adaptations, there were several common gene pathways activated in two or all three populations. These include polyketide and nonribosomal peptide synthesis, Clp proteases, mobile elements, enzymes involved in lipid metabolism, and ATP binding cassette (ABC) transporter systems. In particular, identification of genes that encode polyketide synthases (PKSs) and fatty acid catabolism factors indicates that generation of secondary metabolites, natural products, and complex lipids could be part of the metabolic program that governs the persistence state. We also found that loss-of-function mutations in the PKS-encoding gene locus BTH_I2366, which plays a role in biosynthesis of histone deacetylase (HDAC) inhibitors, resulted in increased sensitivity to antibiotics targeting DNA replication. Furthermore, treatment of multiple bacterial pathogens with a fatty acid synthesis inhibitor, CP-640186, potentiated the efficacy of meropenem against the persister populations. Altogether, our results suggest that bacterial persisters may exhibit an outwardly dormant physiology but maintain active metabolic processes that are required to maintain persistence.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Exploring novel alkane-degradation pathways in uncultured bacteria from the North Atlantic Ocean

Microbes play a significant role in the degradation of petroleum hydrocarbons in the oceans, yet little is known about the native bacteria that metabolize hydrocarbons before an oil spill. The Faroe-Shetland Channel (FSC) is a deepwater subarctic region of the North Atlantic with prominent oil production and a diverse microbial community associated with the degradation of petroleum. Here, we combine DNA-based stable-isotope probing (DNA-SIP) with metagenomics to elucidate the metabolic underpinnings of native alkane-degrading bacteria from the FSC. From two 13C n-hexadecane SIP experiments using seawater from 5 and 700 m depths in the FSC, we obtained 42 metagenome-assembled genomes (MAGs) belonging to 19 genera, including two previously overlooked hydrocarbon-degrading bacteria, Lentibacter (Alphaproteobacteria) and Dokdonia (Bacteroidetes). Diversity surveys indicated Lentibacter were dominant members of the FSC, constituting up to 17% of these communities. Many of the SIP-enriched MAGs (20/42) encoded a complete alkane oxidation pathway, including alkane monooxygenase (AlkB), rubredoxin reductase (AlkT), and rubredoxin-2 (AlkG). Fourteen Aphaproteobacteria MAGs lacked AlkG for electron transfer. Instead, they encoded novel disulfide isomerases with iron-binding cysteine motifs conserved across rubredoxins. Dokdonia lacked AlkT and AlkG, however, their central alkane-degradation catabolic pathways were complete. We describe previously unrecognized bacteria capable of hydrocarbon degradation, including the dominant genera Lentibacter, which may continuously purge hydrocarbons released from oil exploration activities in the FSC. This advances the understanding of the diversity and physiologies of alkane degradation in the North Atlantic and provides evidence of new mechanisms used to metabolize alkanes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Floodplain nitrifiers harbor the genetic potential for utilizing a wide range of organic nitrogen compounds

Organic compounds such as urea and cyanate can serve as nitrogen (N) sources for nitrifying microorganisms, including ammonia-oxidizing archaea (AOA) and bacteria (AOB), complete ammonia-oxidizing (comammox) bacteria, and nitrite-oxidizing bacteria (NOB). Here we investigated metagenome-assembled genomes (MAGs) for all four nitrifier guilds generated from hydrologically variable floodplain sediments of the Wind River Basin (WRB; Riverton, WY, USA) for their genetic potential to utilize organic N compounds. A vast majority of WRB nitrifier MAGs harbored urease (ure) and at least one urea transporter ( utp, urt, dur3 ). AOA were the most abundant and phylogenetically diverse nitrifiers in WRB floodplain sediments. Several AOA MAGs encoded cyanase ( cynS ), nitrilase ( nit1 ), omega-amidase ( nit2 ), nitrile hydratase ( nthA ), and genes related to purine degradation, including biuret hydrolase ( biuH ), oxamic transcarbamylase ( allFGH ), and catabolic carbamate kinase ( allK ). AOA often encoded an uncharacterized amidohydrolase collocated with biuH , rather than allophanate hydrolase ( atzF ). A small number of AOA encoded atzF , functioning in an unknown pathway. AOB and comammox were of relatively low abundance and taxonomic diversity and were present only at certain depths in WRB; however, they encoded triuret/biuret degradation genes ( trtA, biuH , and atzH ), and in comammox, these genes were also collocated with allFGHK . The genetic potential of ammonia oxidizers in the WRB floodplain suggests that organic N may support nitrification in this system. The proposed pathways for utilizing purine degradation products other than urea potentially expand the known metabolic capabilities of AOA, AOB, and comammox bacteria and reveal the possibility for cryptic N cycling between microbial community members.

