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Faculty & Staff

  • Image of Daniel Amador-Noguez

    Daniel Amador-Noguez

    Associate Professor of Bacteriology

    6472 Microbial Sciences Building
    Office: (608) 265-2710
    Lab: (608) 265-4421

Start and Promotion Dates

  • Assistant Professor: 2013
  • Associate Professor: 2020


B.S., Chemistry, Monterrey Institute of Technology 2001
Ph.D., Molecular Genetics, Baylor College of Medicine 2007
Postdoctoral Research: Quantitative Biology, Lewis-Sigler Institute for Integrative Genomics, Princeton University

Areas of Study

Metabolomics and metabolic regulation in biofuel producing bacteria, bacterial biofilms, and the human gut microbiome.

Research Overview

Metabolic regulation in microbial biofuel producers

The production of biofuels from cellulosic biomass holds promise as a source of clean renewable energy that can reduce our dependence on fossil fuels. Attaining this goal will require engineered microorganisms capable of economical conversion of cellulosic biomass into biofuels. Effective microbe design relies on understanding the relevant metabolic pathways and their regulation, including how the integrated networks function as a whole. Current project in our lab integrate systems-level analyses, especially metabolomics, computational modeling, and genetic engineering to advance understanding of metabolism in a variety of emerging biofuel producing microorganisms, including Z. mobilis, C. thermocellum, S. cerevisiae, and others. Our main research objectives in this area are: 1) Systems-level analysis of metabolic regulation in biofuel producing microorganisms and 2) Engineering symbiotic microbial consortia for biofuel production.

Metabolic remodeling during B. subtilis biofilm development

Most bacteria naturally congregate to form complex communities called biofilms through an elaborate process that involves production of secreted polymeric substances, allowing cells to stick to each other and to surfaces while conferring protection against harsh environments. Bacterial biofilms are abundant in natural environments and play an important role in many clinical, industrial, and ecological settings. Due to the ubiquity and significant impacts of biofilms on human activities, there is a clear need to better understand the complex processes that control biofilm formation and development. Current projects in our lab investigate a critical but barely understood aspect of biofilms: cellular metabolism during biofilm development.
We hypothesize that dynamic remodeling of central carbon and nitrogen metabolism constitutes an essential component of the highly coordinated physiological response that takes place during biofilm development. Using Bacillus subtilis as a model organism, we leverage state-of-the-art systems-level metabolomic and proteomic approaches, microscopy, and quantitative computational modeling, to pursue the following objectives: 1) a systems-level quantitative understanding of how metabolism is remodeled during biofilm formation; 2) elucidation of driving regulatory mechanisms controlling metabolic remodeling during biofilm formation; 3) novel insights regarding metabolic heterogeneity within biofilm cell subpopulations; and 4) elucidation of the physiological relevance of major metabolic alterations during biofilm development. This research will advance our understanding of the underlying logic and unifying principles behind the complex signaling systems of biofilm regulatory networks and will provide a holistic and quantitative understanding of the role of metabolism in biofilm development.

Bile acid transformations by the human gut microbe

Within the last decade, the central role the gut microbiota plays in human health has become widely recognized. An important way in which gut microbes affects host physiology is through their ability to chemically modify bile acids produced by the host. Bile acids act as signaling molecules within the host by modulating activity of nuclear hormone receptors in liver and other tissues and can also modulate gut microbiota composition via selective antimicrobial properties. Changes to the bile acid pool by gut microbes therefore has the potential to affect physiology in these organs, nutrient absorption, drug metabolism, and susceptibility to infection by bacterial pathogens. However, fundamental aspects of this process are still poorly understood. In particular, the distribution of bile acid transforming activity within gut microbes remains largely unexplored and the effects on host physiology resulting from modifications in the bile pool resulting from bacterial action remain poorly understood. Projects in our lab aim to generate a systematic and quantitative understanding of bile acid transforming capabilities in gut microbes and advance our understanding of the molecular mechanisms by which modulation of BA pools by host microbes, via production of secondary bile acids, affect liver host physiology and metabolism.


Microbiology 526: Physiology of Microorganisms

Lab Personnel

Picture of Buasakdi
Chavin Buasakdi
Grad Student
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Melanie Callaghan
Picture of Getahun
Fit Getahun
Research Intern
Picture of Jacobson
Tyler Jacobson
Picture of Khana
Daven Khana
Grad Student
Picture of Lucas
Lauren Lucas
Grad Student
Picture of Martien
Julia Martien
Grad Student
Picture of Rivera Vazquez
Julio Rivera Vazquez
Grad Student
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David Stevenson
Sr Research Specialist
Lab Manager
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Jack Williams
Research Intern

Research Papers

  • McDougal CE, Morrow ZT, Christopher T, Kim S, Carter D, Stevenson DM, Amador-Noguez D, Miller MJ, Sauer JD (2021) Phagocytes produce prostaglandin E2 in response to cytosolic Listeria monocytogenes. PLoS pathogens 17((9)):e1009493 PMC8491950 · Pubmed · DOI

    No abstract available.

  • Lucas LN, Barrett K, Kerby RL, Zhang Q, Cattaneo LE, Stevenson D, Rey FE, Amador-Noguez D (2021) Dominant Bacterial Phyla from the Human Gut Show Widespread Ability To Transform and Conjugate Bile Acids. mSystems :e0080521 · Pubmed · DOI

    No abstract available.

  • Lawson CE, Mundinger AB, Koch H, Jacobson TB, Weathersby CA, Jetten MSM, Pabst M, Amador-Noguez D, Noguera DR, McMahon K, Lücker S (2021) Investigating the Chemolithoautotrophic and Formate Metabolism of Nitrospira moscoviensis by Constraint-Based Metabolic Modeling and C-Tracer Analysis. mSystems 6((4)):e0017321 PMC8407350 · Pubmed · DOI

    No abstract available.

  • Jacobson TB, Callaghan MM, Amador-Noguez D (2021) Hostile Takeover: How Viruses Reprogram Prokaryotic Metabolism. Annual review of microbiology 75:515-539 · Pubmed · DOI

    No abstract available.

  • Cook TB, Jacobson TB, Venkataraman MV, Hofstetter H, Amador-Noguez D, Thomas MG, Pfleger BF (2021) Stepwise genetic engineering of Pseudomonas putida enables robust heterologous production of prodigiosin and glidobactin A. Metabolic engineering 67:112-124 PMC8434984 · Pubmed · DOI

    No abstract available.

  • Yang J, Anderson BW, Turdiev A, Turdiev H, Stevenson DM, Amador-Noguez D, Lee VT, Wang JD (2021) Author Correction: The nucleotide pGpp acts as a third alarmone in Bacillus, with functions distinct from those of (p)ppGpp. Nature communications 12((1)):3857 PMC8208980 · Pubmed · DOI

    No abstract available.

  • Clark RL, Connors BM, Stevenson DM, Hromada SE, Hamilton JJ, Amador-Noguez D, Venturelli OS (2021) Design of synthetic human gut microbiome assembly and butyrate production. Nature communications 12((1)):3254 PMC8166853 · Pubmed · DOI

    No abstract available.

  • Updegrove TB, Harke J, Anantharaman V, Yang J, Gopalan N, Wu D, Piszczek G, Stevenson DM, Amador-Noguez D, Wang JD, Aravind L, Ramamurthi KS (2021) Reformulation of an extant ATPase active site to mimic ancestral GTPase activity reveals a nucleotide base requirement for function. eLife 10: PMC7952092 · Pubmed · DOI

    No abstract available.

