Daniel Amador-Noguez · University of Wisconsin-Madison · PhdFit
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Daniel Amador-Noguez is a Professor of Bacteriology at the University of Wisconsin-Madison. His research focuses on metabolomics and metabolic regulation in biofuel producing bacteria, bacterial biofilms, and the human gut microbiome. His work involves investigating the metabolic pathways and regulatory mechanisms that underpin microbial physiology and their implications for biofuel production, human health, and microbial ecology. He has contributed to understanding how gut microbes mediate the effects of dietary components, such as cholesterol and saturated fats, on health, as well as exploring microbial responses to environmental stresses and metabolic engineering of microbes for biotechnological applications.
Through biochemical transformation of host-derived bile acids, gut bacteria mediate host-microbe crosstalk and function at the interface of nutrition and host metabolic regulation. Bile acids play a crucial role in human health by facilitating the absorption of dietary lipophilic nutrients, interacting with hormone receptors to regulate host physiology, and shaping gut microbiota composition through antimicrobial activity. Bile acids deconjugation by bacterial bile salt hydrolase has long been recognized as the first necessary bile acid modification required before further transformations can occur. Here, we show that bile salt hydrolase activity is common among human gut bacterial isolates spanning seven major phyla. However, we observed variation in both the extent and the specificity of deconjugation of bile acids among the tested taxa. Unexpectedly, we discovered that certain strains were capable of directly dehydrogenating conjugated bile acids via hydroxysteroid dehydrogenases to produce conjugated secondary bile acids both in vitro and in vivo. These results challenge the prevailing notion that deconjugation is a prerequisite for further bile acid modifications and lay a foundation for new hypotheses regarding how bacteria act individually or in concert to diversify the bile acid pool and influence host physiology. Here, the authors show that bile salt hydrolase activity is common among human gut bacterial isolates spanning seven major phyla and identify strains capable of directly dehydrogenating conjugated bile acids via hydroxysteroid dehydrogenases to produce conjugated secondary bile acids, challenging the notion that deconjugation is a prerequisite for further bile acid modifications.
ABSTRACT Clostridium thermocellum is a leading candidate for consolidated bioprocessing of lignocellulosic biomass into biofuels due to its native cellulolytic capabilities. Beyond ethanol, C. thermocellum is being developed as a platform for producing higher-chain alcohols such as isobutanol and n -butanol. However, its physiological adaptations to alcohol stress remain poorly understood. Here, we investigate how C. thermocellum remodels its membrane lipid composition in response to exogenous ethanol, n -butanol, isobutanol, and butyrate. Exposure to linear alcohols such as n -butanol or to organic acids like butyrate increased the proportion of straight-chain fatty acids in the membrane at the expense of branched-chain species, whereas exposure to the branched alcohol isobutanol produced the opposite effect. Isotope tracer experiments demonstrated that C. thermocellum directly incorporates the carbon backbones of exogenous alcohols and acids into fatty acids, providing a mechanistic basis for these contrasting shifts. We show that the bifunctional aldehyde/alcohol dehydrogenase AdhE is essential for the assimilation of exogenous alcohols into fatty acids, acting through its oxidative activity by first oxidizing alcohols to aldehydes and then converting them to acyl-CoA intermediates. Deletion of the pyruvate:ferredoxin oxidoreductase isozyme pfor4 abolished branched-chain fatty acid synthesis, but supplementation with isobutanol restored production, indicating that Pfor4 substitutes for the canonical branched-chain α-keto acid dehydrogenase complex. These findings reveal two distinct routes for branched-chain fatty acid production in C. thermocellum : a Pfor4-dependent pathway from α-keto acid intermediates derived from amino acid synthesis, and an AdhE-dependent salvage pathway that assimilates exogenous branched-chain alcohols. IMPORTANCE This study identifies key mechanisms of Clostridium thermocellum membrane remodeling under alcohol stress, showing that AdhE mediates incorporation of exogenous alcohols into fatty acids, while Pfor4 drives branched-chain fatty acid synthesis in the absence of the canonical Bkd complex. These findings highlight actionable targets for metabolic engineering to enhance solvent tolerance and improve the robustness and productivity of C. thermocellum as a biofuel-producing platform.