floodplain↗
DOE OSTI2025

Modification and analysis of context-specific genome-scale metabolic models: methane-utilizing microbial chassis as a case study

ABSTRACT Context-specific genome-scale model (CS-GSM) reconstruction is becoming an efficient strategy for integrating and cross-comparing experimental multi-scale data to explore the relationship between cellular genotypes, facilitating fundamental or applied research discoveries. However, the application of CS modeling for non-conventional microbes is still challenging. Here, we present a graphical user interface that integrates COBRApy, EscherPy, and RIPTiDe, Python-based tools within the BioUML platform, and streamlines the reconstruction and interrogation of the CS genome-scale metabolic frameworks via Jupyter Notebook. The approach was tested using -omics data collected for Methylotuvimicrobium alcaliphilum 20Z R , a prominent microbial chassis for methane capturing and valorization. We optimized the previously reconstructed whole genome-scale metabolic network by adjusting the flux distribution using gene expression data. The outputs of the automatically reconstructed CS metabolic network were comparable to manually optimized i IA409 models for Ca-growth conditions. However, the CS model questions the reversibility of the phosphoketolase pathway and suggests higher flux via primary oxidation pathways. The model also highlighted unresolved carbon partitioning between assimilatory and catabolic pathways at the formaldehyde-formate node. Only a very few genes and only one enzyme with a predicted function in C1 metabolism, a homolog of the formaldehyde oxidation enzyme ( fae1-2 ), showed a significant change in expression in La-growth conditions. The CS-GSM predictions agreed with the experimental measurements under the assumption that the Fae1-2 is a part of the tetrahydrofolate-linked pathway. The cellular roles of the tungsten (W)-dependent formate dehydrogenase ( fdhAB ) and fae homologs ( fae1-2 and fae3 ) were investigated via mutagenesis. The phenotype of the f dhAB mutant followed the model prediction. Furthermore, a more significant reduction of the biomass yield was observed during growth in La-supplemented media, confirming a higher flux through formate. M. alcaliphilum 20Z R mutants lacking fae1-2 did not display any significant defects in methane or methanol-dependent growth. However, contrary to fae1, the fae1-2 homolog failed to restore the formaldehyde-activating enzyme function in complementation tests. Overall, the presented data suggest that the developed computational workflow supports the reconstruction and validation of CS-GSM networks of non-model microbes. IMPORTANCE The interrogation of various types of data is a routine strategy to explore the relationship between genotype and phenotype. An efficient approach for integrating and cross-comparing experimental multi-scale data in the context of whole-genome-based metabolic network reconstruction becomes a powerful tool that facilitates fundamental and applied research discoveries. The present study describes the reconstruction of a context-specific (CS) model for the methane-utilizing bacterium, Methylotuvimicrobium alcaliphilum 20Z R . M. alcaliphilum 20Z R is becoming an attractive microbial platform for the production of biofuels, chemicals, pharmaceuticals, and bio-sorbents for capturing atmospheric methane. We demonstrate that this pipeline can help reconstruct metabolic models that are similar to manually curated networks. Furthermore, the model is able to highlight previously overlooked pathways, thus advancing fundamental knowledge of non-model microbial systems or promoting their development toward biotechnological or environmental implementations.

Kulyashov, M. A.↗
DOE OSTI2020

Symbiont-Mediated Digestion of Plant Biomass in Fungus-Farming Insects

Feeding on living or dead plant material is widespread in insects. Seminal work on termites and aphids has provided profound insights into the critical nutritional role that microbes play in plant-feeding insects. Here, some ants, beetles, and termites, among others, have evolved the ability to use microbes to gain indirect access to plant substrate through the farming of a fungus on which they feed. Recent genomic studies, including studies of insect hosts and fungal and bacterial symbionts, as well as metagenomics and proteomics, have provided important insights into plant biomass digestion across insect–fungal mutualisms. Not only do advances in understanding of the divergent and complementary functions of complex symbionts reveal the mechanism of how these herbivorous insects catabolize plant biomass, but these symbionts also represent a promising reservoir for novel carbohydrate-active enzyme discovery, which is of considerable biotechnological interest.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Skeletal tissue regulation by catalase overexpression in mitochondria