  • Wu MY, Mead ME, Lee MK, Neuhaus GF, Adpressa DA, Martien JI, Son YE, Moon H, Amador-Noguez D, Han KH, Rokas A, Loesgen S, Yu JH, Park HS (2021) Transcriptomic, Protein-DNA Interaction, and Metabolomic Studies of VosA, VelB, and WetA in Aspergillus nidulans Asexual Spores. mBio 12((1)): PMC7885118 · Pubmed · DOI

    No abstract available.

  • Yang J, Anderson BW, Turdiev A, Turdiev H, Stevenson DM, Amador-Noguez D, Lee VT, Wang JD (2020) The nucleotide pGpp acts as a third alarmone in Bacillus, with functions distinct from those of (p) ppGpp. Nature communications 11((1)):5388 PMC7584652 · Pubmed · DOI

    No abstract available.

  • Lawson CE, Nuijten GHL, de Graaf RM, Jacobson TB, Pabst M, Stevenson DM, Jetten MSM, Noguera DR, McMahon KD, Amador-Noguez D, Lücker S (2020) Autotrophic and mixotrophic metabolism of an anammox bacterium revealed by in vivo C and H metabolic network mapping. The ISME journal 15((3)):673-687 PMC8027424 · Pubmed · DOI

    No abstract available.

  • Fung DK, Yang J, Stevenson DM, Amador-Noguez D, Wang JD (2020) Small Alarmone Synthetase SasA Expression Leads to Concomitant Accumulation of pGpp, ppApp, and AppppA in Bacillus subtilis . Frontiers in microbiology 11:2083 PMC7492591 · Pubmed · DOI

    No abstract available.

  • Holwerda EK, Zhou J, Hon S, Stevenson DM, Amador-Noguez D, Lynd LR, van Dijken JP (2020) Metabolic Fluxes of Nitrogen and Pyrophosphate in Chemostat Cultures of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum. Applied and environmental microbiology 86((23)): PMC7657619 · Pubmed · DOI

    No abstract available.

  • Liu Y, Ghosh IN, Martien J, Zhang Y, Amador-Noguez D, Landick R (2020) Regulated redirection of central carbon flux enhances anaerobic production of bioproducts in Zymomonas mobilis. Metabolic engineering 61:261-274 · Pubmed · DOI

    No abstract available.

  • Ortiz BJ, Boursier ME, Barrett KL, Manson DE, Amador-Noguez D, Abbott NL, Blackwell HE, Lynn DM (2020) Liquid Crystal Emulsions That Intercept and Report on Bacterial Quorum Sensing. ACS applied materials & interfaces 12((26)):29056-29065 PMC7343617 · Pubmed · DOI

    No abstract available.

  • Xu J, Martien J, Gilbertson C, Ma J, Amador-Noguez D, Park JO (2020) Metabolic flux analysis and fluxomics-driven determination of reaction free energy using multiple isotopes. Current opinion in biotechnology 64:151-160 · Pubmed · DOI

    No abstract available.

  • Olson WJ, Martorelli Di Genova B, Gallego-Lopez G, Dawson AR, Stevenson D, Amador-Noguez D, Knoll LJ (2020) Dual metabolomic profiling uncovers Toxoplasma manipulation of the host metabolome and the discovery of a novel parasite metabolic capability. PLoS pathogens 16((4)):e1008432 PMC7164669 · Pubmed · DOI

    No abstract available.

  • Jacobson TB, Korosh TK, Stevenson DM, Foster C, Maranas C, Olson DG, Lynd LR, Amador-Noguez D (2020) In Vivo Thermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using C and H Tracers. mSystems 5((2)): PMC7380578 · Pubmed · DOI

    No abstract available.

  • Holwerda EK, Olson DG, Ruppertsberger NM, Stevenson DM, Murphy SJL, Maloney MI, Lanahan AA, Amador-Noguez D, Lynd LR (2020) Metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production. Biotechnology for biofuels 13:40 PMC7063780 · Pubmed · DOI

    No abstract available.

  • Koendjbiharie JG, Hon S, Pabst M, Hooftman R, Stevenson DM, Cui J, Amador-Noguez D, Lynd LR, Olson DG, van Kranenburg R (2019) The pentose phosphate pathway of cellulolytic clostridia relies on 6-phosphofructokinase instead of transaldolase. The Journal of biological chemistry 295((7)):1867-1878 PMC7029132 · Pubmed · DOI

    No abstract available.

  • Felczak MM, Jacobson TB, Ong WK, Amador-Noguez D, TerAvest MA (2019) Expression of Phosphofructokinase Is Not Sufficient to Enable Embden-Meyerhof-Parnas Glycolysis in ZM4. Frontiers in microbiology 10:2270 PMC6777484 · Pubmed · DOI

    is a bacterium that produces ethanol from glucose at up to 97% of theoretical efficiency on a carbon basis. One factor contributing to the high efficiency of ethanol production is that has a low biomass yield. The low biomass yield may be caused partly by the low ATP yield of the Entner-Doudoroff (ED) glycolytic pathway used by , which produces only one ATP per glucose consumed. To test the hypothesis that ATP yield limits biomass yield in we attempted to introduce the Embden-Meyerhof-Parnas (EMP) glycolytic pathway (with double the ATP yield) by expressing phosphofructokinase (Pfk I) from Expression of Pfk I caused growth inhibition and resulted in accumulation of mutations in the gene. Co-expression of additional EMP enzymes, fructose bisphosphate aldolase (Fba) and triose phosphate isomerase (Tpi), with Pfk I did not enable EMP flux, and resulted in production of glycerol as a side product. Further analysis indicated that heterologous reactions may have operated in the reverse direction because of native metabolite concentrations. This study reveals how the metabolomic context of a chassis organism influences the range of pathways that can be added by heterologous expression.

  • Tatli M, Hebert AS, Coon JJ, Amador-Noguez D (2019) Genome Wide Phosphoproteome Analysis of Under Anaerobic, Aerobic, and N-Fixing Conditions. Frontiers in microbiology 10:1986 PMC6737584 · Pubmed · DOI

    Protein phosphorylation is a post-translational modification with widespread regulatory roles in both eukaryotes and prokaryotes. Using mass spectrometry, we performed a genome wide investigation of protein phosphorylation in the non-model organism and biofuel producer under anaerobic, aerobic, and N-fixing conditions. Our phosphoproteome analysis revealed 125 unique phosphorylated proteins, belonging to major pathways such as glycolysis, TCA cycle, electron transport, nitrogen metabolism, and protein synthesis. Quantitative analysis revealed significant and widespread changes in protein phosphorylation across growth conditions. For example, we observed increased phosphorylation of nearly all glycolytic enzymes and a large fraction of ribosomal proteins during aerobic and N-fixing conditions. We also observed substantial changes in the phosphorylation status of enzymes and regulatory proteins involved in nitrogen fixation and ammonia assimilation during N-fixing conditions, including nitrogenase, the Rnf electron transport complex, the transcription factor NifA, GS-GOGAT cycle enzymes, and the P regulatory protein. This suggested that protein phosphorylation may play an important role at regulating all aspects of nitrogen metabolism in . This study provides new knowledge regarding the specific pathways and cellular processes that may be regulated by protein phosphorylation in this important industrial organism and provides a useful road map for future experiments that investigate the physiological role of specific phosphorylation events in .