bioRxiv (Cold Spring Harbor Laboratory) · 2025-05-29
preprintOpen access
ABSTRACT To survive within restrictive host niches, bacterial pathogens must possess finely tuned physiological adaptations. One such niche inhabited by Listeria monocytogenes ( L. monocytogenes ) is the host cell cytosol—a compartment characterized by significant barriers to entry, metabolic limitation, and immune surveillance. Previously, we identified L. monocytogenes transposon mutants defective for intracellular survival due to disruptions in key metabolic pathways, including cell wall biosynthesis, menaquinone production, and pyruvate metabolism. One of these mutants mapped to a central component of the pyruvate dehydrogenase (PDH) complex, pdhC ::Tn. Notably, this mutant exhibits pronounced survival defects during infection, despite retaining robust growth and survival in nutrient-rich media. We go on to show that disruption of pdhA ::Tn and pdhD ::Tn similarly led to virulence attenuation during intra-macrophage growth, plaquing assays, and murine infections. Respiro-fermentative metabolic profiling revealed that pdhC ::Tn mutants have an altered respiro-fermentative metabolism with more prominent secretion of lactate. Further, unbiased metabolomic profiling revealed a global starvation phenotype with lower levels of upper glycolytic intermediates and TCA cycle intermediates coupled with elevated intra-bacterial levels of pyruvate and lactate. We then demonstrate that PDH mutants are unable to efficiently utilize phosphotransferase (PTS)-dependent carbon sources and that their growth can be rescued using non-PTS-mediated carbon sources such as hexose phosphates. To identify genetic suppressors of PDH deficiency, we performed an EMS mutagenesis screen using fructose—a PTS-transported carbon source—as the sole carbon source. Five suppressors each contained a single independent mutation in the redox sensing regulator rex . Subsequently, we show that loss of rex restores pdhC ::Tn’s ability to consume PTS-mediated carbon sources through the alleviation of fermentative repression. Further, pdhC ::Tn suppressor mutants show restored intracellular growth, but not virulence in vivo . Together, these findings indicate that a key defect in PDH mutants is the inability to import and metabolize PTS-dependent carbon sources in the host cytosol. We posit this impairment leads to disruptions in redox balance and a shift in respiro-fermentative metabolism, ultimately contributing to the loss of intracellular fitness and virulence.
bioRxiv (Cold Spring Harbor Laboratory) · 2025-02-06 · 2 citations
preprintOpen accessSenior authorCorresponding
Abstract Thermodynamically constrained reactions and pathways are hypothesized to impose greater protein demands on cells, requiring higher enzyme amounts to sustain a given flux compared to those with stronger thermodynamics. To test this, we quantified the absolute concentrations of glycolytic enzymes in three bacterial species — Zymomonas mobilis , Escherichia coli , and Clostridium thermocellum — which employ distinct glycolytic pathways with varying thermodynamic driving forces. By integrating enzyme concentration data with corresponding in vivo metabolic fluxes and ΔG measurements, we found that the highly favorable Entner-Doudoroff (ED) pathway in Z. mobilis requires only one-fourth the amount of enzymatic protein to sustain the same flux as the thermodynamically constrained pyrophosphate-dependent glycolytic pathway in C. thermocellum , with the Embden-Meyerhof-Parnas (EMP) pathway in E. coli exhibiting intermediate thermodynamic favorability and enzyme demand. Across all three pathways, early reactions with stronger thermodynamic driving forces generally required lower enzyme investment than later, less favorable steps. Additionally, reflecting differences in glycolytic strategies, the highly reversible ethanol fermentation pathway in C. thermocellum requires 10-fold more protein to maintain the same flux as the irreversible, forward-driven ethanol fermentation pathway in Z. mobilis . Thus, thermodynamic driving forces constitute a major in vivo determinant of the enzyme burden in metabolic pathways.
ABSTRACT Gluconeogenesis, the reciprocal pathway of glycolysis, is an energy-consuming process that generates glycolytic intermediates from non-carbohydrate sources. In this study, we demonstrate that robust and efficient gluconeogenesis in bacteria relies on the allosteric inactivation of pyruvate kinase, the enzyme responsible for the irreversible final step of glycolysis. Using the model bacterium Bacillus subtilis as an example, we discovered that pyruvate kinase activity is inhibited during gluconeogenesis via its extra C-terminal domain (ECTD), which is essential for autoinhibition and metabolic regulation. Physiologically, a B. subtilis mutant lacking the ECTD in pyruvate kinase displayed multiple defects under gluconeogenic conditions, including inefficient carbon utilization, slower growth, and decreased resistance to the herbicide glyphosate. These defects were not caused by the phosphoenolpyruvate–pyruvate–oxaloacetate futile cycle. Instead, we identified two major metabolic consequences of pyruvate kinase dysregulation during gluconeogenesis: failure to establish high phosphoenolpyruvate (PEP) concentrations necessary for robust gluconeogenesis and increased carbon overflow into the medium. In silico analysis revealed that, in wild-type cells, an expanded PEP pool enabled by pyruvate kinase inactivation is critical for maintaining the thermodynamic feasibility of gluconeogenesis. Additionally, we discovered that B. subtilis exhibits glyphosate resistance specifically under gluconeogenic conditions, and this resistance depends on the PEP pool expansion resulting from pyruvate kinase inactivation. Our findings underscore the importance of allosteric regulation during gluconeogenesis in coordinating metabolic flux, efficient carbon utilization, and antimicrobial resistance. IMPORTANCE Pyruvate kinase catalyzes the final irreversible step in glycolysis and is commonly thought to play a critical role in regulating this pathway. In this study, we identified a constitutively active variant of pyruvate kinase, which did not impact glycolysis but instead led to multiple metabolic defects during gluconeogenesis. Contrary to conventional understanding, these defects were not due to the phosphoenolpyruvate–pyruvate–oxaloacetate futile cycle. Our findings suggest that the defects arose from an insufficient buildup of the phosphoenolpyruvate pool and an increase in carbon overflow metabolism. Overall, this study demonstrates the essential role of pyruvate kinase allosteric regulation during gluconeogenesis in maintaining adequate phosphoenolpyruvate levels, which helps prevent overflow metabolism and enhances the thermodynamic favorability of the pathway. This study also provides a novel link between glyphosate resistance and gluconeogenesis.