Accumulation of oxidative damage from excess reactive oxygen species (ROS) may contribute to skeletal aging and mediate adverse responses to physiological challenges. Wild-type (WT) mice and transgenic mice (male, 16 wk of age) with human catalase targeted to the mitochondria (mCAT) were analyzed for skeletal responses to the remodeling stimuli of combined hind-limb unloading and exposure to ionizing radiation ( 137 Cs, 2 Gy). Treatment for 2 wk caused lipid peroxidation in the bones WT but not mCAT mice, showing that transgene expression mitigated oxidative stress. Ex vivo osteoblast colony growth rate was 95% greater in mCAT than WT mice and correlated with catalase activity levels ( P < 0.005, r = 0.67), although terminal osteoblast and osteoclast differentiation were unaffected. mCAT mice had lower cancellous bone volume and cortical size than WT mice. Ambulatory control mCAT animals also displayed reduced cancellous and cortical structural properties compared with control WT mice. In mCAT but not WT mice, treatment caused an unexpectedly rapid radial expansion (+8% cortical area, +22% moment of inertia), reminiscent of compensatory bone growth during advancing age. In contrast, treatment caused similar structural deficits in cancellous tissue of mCAT and WT mice. In sum, mitochondrial ROS signaling via H 2 O 2 was important for the acquisition of adult bone structure and catalase overexpression failed to protect cancellous tissue from treatment. In contrast, catabolic stimuli caused radial expansion in mCAT not WT mice, suggesting that mitochondrial ROS in skeletal cells act to suppress tissue turnover in response to remodeling challenges.

Cell Biology↗
DOE OSTI2021

Metabolomic profiles are reflective of hypoxia-induced insulin resistance during exercise in healthy young adult males

Hypoxia-induced insulin resistance appears to suppress exogenous glucose oxidation during metabolically matched aerobic exercise during acute (<8 h) high-altitude (HA) exposure. However, a better understanding of this metabolic dysregulation is needed to identify interventions to mitigate these effects. The objective of this study was to determine if differences in metabolomic profiles during exercise at sea level (SL) and HA are reflective of hypoxia-induced insulin resistance. Native lowlanders ( n = 8 males) consumed 145 g (1.8 g/min) of glucose while performing 80-min of metabolically matched treadmill exercise at SL (757 mmHg) and HA (460 mmHg) after 5-h exposure. Exogenous glucose oxidation and glucose turnover were determined using indirect calorimetry and dual tracer technique ([ 13 C]glucose and [6,6- 2 H 2 ]glucose). Metabolite profiles were analyzed in serum as change (Δ), calculated by subtracting postprandial/exercised state SL (ΔSL) and HA (ΔHA) from fasted, rested conditions at SL. Compared with SL, exogenous glucose oxidation, glucose rate of disappearance, and glucose metabolic clearance rate (MCR) were lower ( P < 0.05) during exercise at HA. One hundred and eighteen metabolites differed between ΔSL and ΔHA ( P < 0.05, Q < 0.10). Differences in metabolites indicated increased glycolysis, tricarboxylic acid cycle, amino acid catabolism, oxidative stress, and fatty acid storage, and decreased fatty acid mobilization for ΔHA. Branched-chain amino acids and oxidative stress metabolites, Δ3-methyl-2-oxobutyrate ( r = -0.738) and Δγ-glutamylalanine ( r = -0.810), were inversely associated ( P < 0.05) with Δexogenous glucose oxidation. Δ3-Hydroxyisobutyrate ( r = -0.762) and Δ2-hydroxybutyrate/2-hydroxyisobutyrate ( r = -0.738) were inversely associated ( P < 0.05) with glucose MCR. Coupling global metabolomics and glucose kinetic data suggest that the underlying cause for diminished exogenous glucose oxidative capacity during aerobic exercise is acute hypoxia-mediated peripheral insulin resistance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Metabolic engineering of the oleaginous yeast Yarrowia lipolytica PO1f for production of erythritol from glycerol