  • Park JO, Tanner LB, Wei MH, Khana DB, Jacobson TB, Zhang Z, Rubin SA, Li SH, Higgins MB, Stevenson DM, Amador-Noguez D, Rabinowitz JD (2019) Near-equilibrium glycolysis supports metabolic homeostasis and energy yield. Nature chemical biology 15((10)):1001-1008 · Pubmed · DOI

    Glycolysis plays a central role in producing ATP and biomass. Its control principles, however, remain incompletely understood. Here, we develop a method that combines H and C tracers to determine glycolytic thermodynamics. Using this method, we show that, in conditions and organisms with relatively slow fluxes, multiple steps in glycolysis are near to equilibrium, reflecting spare enzyme capacity. In Escherichia coli, nitrogen or phosphorus upshift rapidly increases the thermodynamic driving force, deploying the spare enzyme capacity to increase flux. Similarly, respiration inhibition in mammalian cells rapidly increases both glycolytic flux and the thermodynamic driving force. The thermodynamic shift allows flux to increase with only small metabolite concentration changes. Finally, we find that the cellulose-degrading anaerobe Clostridium cellulolyticum exhibits slow, near-equilibrium glycolysis due to the use of pyrophosphate rather than ATP for fructose-bisphosphate production, resulting in enhanced per-glucose ATP yield. Thus, near-equilibrium steps of glycolysis promote both rapid flux adaptation and energy efficiency.

  • Kemis JH, Linke V, Barrett KL, Boehm FJ, Traeger LL, Keller MP, Rabaglia ME, Schueler KL, Stapleton DS, Gatti DM, Churchill GA, Amador-Noguez D, Russell JD, Yandell BS, Broman KW, Coon JJ, Attie AD, Rey FE (2019) Genetic determinants of gut microbiota composition and bile acid profiles in mice. PLoS genetics 15((8)):e1008073 PMC6715156 · Pubmed · DOI

    The microbial communities that inhabit the distal gut of humans and other mammals exhibit large inter-individual variation. While host genetics is a known factor that influences gut microbiota composition, the mechanisms underlying this variation remain largely unknown. Bile acids (BAs) are hormones that are produced by the host and chemically modified by gut bacteria. BAs serve as environmental cues and nutrients to microbes, but they can also have antibacterial effects. We hypothesized that host genetic variation in BA metabolism and homeostasis influence gut microbiota composition. To address this, we used the Diversity Outbred (DO) stock, a population of genetically distinct mice derived from eight founder strains. We characterized the fecal microbiota composition and plasma and cecal BA profiles from 400 DO mice maintained on a high-fat high-sucrose diet for ~22 weeks. Using quantitative trait locus (QTL) analysis, we identified several genomic regions associated with variations in both bacterial and BA profiles. Notably, we found overlapping QTL for Turicibacter sp. and plasma cholic acid, which mapped to a locus containing the gene for the ileal bile acid transporter, Slc10a2. Mediation analysis and subsequent follow-up validation experiments suggest that differences in Slc10a2 gene expression associated with the different strains influences levels of both traits and revealed novel interactions between Turicibacter and BAs. This work illustrates how systems genetics can be utilized to generate testable hypotheses and provide insight into host-microbe interactions.

  • Dash S, Olson DG, Joshua Chan SH, Amador-Noguez D, Lynd LR, Maranas CD (2019) Thermodynamic analysis of the pathway for ethanol production from cellobiose in Clostridium thermocellum. Metabolic engineering 55:161-169 · Pubmed · DOI

    Clostridium thermocellum is a candidate for consolidated bioprocessing by carrying out both cellulose solubilization and fermentation. However, despite significant efforts the maximum ethanol titer achieved to date remains below industrially required targets. Several studies have analyzed the impact of increasing ethanol concentration on C. thermocellum's membrane properties, cofactor pool ratios, and altered enzyme regulation. In this study, we explore the extent to which thermodynamic equilibrium limits maximum ethanol titer. We used the max-min driving force (MDF) algorithm (Noor et al., 2014) to identify the range of allowable metabolite concentrations that maintain a negative free energy change for all reaction steps in the pathway from cellobiose to ethanol. To this end, we used a time-series metabolite concentration dataset to flag five reactions (phosphofructokinase (PFK), fructose bisphosphate aldolase (FBA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH)) which become thermodynamic bottlenecks under high external ethanol concentrations. Thermodynamic analysis was also deployed in a prospective mode to evaluate genetic interventions which can improve pathway thermodynamics by generating minimal set of reactions or elementary flux modes (EFMs) which possess unique genetic variations while ensuring mass and redox balance with ethanol production. MDF evaluation of all generated (336) EFMs indicated that, i) pyruvate phosphate dikinase (PPDK) has a higher pathway MDF than the malate shunt alternative due to limiting CO concentrations under physiological conditions, and ii) NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) can alleviate thermodynamic bottlenecks at high ethanol concentrations due to cofactor modification and reduction in ATP generation. The combination of ATP linked phosphofructokinase (PFK-ATP) and NADPH linked alcohol dehydrogenase (ADH-NADPH) with NADPH linked aldehyde dehydrogenase (ALDH-NADPH) or ferredoxin: NADP ​+ ​oxidoreductase (NADPH-FNOR) emerges as the best intervention strategy for ethanol production that balances MDF improvements with ATP generation, and appears to functionally reproduce the pathway employed by the ethanologen Thermoanaerobacterium saccharolyticum. Expanding the list of measured intracellular metabolites and improving the quantification accuracy of measurements was found to improve the fidelity of pathway thermodynamics analysis in C. thermocellum. This study demonstrates even before addressing an organism's enzyme kinetics and allosteric regulations, pathway thermodynamics can flag pathway bottlenecks and identify testable strategies for enhancing pathway thermodynamic feasibility and function.

  • Ostrem Loss EM, Lee MK, Wu MY, Martien J, Chen W, Amador-Noguez D, Jefcoate C, Remucal C, Jung S, Kim SC, Yu JH (2019) Cytochrome P450 Monooxygenase-Mediated Metabolic Utilization of Benzo[]Pyrene by Species. mBio 10((3)): PMC6538779 · Pubmed · DOI

    Soil-dwelling fungal species possess the versatile metabolic capability to degrade complex organic compounds that are toxic to humans, yet the mechanisms they employ remain largely unknown. Benzo[]pyrene (BaP) is a pervasive carcinogenic contaminant, posing a significant concern for human health. Here, we report that several species are capable of degrading BaP. Exposing cells to BaP results in transcriptomic and metabolic changes associated with cellular growth and energy generation, implying that the fungus utilizes BaP as a growth substrate. Importantly, we identify and characterize the conserved gene encoding a cytochrome P450 monooxygenase that is necessary for the metabolic utilization of BaP in We further demonstrate that the fungal NF-κB-type regulators VeA and VelB are required for proper expression of in response to nutrient limitation and BaP degradation in Our study illuminates fundamental knowledge of fungal BaP metabolism and provides novel insights into enhancing bioremediation potential. We are increasingly exposed to environmental pollutants, including the carcinogen benzo[]pyrene (BaP), which has prompted extensive research into human metabolism of toxicants. However, little is known about metabolic mechanisms employed by fungi that are able to use some toxic pollutants as the substrates for growth, leaving innocuous by-products. This study systemically demonstrates that a common soil-dwelling fungus is able to use benzo[]pyrene as food, which results in expression and metabolic changes associated with growth and energy generation. Importantly, this study reveals key components of the metabolic utilization of BaP, notably a cytochrome P450 monooxygenase and the fungal NF-κB-type transcriptional regulators. Our study advances fundamental knowledge of fungal BaP metabolism and provides novel insight into designing and implementing enhanced bioremediation strategies.