Background/Study Objective: Fatty acid β-oxidation (FAO) is a central catabolic pathway with broad implications for organismal health. However, various fatty acids are largely incompatible with FAO machinery before being modified by other enzymes. Such lipids include 4-hydroxy acids (4-HAs)-fatty acids hydroxylated at the 4 (γ) position-which can be provided from dietary intake, lipid peroxidation, and certain drugs of abuse. While the core enzymes of FAO were discovered several decades ago, the enzymes responsible for 4-HA catabolism in mammals remain unknown.
Journal of Bacteriology · 2025-06-02 · 2 citations
articleOpen access
ABSTRACT Intracellular pools of deoxynucleoside triphosphates (dNTPs) are strictly maintained throughout the cell cycle to ensure accurate and efficient DNA replication. DNA synthesis requires an abundance of dNTPs, but elevated dNTP concentrations in nonreplicating cells delay entry into S phase. Enzymes known as deoxyguanosine triphosphate triphosphohydrolases (Dgts) hydrolyze dNTPs into deoxynucleosides and triphosphates, and we propose that Dgts restrict dNTP concentrations to promote the G1 to S phase transition. We characterized a Dgt from the bacterium Caulobacter crescentus termed flagellar signaling suppressor C ( fssC ) to clarify the role of Dgts in cell cycle regulation. Deleting fssC increases dNTP levels and extends the G1 phase of the cell cycle through a mechanism independent of the response regulator CtrA. Segregation and duplication of the chromosomal origin of replication ( oriC ) are delayed in ∆ fssC , but the rate of replication elongation is unchanged. We conclude that dNTP hydrolysis by FssC promotes the initiation of DNA replication. This work further establishes Dgts as important regulators of the G1 to S phase transition, and the high conservation of Dgts across all domains of life implies that Dgt-dependent cell cycle control may be widespread in many organisms. IMPORTANCE Cells must faithfully replicate their genetic material in order to proliferate. Studying the regulatory pathways that determine when a cell initiates DNA replication is important for understanding fundamental biological processes, and it can also improve the strategies used to treat diseases that affect the cell cycle. Here, we identify a nucleotide signaling pathway that influences when cells begin DNA replication. We show that this pathway promotes the transition from the G1 to the S phase of the cell cycle in the bacterium Caulobacter crescentus and propose that this pathway is prevalent in all domains of life.
Microbes in the intestine transform bile acids during transit, altering their functional and signaling capacities before recirculation via the portal vein. Sex differences in the gut microbiota have been noted, but their consequence on bile acid composition is unclear. Here, we investigated the composition and functional potential of microbes in the small and large intestines together with portal and systemic bile acid levels. Female and male mice exhibit distinct microbial diversity throughout the length of the intestine, leading to dimorphism in genes related to bile acid transformation. Of note, genes linked to bacterial oxidative properties were abundant in males, and consistently, we found 3X higher oxo-bile acids in portal circulation in males than females. Conversely, conjugated primary bile acids were 1.8X more abundant in the portal bile acid pool of female mice. Oxidized and deconjugated bile acids were absent in germ-free mice consistent with microbe-mediated bile acid transformation. More importantly, gnotobiotic mice do not show sex differences in portal bile acids. Taken together, we demonstrate that sex differences in gut microbiota with subsequent changes in microbially transformed bile acid levels contribute to distinct sex-specific bile acid pools within the enterohepatic loop.