Abstract Background Sugar alcohols are widely used as low-calorie sweeteners in the food and pharmaceutical industries. They can also be transformed into platform chemicals. Yarrowia lipolytica , an oleaginous yeast, is a promising host for producing many sugar alcohols. In this work, we tested whether heterologous expression of a recently identified sugar alcohol phosphatase (PYP) from Saccharomyces cerevisiae would increase sugar alcohol production in Y. lipolytica . Results Y. lipolytica was found natively to produce erythritol, mannitol, and arabitol during growth on glucose, fructose, mannose, and glycerol. Osmotic stress is known to increase sugar alcohol production, and was found to significantly increase erythritol production during growth on glycerol. To better understand erythritol production from glycerol, since it was the most promising sugar alcohol, we measured the expression of key genes and intracellular metabolites. Osmotic stress increased the expression of several key genes in the glycerol catabolic pathway and the pentose phosphate pathway. Analysis of intracellular metabolites revealed that amino acids, sugar alcohols, and polyamines are produced at higher levels in response to osmotic stress. Heterologous overexpression of the sugar alcohol phosphatase increased erythritol production and glycerol utilization in Y. lipolytica . We further increased erythritol production by increasing the expression of native glycerol kinase (GK), and transketolase (TKL). This strain was able to produce 27.5 ± 0.7 g/L erythritol from glycerol during batch growth and 58.8 ± 1.68 g/L erythritol during fed-batch growth in shake-flasks experiments. In addition, the glycerol utilization was increased by 2.5-fold. We were also able to demonstrate that this strain efficiently produces erythritol from crude glycerol, a major byproduct of the biodiesel production. Conclusions We demonstrated the application of a promising enzyme for increasing erythritol production in Y. lipolytica . We were further able to boost production by combining the expression of this enzyme with other approaches known to increase erythritol production in Y. lipolytica . This suggest that this new enzyme provides an orthogonal route for boosting production and can be stacked with existing designs known to increase sugar alcohol production in yeast such as Y. lipolytica . Collectively, this work establishes a new route for increasing sugar alcohol production and further develops Y. lipolytica as a promising host for erythritol production from cheap substrates such as glycerol.

09 BIOMASS FUELS↗
DOE OSTI2025

Sulfoquinovose is exclusively metabolized by the gut microbiota and degraded differently in mice and humans

Abstract Background Sulfoquinovose (SQ) is a green-diet-derived sulfonated glucose and a selective substrate for a limited number of human gut bacteria. Complete anaerobic SQ degradation via interspecies metabolite transfer to sulfonate-respiring bacteria produces hydrogen sulfide, which has dose- and context-dependent health effects. Here, we studied potential SQ degradation by the mammalian host and the impact of SQ supplementation on human and murine gut microbiota diversity and metabolism. Results 13 CO 2 breath tests with germ-free C57BL/6 mice gavaged with 13 C-SQ were negative. Also, SQ was not degraded by human intestinal cells in vitro, indicating that SQ is not directly metabolized by mice and humans. Addition of increasing SQ concentrations to human fecal microcosms revealed dose-dependent responses of the microbiota and corroborated the relevance ofAgathobacter rectalisandBilophila wadsworthiain cooperative degradation of SQ to hydrogen sulfide via interspecies transfer of 2,3-dihydroxy-1-propanesulfonate (DHPS). Similar to the human gut microbiome, the genetic capacity for SQ or DHPS degradation is sparsely distributed among bacterial species in the gut of conventional laboratory mice.Escherichia coliandEnterocloster clostridioformiswere identified as primary SQ degraders in the mouse gut. SQ and DHPS supplementation experiments with conventional laboratory mice and their intestinal contents showed that SQ was incompletely catabolized to DHPS. Although someE. clostridioformisgenomes encode an extended sulfoglycolytic pathway for both SQ and DHPS fermentation, SQ was only degraded to DHPS by a mouse-derivedE. clostridioformisstrain. Conclusions Our findings suggest that SQ is solely a nutrient for the gut microbiota and not for mice and humans, emphasizing its potential as a prebiotic. SQ degradation by the microbiota of conventional laboratory mice differs from the human gut microbiota by absence of DHPS degradation activity. Hence, the microbiota of conventional laboratory mice does not fully represent the SQ metabolism in humans, indicating the need for alternative model systems to assess the impact of SQ on human health. This study advances our understanding of how individual dietary compounds shape the microbial community structure and metabolism in the gut and thereby potentially influence host health.