  • Pisithkul T, Schroeder JW, Trujillo EA, Yeesin P, Stevenson DM, Chaiamarit T, Coon JJ, Wang JD, Amador-Noguez D (2019) Metabolic Remodeling during Biofilm Development of Bacillus subtilis. mBio 10((3)): PMC6529636 · Pubmed · DOI

    Biofilms are structured communities of tightly associated cells that constitute the predominant state of bacterial growth in natural and human-made environments. Although the core genetic circuitry that controls biofilm formation in model bacteria such as has been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, we performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development in by combining metabolomic, transcriptomic, and proteomic analyses. We report surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hitherto unrecognized as biofilm associated. For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provide insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following upon these results, we demonstrated that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This report represents a comprehensive systems-level investigation of the metabolic remodeling occurring during biofilm development that will serve as a useful road map for future studies on biofilm physiology. Bacterial biofilms are ubiquitous in natural environments and play an important role in many clinical, industrial, and ecological settings. Although much is known about the transcriptional regulatory networks that control biofilm formation in model bacteria such as , very little is known about the role of metabolism in this complex developmental process. To address this important knowledge gap, we performed a time-resolved analysis of the metabolic changes associated with bacterial biofilm development in by combining metabolomic, transcriptomic, and proteomic analyses. Here, we report a widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. This report serves as a unique hypothesis-generating resource for future studies on bacterial biofilm physiology. Outside the biofilm research area, this work should also prove relevant to any investigators interested in microbial physiology and metabolism.

  • Scott IM, Rubinstein GM, Poole FL, Lipscomb GL, Schut GJ, Williams-Rhaesa AM, Stevenson DM, Amador-Noguez D, Kelly RM, Adams MWW (2019) The thermophilic biomass-degrading bacterium utilizes two enzymes to oxidize glyceraldehyde 3-phosphate during glycolysis. The Journal of biological chemistry 294((25)):9995-10005 PMC6597818 · Pubmed · DOI

    is an extremely thermophilic, cellulolytic bacterium with a growth optimum at 78 °C and is the most thermophilic cellulose degrader known. It is an attractive target for biotechnological applications, but metabolic engineering will require an in-depth understanding of its primary pathways. A previous analysis of its genome uncovered evidence that may have a completely uncharacterized aspect to its redox metabolism, involving a tungsten-containing oxidoreductase of unknown function. Herein, we purified and characterized this new member of the aldehyde ferredoxin oxidoreductase family of tungstoenzymes. We show that it is a heterodimeric glyceraldehyde-3-phosphate (GAP) ferredoxin oxidoreductase (GOR) present not only in all known species, but also in 44 mostly anaerobic bacterial genera. GOR is phylogenetically distinct from the monomeric GAP-oxidizing enzyme found previously in several Archaea. We found that its large subunit (GOR-L) contains a single tungstopterin site and one iron-sulfur [4Fe-4S] cluster, that the small subunit (GOR-S) contains four [4Fe-4S] clusters, and that GOR uses ferredoxin as an electron acceptor. Deletion of either subunit resulted in a distinct growth phenotype on both C and C sugars, with an increased lag phase, but higher cell densities. Using metabolomics and kinetic analyses, we show that GOR functions in parallel with the conventional GAP dehydrogenase, providing an alternative ferredoxin-dependent glycolytic pathway. These two pathways likely facilitate the recycling of reduced redox carriers (NADH and ferredoxin) in response to environmental H concentrations. This metabolic flexibility has important implications for the future engineering of this and related species.

  • Jacobson TB, Adamczyk PA, Stevenson DM, Regner M, Ralph J, Reed JL, Amador-Noguez D (2019) H and C metabolic flux analysis elucidates in vivo thermodynamics of the ED pathway in Zymomonas mobilis. Metabolic engineering 54:301-316 · Pubmed · DOI

    Zymomonas mobilis is an industrially relevant bacterium notable for its ability to rapidly ferment simple sugars to ethanol using the Entner-Doudoroff (ED) glycolytic pathway, an alternative to the well-known Embden-Meyerhof-Parnas (EMP) pathway used by most organisms. Recent computational studies have predicted that the ED pathway is substantially more thermodynamically favorable than the EMP pathway, a potential factor explaining the high glycolytic rate in Z. mobilis. Here, to investigate the in vivo thermodynamics of the ED pathway and central carbon metabolism in Z. mobilis, we implemented a network-level approach that integrates quantitative metabolomics with H and C metabolic flux analysis to estimate reversibility and Gibbs free energy (ΔG) of metabolic reactions. This analysis revealed a strongly thermodynamically favorable ED pathway in Z. mobilis that is nearly twice as favorable as the EMP pathway in E. coli or S. cerevisiae. The in vivo step-by-step thermodynamic profile of the ED pathway was highly similar to previous in silico predictions, indicating that maximizing ΔG for each pathway step likely constitutes a cellular objective in Z. mobilis. Our analysis also revealed novel features of Z. mobilis metabolism, including phosphofructokinase-like enzyme activity, tricarboxylic acid cycle anaplerosis via PEP carboxylase, and a metabolic imbalance in the pentose phosphate pathway resulting in excretion of shikimate pathway intermediates. The integrated approach we present here for in vivo ΔG quantitation may be applied to the thermodynamic profiling of pathways and metabolic networks in other microorganisms and will contribute to the development of quantitative models of metabolism.

  • Martien JI, Hebert AS, Stevenson DM, Regner MR, Khana DB, Coon JJ, Amador-Noguez D (2019) Systems-Level Analysis of Oxygen Exposure in : Implications for Isoprenoid Production. mSystems 4((1)): PMC6372839 · Pubmed · DOI

    Zymomonas mobilis is an aerotolerant anaerobe and prolific ethanologen with attractive characteristics for industrial bioproduct generation. However, there is currently insufficient knowledge of the impact that environmental factors have on flux through industrially relevant biosynthetic pathways. Here, we examined the effect of oxygen exposure on metabolism and gene expression in Z. mobilis by combining targeted metabolomics, mRNA sequencing, and shotgun proteomics. We found that exposure to oxygen profoundly influenced metabolism, inducing both transient metabolic bottlenecks and long-term metabolic remodeling. In particular, oxygen induced a severe but temporary metabolic bottleneck in the methyl erythritol 4-phosphate pathway for isoprenoid biosynthesis caused by oxidative damage to the iron-sulfur cofactors of the final two enzymes in the pathway. This bottleneck was resolved with minimal changes in expression of isoprenoid biosynthetic enzymes. Instead, it was associated with pronounced upregulation of enzymes related to iron-sulfur cluster maintenance and biogenesis (i.e., flavodoxin reductase and the operon). We also detected major changes in glucose utilization in the presence of oxygen. Specifically, we observed increased gluconate production following exposure to oxygen, accounting for 18% of glucose uptake. Our results suggest that under aerobic conditions, electrons derived from the oxidation of glucose to gluconate are diverted to the electron transport chain, where they can minimize oxidative damage by reducing reactive oxygen species such as HO. This model is supported by the simultaneous upregulation of three membrane-bound dehydrogenases, cytochrome peroxidase, and a cytochrome oxidase following exposure to oxygen. Microbially generated biofuels and bioproducts have the potential to provide a more environmentally sustainable alternative to fossil-fuel-derived products. In particular, isoprenoids, a diverse class of natural products, are chemically suitable for use as high-grade transport fuels and other commodity molecules. However, metabolic engineering for increased production of isoprenoids and other bioproducts is limited by an incomplete understanding of factors that control flux through biosynthetic pathways. Here, we examined the native regulation of the isoprenoid biosynthetic pathway in the biofuel producer Zymomonas mobilis. We leveraged oxygen exposure as a means to perturb carbon flux, allowing us to observe the formation and resolution of a metabolic bottleneck in the pathway. Our multi-omics analysis of this perturbation enabled us to identify key auxiliary enzymes whose expression correlates with increased production of isoprenoid precursors, which we propose as potential targets for future metabolic engineering.