Microbiology↗
DOE OSTI2021

Metabolic Engineering of the Oleaginous Yeast Yarrowia lipolytica PO1f for Production of Erythritol from Glycerol

Yarrowia lipolytica was found natively to produce erythritol, mannitol, and arabitol during growth on glucose, fructose, mannose, and glycerol. Osmotic stress is known to increase sugar alcohol production, and was found to significantly increase erythritol production during growth on glycerol. To better understand erythritol production from glycerol, since it was the most promising sugar alcohol, we measured the expression of key genes and intracellular metabolites. Osmotic stress increased the expression of several key genes in the glycerol catabolic pathway and the pentose phosphate pathway. Analysis of intracellular metabolites revealed that amino acids, sugar alcohols, and polyamines are produced at higher levels in response to osmotic stress. Heterologous overexpression of the sugar alcohol phosphatase increased erythritol production and glycerol utilization in Y. lipolytica . We further increased erythritol production by increasing the expression of native glycerol kinase (GK), and transketolase (TKL). These data show the growth and titers produced.

Conversion↗
DOE OSTI2022

Data for Glucose Feeds the Tricarboxylic Acid Cycle via Excreted Ethanol in Fermenting Yeast

Ethanol and lactate are typical waste products of glucose fermentation. In mammals, glucose is catabolized by glycolysis into circulating lactate, which is broadly used throughout the body as a carbohydrate fuel. Individual cells can both uptake and excrete lactate, uncoupling glycolysis from glucose oxidation. Here we show that similar uncoupling occurs in budding yeast batch cultures of Saccharomyces cerevisiae and Issatchenkia orientalis . Even in fermenting S. cerevisiae that is net releasing ethanol, media 13C-ethanol rapidly enters and is oxidized to acetaldehyde and acetyl-CoA. This is evident in exogenous ethanol being a major source of both cytosolic and mitochondrial acetyl units. 2H-tracing reveals that ethanol is also a major source of both NADH and NADPH high-energy electrons, and this role is augmented under oxidative stress conditions. Thus, uncoupling of glycolysis from the oxidation of glucose-derived carbon via rapidly reversible reactions is a conserved feature of eukaryotic metabolism.

Conversion↗
DOE OSTI2025

A genome-scale metabolic model for the denitrifying bacterium Thauera sp. MZ1T accurately predicts degradation of pollutants and production of polymers

The denitrifying bacterium Thauera sp . MZ1T, a common member of microbial communities in wastewater treatment facilities, can produce different compounds from a range of carbon (C) and nitrogen (N) sources under aerobic and anaerobic conditions. In these different conditions, Thauera modifies its metabolism to produce different compounds that influence the microbial community. In particular, Thauera sp . MZ1T produces different exopolysaccharides with floc-forming properties, impacting the physical disposition of wastewater consortia and the efficiency of nutrient assimilation by the microbial community. Under N-limiting conditions, Thauera sp . MZ1T decreases its growth rate and accelerates the accumulation of polyhydroxyalkanoate-related (PHA) compounds including polyhydroxybutyrate (PHB), which plays a fundamental role as C and energy storage in this β-proteobacterium. However, the metabolic mechanisms employed by Thauera sp . MZ1T to assimilate and catabolize many of the different C and N sources under aerobic and anaerobic conditions remain unknown. Systems biology approaches such as genome-scale metabolic modeling have been successfully used to unveil complex metabolic mechanisms for various microorganisms. Here, we developed a comprehensive metabolic model (M-model) for Thauera sp . MZ1T ( i Thauera861), consisting of 1,744 metabolites, 2,384 reactions, and 861 genes. We validated the model experimentally using over 70 different C and N sources under both aerobic and anaerobic conditions. i Thauera861 achieved a prediction accuracy of 95% for growth on various C and N sources and close to 85% for assimilation of aromatic compounds under denitrifying conditions. The M-model was subsequently deployed to determine the effects of substrates, oxygen presence, and the C:N ratio on the production of PHB and exopolysaccharides (EPS), showing the highest polymer yields are achieved with nucleotides and amino acids under aerobic conditions. This comprehensive M-model will help reveal the metabolic processes by which this ubiquitous species influences communities in wastewater treatment systems and natural environments.

Tec-Campos, Diego (ORCID:0000000188194150)↗
DOE OSTI2021

Effect of biochanin A on the rumen microbial community of Holstein steers consuming a high fiber diet and subjected to a subacute acidosis challenge

Subacute rumen acidosis (SARA) occurs when highly fermentable carbohydrates are introduced into the diet, decreasing pH and disturbing the microbial ecology of the rumen. Rumen amylolytic bacteria rapidly catabolize starch, fermentation acids accumulate in the rumen and reduce environmental pH. Historically, antibiotics ( e . g ., monensin, MON) have been used in the prevention and treatment of SARA. Biochanin A (BCA), an isoflavone produced by red clover ( Trifolium pratense ), mitigates changes associated with starch fermentation ex vivo . The objective of the study was to determine the effect of BCA on amylolytic bacteria and rumen pH during a SARA challenge. Twelve rumen fistulated steers were assigned to 1 of 4 treatments: HF CON (high fiber control), SARA CON, MON (200 mg d -1 ), or BCA (6 g d -1 ). The basal diet consisted of corn silage and dried distiller’s grains ad libitum . The study consisted of a 2-wk adaptation, a 1-wk HF period, and an 8-d SARA challenge (d 1–4: 40% corn; d 5–8: 70% cracked corn). Samples for pH and enumeration were taken on the last day of each period (4 h). Amylolytic, cellulolytic, and amino acid/peptide-fermenting bacteria (APB) were enumerated. Enumeration data were normalized by log transformation and data were analyzed by repeated measures ANOVA using the MIXED procedure of SAS. The SARA challenge increased total amylolytics and APB, but decreased pH, cellulolytics, and in situ DMD of hay (P < 0.05). BCA treatment counteracted the pH, microbiological, and fermentative changes associated with SARA challenge (P < 0.05). Similar results were also observed with MON (P < 0.05). These results indicate that BCA may be an effective alternative to antibiotics for mitigating SARA in cattle production systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Biophysical and biochemical studies support TP0094 as a phosphotransacetylase in an acetogenic energy-conservation pathway in Treponema pallidum

The mechanisms of energy generation and carbon-source utilization in the syphilis spirochete Treponema pallidum have remained enigmatic despite complete genomic sequence information. Whereas the bacterium harbors enzymes for glycolysis, the apparatus for more efficient use of glucose catabolites, namely the citric-acid cycle, is apparently not present. Yet, the organism’s energy needs likely exceed the modest output from glycolysis alone. Recently, building on our structure-function studies of T . pallidum lipoproteins, we proposed a “flavin-centric” metabolic lifestyle for the organism that partially resolves this conundrum. As a part of the hypothesis, we have proposed that T . pallidum contains an acetogenic energy-conservation pathway that catabolizes D-lactate, yielding acetate, reducing equivalents for the generation and maintenance of chemiosmotic potential, and ATP. We already have confirmed the D-lactate dehydrogenase activity in T . pallidum necessary for this pathway to operate. In the current study, we focused on another enzyme ostensibly involved in treponemal acetogenesis, phosphotransacetylase (Pta). This enzyme is putatively identified as TP0094 and, in this study, we determined a high-resolution (1.95 Å) X-ray crystal structure of the protein, finding that its fold comports with other known Pta enzymes. Further studies on its solution behavior and enzyme activity confirmed that it has the properties of a Pta. These results are consistent with the proposed acetogenesis pathway in T . pallidum , and we propose that the protein be referred to henceforth as TpPta.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Bacteria isolated from the grape phyllosphere capable of degrading guaiacol, a main volatile phenol associated with smoke taint in wine

Recent wildfires near vineyards in the Pacific United States have caused devastating financial losses due to smoke taint in wine. When wine grapes (Vitis vinifera) are exposed to wildfire smoke, their berries absorb volatile phenols derived from the lignin of burning plant material. Volatile phenols are released during the winemaking process giving the finished wine an unpleasant, smokey, and ashy taste known as smoke taint. Bacteria are capable of undergoing a wide variety of metabolic processes and therefore present great potential for bioremediation applications in many industries. In this study, we identify two strains of the same species that colonize the grape phyllosphere and are able to degrade guaiacol, a main volatile phenol responsible for smoke taint in wine. We identify the suite of genes that enable guaiacol degradation in Gordonia alkanivorans via RNAseq of cells growing on guaiacol as a sole carbon source. Additionally, we knockout guaA, a cytochrome P450 gene involved in the conversion of guaiacol to catechol; ΔguaA cells cannot catabolize guaiacol in vitro, providing evidence that GuaA is necessary for this process. Furthermore, we analyze the microbiome of berries and leaves exposed to smoke in the vineyard to investigate the impact of smoke on the grape microbial community. We found smoke has a significant but small effect on the microbial community, leading to an enrichment of several genera belonging to the Bacilli class. Collectively, this research shows that studying microbes and their enzymes has the potential to identify novel tools for alleviating smoke taint.

Castro, Claudia [United States Department of Agric↗