  • Ghosh IN, Martien J, Hebert AS, Zhang Y, Coon JJ, Amador-Noguez D, Landick R (2018) OptSSeq explores enzyme expression and function landscapes to maximize isobutanol production rate. Metabolic engineering 52:324-340 · Pubmed · DOI

    Efficient microbial production of the next-generation biofuel isobutanol (IBA) is limited by metabolic bottlenecks. Overcoming these bottlenecks will be aided by knowing the optimal ratio of enzymes for efficient flux through the IBA biosynthetic pathway. OptSSeq (Optimization by Selection and Sequencing) accomplishes this goal by tracking growth rate-linked selection of optimal expression elements from a combinatorial library. The 5-step pathway to IBA consists of Acetolactate synthase (AlsS), Keto-acid reductoisomerase (KARI), Di-hydroxy acid dehydratase (DHAD), Ketoisovalerate decarboxylase (Kivd) and Alcohol dehydrogenase (Adh). Using OptSSeq, we identified gene expression elements leading to optimal enzyme levels that enabled theoretically maximal productivities per cell biomass in Escherichia coli. We identified KARI as the rate-limiting step, requiring the highest levels of enzymes expression, followed by AlsS and AdhA. DHAD and Kivd required relatively lower levels of expression for optimal IBA production. OptSSeq also enabled the identification of an Adh enzyme variant capable of an improved rate of IBA production. Using models that predict impacts of enzyme synthesis costs on cellular growth rates, we found that optimum levels of pathway enzymes led to maximal IBA production, and that additional limitations lie in the E. coli metabolic network. Our optimized constructs enabled the production of ~3 g IBA per hour per gram dry cell weight and was achieved with 20 % of the total cell protein devoted to IBA-pathway enzymes in the molar ratio 2.5:6.7:2:1:5.2 (AlsS:IlvC:IlvD:Kivd:AdhA). These enzyme levels and ratios optimal for IBA production in E. coli provide a useful starting point for optimizing production of IBA in diverse microbes and fermentation conditions.

  • Dwulit-Smith JR, Hamilton JJ, Stevenson DM, He S, Oyserman BO, Moya-Flores F, Garcia SL, Amador-Noguez D, McMahon KD, Forest KT (2018) acI Actinobacteria Assemble a Functional Actinorhodopsin with Natively Synthesized Retinal. Appl. Environ. Microbiol. 84(24): (PMC6275354) · Pubmed · DOI

    Freshwater lakes harbor complex microbial communities, but these ecosystems are often dominated by acI Members of this cosmopolitan lineage are proposed to bolster heterotrophic growth using phototrophy because their genomes encode actino-opsins (). This model has been difficult to validate experimentally because acI are not consistently culturable. Based primarily on genomes from single cells and metagenomes, we provide a detailed biosynthetic route for members of acI clades A and B to synthesize retinal and its carotenoid precursors. Consequently, acI cells should be able to natively assemble light-driven actinorhodopsins (holo-ActR) to pump protons, unlike many bacteria that encode opsins but may need to exogenously obtain retinal because they lack retinal machinery. Moreover, we show that all acI clades contain genes for a secondary branch of the carotenoid pathway, implying synthesis of a complex carotenoid. Transcription analysis of acI in a eutrophic lake shows that all retinal and carotenoid pathway operons are transcribed and that is among the most highly transcribed of all acI genes. Furthermore, heterologous expression of acI retinal pathway genes showed that lycopene, retinal, and ActR can be made using the genes encoded in these organisms. Model cells producing ActR and the key acI retinal-producing β-carotene oxygenase formed holo-ActR and acidified solution during illumination. Taken together, our results prove that acI containing both ActR and acI retinal production machinery have the capacity to natively synthesize a green light-dependent outward proton-pumping rhodopsin. Microbes play critical roles in determining the quality of freshwater ecosystems, which are vital to human civilization. Because acI are ubiquitous and abundant in freshwater lakes, clarifying their ecophysiology is a major step in determining the contributions that they make to nitrogen and carbon cycling. Without accurate knowledge of these cycles, freshwater systems cannot be incorporated into climate change models, ecosystem imbalances cannot be predicted, and policy for service disruption cannot be planned. Our work fills major gaps in microbial light utilization, secondary metabolite production, and energy cycling in freshwater habitats.

  • Kwan G, Plagenz B, Cowles K, Pisithkul T, Amador-Noguez D, Barak JD (2018) Few Differences in Metabolic Network Use Found Between Colonization of Plants and Typhoidal Mice. Front Microbiol 9:695 (PMC5951976) · Pubmed · DOI

    The human enteric pathogen leads a cross-kingdom lifestyle, actively colonizing and persisting on plants in between animal hosts. One of the questions that arises from this dual lifestyle is how is able to adapt to such divergent hosts. Metabolic pathways required for animal colonization and virulence have been previously identified, but the metabolism of this bacterium on plants is poorly understood. To determine the requirements for plant colonization by , we first screened a library of metabolic mutants, previously examined in a systemic mouse typhoidal model, for competitive plant colonization fitness on alfalfa seedlings. By comparing our results to those reported in -infected murine spleens, we found that the presence of individual nutrients differed between the two host niches. Yet, similar metabolic pathways contributed to colonization of both plants and animals, such as the biosynthesis of amino acids, purines, and vitamins and the catabolism of glycerol and glucose. However, utilization of at least three metabolic networks differed during the bacterium's plant- and animal-associated lifestyles. Whereas both fatty acid biosynthesis and degradation contributed to animal colonization, only fatty acid biosynthesis was required during plant colonization. Though serine biosynthesis was required in both hosts, used different pathways within the serine metabolic network to achieve this outcome. Lastly, the metabolic network surrounding played different roles during colonization of each host. In animal models of infection, O-antigen production downstream of facilitates immune evasion. We discovered that contributed to attachment, to seeds and germinated seedlings, and was essential for growth in early seedling exudates, when mannose is limited. However, only seedling attachment was linked to O-antigen production, indicating that played additional roles critical for plant colonization that were independent of surface polysaccharide production. The integrated view of metabolism throughout its life cycle presented here provides insight on how metabolic versatility and adaption of known physiological pathways for alternate functions enable a zoonotic pathogen to thrive in niches spanning across multiple kingdoms of life.

  • Romano KA, Dill-McFarland KA, Kasahara K, Kerby RL, Vivas EI, Amador-Noguez D, Herd P, Rey FE (2018) Fecal Aliquot Straw Technique (FAST) allows for easy and reproducible subsampling: assessing interpersonal variation in trimethylamine-N-oxide (TMAO) accumulation. Microbiome 6(1):91 (PMC5960144) · Pubmed · DOI

    Convenient, reproducible, and rapid preservation of unique biological specimens is pivotal to their use in microbiome analyses. As an increasing number of human studies incorporate the gut microbiome in their design, there is a high demand for streamlined sample collection and storage methods that are amenable to different settings and experimental needs. While several commercial kits address collection/shipping needs for sequence-based studies, these methods do not preserve samples properly for studies that require viable microbes. We describe the Fecal Aliquot Straw Technique (FAST) of fecal sample processing for storage and subsampling. This method uses a straw to collect fecal material from samples recently voided or preserved at low temperature but not frozen (i.e., 4 °C). Different straw aliquots collected from the same sample yielded highly reproducible communities as disclosed by 16S rRNA gene sequencing; operational taxonomic units that were lost, or gained, between the two aliquots represented very low-abundance taxa (i.e., < 0.3% of the community). FAST-processed samples inoculated into germ-free animals resulted in gut communities that retained on average ~ 80% of the donor's bacterial community. Assessment of choline metabolism and trimethylamine-N-oxide accumulation in transplanted mice suggests large interpersonal variation. Overall, FAST allows for repetitive subsampling without thawing of the specimens and requires minimal supplies and storage space, making it convenient to utilize both in the lab and in the field. FAST has the potential to advance microbiome research through easy, reproducible sample processing.

  • Theisen E, McDougal CE, Nakanishi M, Stevenson DM, Amador-Noguez D, Rosenberg DW, Knoll LJ, Sauer JD (2018) Cyclooxygenase-1 and -2 Play Contrasting Roles in -Stimulated Immunity. J. Immunol. 200(11):3729-3738 (PMC5964023) · Pubmed · DOI

    Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (COX) activity and are commonly used for pain relief and fever reduction. NSAIDs are used following childhood vaccinations and cancer immunotherapies; however, how NSAIDs influence the development of immunity following these therapies is unknown. We hypothesized that NSAIDs would modulate the development of an immune response to -based immunotherapy. Treatment of mice with the nonspecific COX inhibitor indomethacin impaired the generation of cell-mediated immunity. This phenotype was due to inhibition of the inducible COX-2 enzyme, as treatment with the COX-2-selective inhibitor celecoxib similarly inhibited the development of immunity. In contrast, loss of COX-1 activity improved immunity to Impairments in immunity were independent of bacterial burden, dendritic cell costimulation, or innate immune cell infiltrate. Instead, we observed that PGE production following is critical for the formation of an Ag-specific CD8 T cell response. Use of the alternative analgesic acetaminophen did not impair immunity. Taken together, our results suggest that COX-2 is necessary for optimal CD8 T cell responses to , whereas COX-1 is detrimental. Use of pharmacotherapies that spare COX-2 activity and the production of PGE like acetaminophen will be critical for the generation of optimal antitumor responses using .

  • Tian L, Perot SJ, Stevenson D, Jacobson T, Lanahan AA, Amador-Noguez D, Olson DG, Lynd LR (2017) Metabolome analysis reveals a role for glyceraldehyde 3-phosphate dehydrogenase in the inhibition of by ethanol. Biotechnol Biofuels 10:276 (PMC5708176) · Pubmed · DOI

    is a promising microorganism for conversion of cellulosic biomass to biofuel, without added enzymes; however, the low ethanol titer produced by strains developed thus far is an obstacle to industrial application. Here, we analyzed changes in the relative concentration of intracellular metabolites in response to gradual addition of ethanol to growing cultures. For , we observed that ethanol tolerance, in experiments with gradual ethanol addition, was twofold higher than previously observed in response to a stepwise increase in the ethanol concentration, and appears to be due to a mechanism other than mutation. As ethanol concentrations increased, we found accumulation of metabolites upstream of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction and depletion of metabolites downstream of that reaction. This pattern was not observed in the more ethanol-tolerant organism . We hypothesize that the Gapdh enzyme may have different properties in the two organisms. Our hypothesis is supported by enzyme assays showing greater sensitivity of the enzyme to high levels of NADH, and by the increase in ethanol tolerance and production when the was expressed in . We have demonstrated that a metabolic bottleneck occurs at the GAPDH reaction when the growth of is inhibited by high levels of ethanol. We then showed that this bottleneck could be relieved by expression of the gene from . This enzyme is a promising target for future metabolic engineering work.

  • Rand JM, Pisithkul T, Clark RL, Thiede JM, Mehrer CR, Agnew DE, Campbell CE, Markley AL, Price MN, Ray J, Wetmore KM, Suh Y, Arkin AP, Deutschbauer AM, Amador-Noguez D, Pfleger BF (2017) A metabolic pathway for catabolizing levulinic acid in bacteria. Nat Microbiol 2(12):1624-1634 (PMC5705400) · Pubmed · DOI

    Microorganisms can catabolize a wide range of organic compounds and therefore have the potential to perform many industrially relevant bioconversions. One barrier to realizing the potential of biorefining strategies lies in our incomplete knowledge of metabolic pathways, including those that can be used to assimilate naturally abundant or easily generated feedstocks. For instance, levulinic acid (LA) is a carbon source that is readily obtainable as a dehydration product of lignocellulosic biomass and can serve as the sole carbon source for some bacteria. Yet, the genetics and structure of LA catabolism have remained unknown. Here, we report the identification and characterization of a seven-gene operon that enables LA catabolism in Pseudomonas putida KT2440. When the pathway was reconstituted with purified proteins, we observed the formation of four acyl-CoA intermediates, including a unique 4-phosphovaleryl-CoA and the previously observed 3-hydroxyvaleryl-CoA product. Using adaptive evolution, we obtained a mutant of Escherichia coli LS5218 with functional deletions of fadE and atoC that was capable of robust growth on LA when it expressed the five enzymes from the P. putida operon. This discovery will enable more efficient use of biomass hydrolysates and metabolic engineering to develop bioconversions using LA as a feedstock.

  • Romano KA, Martinez-Del Campo A, Kasahara K, Chittim CL, Vivas EI, Amador-Noguez D, Balskus EP, Rey FE (2017) Metabolic, Epigenetic, and Transgenerational Effects of Gut Bacterial Choline Consumption. Cell Host Microbe 22(3):279-290.e7 (PMC5599363) · Pubmed · DOI

    Choline is an essential nutrient and methyl donor required for epigenetic regulation. Here, we assessed the impact of gut microbial choline metabolism on bacterial fitness and host biology by engineering a microbial community that lacks a single choline-utilizing enzyme. Our results indicate that choline-utilizing bacteria compete with the host for this nutrient, significantly impacting plasma and hepatic levels of methyl-donor metabolites and recapitulating biochemical signatures of choline deficiency. Mice harboring high levels of choline-consuming bacteria showed increased susceptibility to metabolic disease in the context of a high-fat diet. Furthermore, bacterially induced reduction of methyl-donor availability influenced global DNA methylation patterns in both adult mice and their offspring and engendered behavioral alterations. Our results reveal an underappreciated effect of bacterial choline metabolism on host metabolism, epigenetics, and behavior. This work suggests that interpersonal differences in microbial metabolism should be considered when determining optimal nutrient intake requirements.

  • Rydzak T, Garcia D, Stevenson DM, Sladek M, Klingeman DM, Holwerda EK, Amador-Noguez D, Brown SD, Guss AM (2017) Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellum. Metab. Eng. 41:182-191 · Pubmed · DOI

    Clostridium thermocellum rapidly deconstructs cellulose and ferments resulting hydrolysis products into ethanol and other products, and is thus a promising platform organism for the development of cellulosic biofuel production via consolidated bioprocessing. While recent metabolic engineering strategies have targeted eliminating canonical fermentation products (acetate, lactate, formate, and H), C. thermocellum also secretes amino acids, which has limited ethanol yields in engineered strains to approximately 70% of the theoretical maximum. To investigate approaches to decrease amino acid secretion, we attempted to reduce ammonium assimilation by deleting the Type I glutamine synthetase (glnA) in an essentially wild type strain of C. thermocellum. Deletion of glnA reduced levels of secreted valine and total amino acids by 53% and 44% respectively, and increased ethanol yields by 53%. RNA-seq analysis revealed that genes encoding the RNF-complex were more highly expressed in ΔglnA and may have a role in improving NADH-availability for ethanol production. While a significant up-regulation of genes involved in nitrogen assimilation and urea uptake suggested that deletion of glnA induces a nitrogen starvation response, metabolomic analysis showed an increase in intracellular glutamine levels indicative of nitrogen-rich conditions. We propose that deletion of glnA causes deregulation of nitrogen metabolism, leading to overexpression of nitrogen metabolism genes and, in turn, elevated glutamine levels. Here we demonstrate that perturbation of nitrogen assimilation is a promising strategy to redirect flux from the production of nitrogenous compounds toward biofuels in C. thermocellum.

  • Olson DG, Hörl M, Fuhrer T, Cui J, Zhou J, Maloney MI, Amador-Noguez D, Tian L, Sauer U, Lynd LR (2016) Glycolysis without pyruvate kinase in Clostridium thermocellum. Metab. Eng. 39:169-180 · Pubmed · DOI

    The metabolism of Clostridium thermocellum is notable in that it assimilates sugar via the EMP pathway but does not possess a pyruvate kinase enzyme. In the wild type organism, there are three proposed pathways for conversion of phosphoenolpyruvate (PEP) to pyruvate, which differ in their cofactor usage. One path uses pyruvate phosphate dikinase (PPDK), another pathway uses the combined activities of PEP carboxykinase (PEPCK) and oxaloacetate decarboxylase (ODC). Yet another pathway, the malate shunt, uses the combined activities of PEPCK, malate dehydrogenase and malic enzyme. First we showed that there is no flux through the ODC pathway by enzyme assay. Flux through the remaining two pathways (PPDK and malate shunt) was determined by dynamic C labeling. In the wild-type strain, the malate shunt accounts for about 33±2% of the flux to pyruvate, with the remainder via the PPDK pathway. Deletion of the ppdk gene resulted in a redirection of all pyruvate flux through the malate shunt. This provides the first direct evidence of the in-vivo function of the malate shunt.

  • Martien JI, Amador-Noguez D (2016) Recent applications of metabolomics to advance microbial biofuel production. Curr. Opin. Biotechnol. 43:118-126 · Pubmed · DOI

    Biofuel production from plant biomass is a promising source of renewable energy [1]. However, efficient biofuel production involves the complex task of engineering high-performance microorganisms, which requires detailed knowledge of metabolic function and regulation. This review highlights the potential of mass-spectrometry-based metabolomic analysis to guide rational engineering of biofuel-producing microbes. We discuss recent studies that apply knowledge gained from metabolomic analyses to increase the productivity of engineered pathways, characterize the metabolism of emerging biofuel producers, generate novel bioproducts, enable utilization of lignocellulosic feedstock, and improve the stress tolerance of biofuel producers.

  • Patel NM, Moore JD, Blackwell HE, Amador-Noguez D (2016) Identification of Unanticipated and Novel N-Acyl L-Homoserine Lactones (AHLs) Using a Sensitive Non-Targeted LC-MS/MS Method. PLoS ONE 11(10):e0163469 (PMC5051804) · Pubmed

    N-acyl L-homoserine lactones (AHLs) constitute a predominant class of quorum-sensing signaling molecules used by Gram-negative bacteria. Here, we report a sensitive and non-targeted HPLC-MS/MS method based on parallel reaction monitoring (PRM) to identify and quantitate known, unanticipated, and novel AHLs in microbial samples. Using a hybrid quadrupole-high resolution mass analyzer, this method integrates MS scans and all-ion fragmentation MS/MS scans to allow simultaneous detection of AHL parent-ion masses and generation of full mass spectra at high resolution and high mass accuracy in a single chromatographic run. We applied this method to screen for AHL production in a variety of Gram-negative bacteria (i.e. B. cepacia, E. tarda, E. carotovora, E. herbicola, P. stewartii, P. aeruginosa, P. aureofaciens, and R. sphaeroides) and discovered that nearly all of them produce a larger set of AHLs than previously reported. Furthermore, we identified production of an uncommon AHL (i.e. 3-oxo-C7-HL) in E. carotovora and P. stewartii, whose production has only been previously observed within the genera Serratia and Yersinia. Finally, we used our method to quantitate AHL degradation in B. cepacia, E. carotovora, E. herbicola, P. stewartii, P. aeruginosa, P. aureofaciens, the non-AHL producer E. coli, and the Gram-positive bacterium B. subtilis. We found that AHL degradation ability varies widely across these microbes, of which B. subtilis and E. carotovora are the best degraders, and observed that there is a general trend for AHLs containing long acyl chains (≥10 carbons) to be degraded at faster rates than AHLs with short acyl chains (≤6 carbons).

  • Park JO, Rubin SA, Xu YF, Amador-Noguez D, Fan J, Shlomi T, Rabinowitz JD (2016) Metabolite concentrations, fluxes and free energies imply efficient enzyme usage. Nat. Chem. Biol. 12(7):482-9 (PMC4912430) · Pubmed

    In metabolism, available free energy is limited and must be divided across pathway steps to maintain a negative ΔG throughout. For each reaction, ΔG is log proportional both to a concentration ratio (reaction quotient to equilibrium constant) and to a flux ratio (backward to forward flux). Here we use isotope labeling to measure absolute metabolite concentrations and fluxes in Escherichia coli, yeast and a mammalian cell line. We then integrate this information to obtain a unified set of concentrations and ΔG for each organism. In glycolysis, we find that free energy is partitioned so as to mitigate unproductive backward fluxes associated with ΔG near zero. Across metabolism, we observe that absolute metabolite concentrations and ΔG are substantially conserved and that most substrate (but not inhibitor) concentrations exceed the associated enzyme binding site dissociation constant (Km or Ki). The observed conservation of metabolite concentrations is consistent with an evolutionary drive to utilize enzymes efficiently given thermodynamic and osmotic constraints.

  • Spero MA, Brickner JR, Mollet JT, Pisithkul T, Amador-Noguez D, Donohue TJ (2016) Different Functions of Phylogenetically Distinct Bacterial Complex I Isozymes. J. Bacteriol. 198(8):1268-80 (PMC4859585) · Pubmed

    NADH:quinone oxidoreductase (complex I) is a bioenergetic enzyme that transfers electrons from NADH to quinone, conserving the energy of this reaction by contributing to the proton motive force. While the importance of NADH oxidation to mitochondrial aerobic respiration is well documented, the contribution of complex I to bacterial electron transport chains has been tested in only a few species. Here, we analyze the function of two phylogenetically distinct complex I isozymes in Rhodobacter sphaeroides, an alphaproteobacterium that contains well-characterized electron transport chains. We found that R. sphaeroides complex I activity is important for aerobic respiration and required for anaerobic dimethyl sulfoxide (DMSO) respiration (in the absence of light), photoautotrophic growth, and photoheterotrophic growth (in the absence of an external electron acceptor). Our data also provide insight into the functions of the phylogenetically distinct R. sphaeroidescomplex I enzymes (complex IA and complex IE) in maintaining a cellular redox state during photoheterotrophic growth. We propose that the function of each isozyme during photoheterotrophic growth is either NADH synthesis (complex IA) or NADH oxidation (complex IE). The canonical alphaproteobacterial complex I isozyme (complex IA) was also shown to be important for routing electrons to nitrogenase-mediated H2 production, while the horizontally acquired enzyme (complex IE) was dispensable in this process. Unlike the singular role of complex I in mitochondria, we predict that the phylogenetically distinct complex I enzymes found across bacterial species have evolved to enhance the functions of their respective electron transport chains. Cells use a proton motive force (PMF), NADH, and ATP to support numerous processes. In mitochondria, complex I uses NADH oxidation to generate a PMF, which can drive ATP synthesis. This study analyzed the function of complex I in bacteria, which contain more-diverse and more-flexible electron transport chains than mitochondria. We tested complex I function in Rhodobacter sphaeroides, a bacterium predicted to encode two phylogenetically distinct complex I isozymes. R. sphaeroides cells lacking both isozymes had growth defects during all tested modes of growth, illustrating the important function of this enzyme under diverse conditions. We conclude that the two isozymes are not functionally redundant and predict that phylogenetically distinct complex I enzymes have evolved to support the diverse lifestyles of bacteria.

  • Wang PM, Choera T, Wiemann P, Pisithkul T, Amador-Noguez D, Keller NP (2015) TrpE feedback mutants reveal roadblocks and conduits toward increasing secondary metabolism in Aspergillus fumigatus. Fungal Genet. Biol. 89:102-113 (PMC4789178) · Pubmed

    Small peptides formed from non-ribosomal peptide synthetases (NRPS) are bioactive molecules produced by many fungi including the genus Aspergillus. A subset of NRPS utilizes tryptophan and its precursor, the non-proteinogenic amino acid anthranilate, in synthesis of various metabolites such as Aspergillus fumigatus fumiquinazolines (Fqs) produced by the fmq gene cluster. The A. fumigatus genome contains two putative anthranilate synthases - a key enzyme in conversion of anthranilic acid to tryptophan - one beside the fmq cluster and one in a region of co-linearity with other Aspergillus spp. Only the gene found in the co-linear region, trpE, was involved in tryptophan biosynthesis. We found that site-specific mutations of the TrpE feedback domain resulted in significantly increased production of anthranilate, tryptophan, p-aminobenzoate and fumiquinazolines FqF and FqC. Supplementation with tryptophan restored metabolism to near wild type levels in the feedback mutants and suggested that synthesis of the tryptophan degradation product kynurenine could negatively impact Fq synthesis. The second putative anthranilate synthase gene next to the fmq cluster was termed icsA for its considerable identity to isochorismate synthases in bacteria. Although icsA had no impact on A. fumigatus Fq production, deletion and over-expression of icsA increased and decreased respectively aromatic amino acid levels suggesting that IcsA can draw from the cellular chorismate pool.

  • Pisithkul T, Jacobson TB, O'Brien TJ, Stevenson DM, Amador-Noguez D (2015) Phenolic Amides Are Potent Inhibitors of De Novo Nucleotide Biosynthesis. Appl. Environ. Microbiol. 81(17):5761-72 (PMC4551265) · Pubmed

    An outstanding challenge toward efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using Escherichia coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl amide and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we showed that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposure leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using (13)C-labeled sugars and [(15)N]ammonia demonstrated that carbon and nitrogen fluxes into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. The results presented here will help in the development of strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals.

  • Pisithkul T, Patel NM, Amador-Noguez D (2015) Post-translational modifications as key regulators of bacterial metabolic fluxes. Curr. Opin. Microbiol. 24:29-37 · Pubmed

    In order to survive and compete in natural settings, bacteria must excel at quickly adapting their metabolism to fluctuations in nutrient availability and other environmental variables. This necessitates fast-acting post-translational regulatory mechanisms, that is, allostery or covalent modification, to control metabolic flux. While allosteric regulation has long been a well-established strategy for regulating metabolic enzyme activity in bacteria, covalent post-translational modes of regulation, such as phosphorylation or acetylation, have previously been regarded as regulatory mechanisms employed primarily by eukaryotic organisms. Recent findings, however, have shifted this perception and point to a widespread role for covalent posttranslational modification in the regulation of metabolic enzymes and fluxes in bacteria. This review provides an outline of the exciting recent advances in this area.

  • Romano KA, Vivas EI, Amador-Noguez D, Rey FE (2015) Intestinal microbiota composition modulates choline bioavailability from diet and accumulation of the proatherogenic metabolite trimethylamine-N-oxide. MBio 6(2):e02481 (PMC4453578) · Pubmed

    Choline is a water-soluble nutrient essential for human life. Gut microbial metabolism of choline results in the production of trimethylamine (TMA), which upon absorption by the host is converted in the liver to trimethylamine-N-oxide (TMAO). Recent studies revealed that TMAO exacerbates atherosclerosis in mice and positively correlates with the severity of this disease in humans. However, which microbes contribute to TMA production in the human gut, the extent to which host factors (e.g., genotype) and diet affect TMA production and colonization of these microbes, and the effects TMA-producing microbes have on the bioavailability of dietary choline remain largely unknown. We screened a collection of 79 sequenced human intestinal isolates encompassing the major phyla found in the human gut and identified nine strains capable of producing TMA from choline in vitro. Gnotobiotic mouse studies showed that TMAO accumulates in the serum of animals colonized with TMA-producing species, but not in the serum of animals colonized with intestinal isolates that do not generate TMA from choline in vitro. Remarkably, low levels of colonization by TMA-producing bacteria significantly reduced choline levels available to the host. This effect was more pronounced as the abundance of TMA-producing bacteria increased. Our findings provide a framework for designing strategies aimed at changing the representation or activity of TMA-producing bacteria in the human gut and suggest that the TMA-producing status of the gut microbiota should be considered when making recommendations about choline intake requirements for humans. Cardiovascular disease (CVD) is the leading cause of death and disability worldwide, and increased trimethylamine N-oxide (TMAO) levels have been causally linked with CVD development. This work identifies members of the human gut microbiota responsible for both the accumulation of trimethylamine (TMA), the precursor of the proatherogenic compound TMAO, and subsequent decreased choline bioavailability to the host. Understanding how to manipulate the representation and function of choline-consuming, TMA-producing species in the intestinal microbiota could potentially lead to novel means for preventing or treating atherosclerosis and choline deficiency-associated diseases.

  • Kwan G, Pisithkul T, Amador-Noguez D, Barak J (2015) De novo amino acid biosynthesis contributes to salmonella enterica growth in Alfalfa seedling exudates. Appl. Environ. Microbiol. 81(3):861-73 (PMC4292483) · Pubmed

    Salmonella enterica is a member of the plant microbiome. Growth of S. enterica in sprouting-seed exudates is rapid; however, the active metabolic networks essential in this environment are unknown. To examine the metabolic requirements of S. enterica during growth in sprouting-seed exudates, we inoculated alfalfa seeds and identified 305 S. enterica proteins extracted 24 h postinoculation from planktonic cells. Over half the proteins had known metabolic functions, and they are involved in over one-quarter of the known metabolic reactions. Ion and metabolite transport accounted for the majority of detected reactions. Proteins involved in amino acid transport and metabolism were highly represented, suggesting that amino acid metabolic networks may be important for S. enterica growth in association with roots. Amino acid auxotroph growth phenotypes agreed with the proteomic data; auxotrophs in amino acid-biosynthetic pathways that were detected in our screen developed growth defects by 48 h. When the perceived sufficiency of each amino acid was expressed as a ratio of the calculated biomass requirement to the available concentration and compared to growth of each amino acid auxotroph, a correlation between nutrient availability and bacterial growth was found. Furthermore, glutamate transport acted as a fitness factor during S. enterica growth in association with roots. Collectively, these data suggest that S. enterica metabolism is robust in the germinating-alfalfa environment; that single-amino-acid metabolic pathways are important but not essential; and that targeting central metabolic networks, rather than dedicated pathways, may be necessary to achieve dramatic impacts on bacterial growth.

  • Liu K, Myers AR, Pisithkul T, Claas KR, Satyshur KA, Amador-Noguez D, Keck JL, Wang JD (2015) Molecular mechanism and evolution of guanylate kinase regulation by (p)ppGpp. Mol. Cell 57(4):735-49 (PMC4336630) · Pubmed

    The nucleotide (p)ppGpp mediates bacterial stress responses, but its targets and underlying mechanisms of action vary among bacterial species and remain incompletely understood. Here, we characterize the molecular interaction between (p)ppGpp and guanylate kinase (GMK), revealing the importance of this interaction in adaptation to starvation. Combining structural and kinetic analyses, we show that (p)ppGpp binds the GMK active site and competitively inhibits the enzyme. The (p)ppGpp-GMK interaction prevents the conversion of GMP to GDP, resulting in GMP accumulation upon amino acid downshift. Abolishing this interaction leads to excess (p)ppGpp and defective adaptation to amino acid starvation. A survey of GMKs from phylogenetically diverse bacteria shows that the (p)ppGpp-GMK interaction is conserved in members of Firmicutes, Actinobacteria, and Deinococcus-Thermus, but not in Proteobacteria, where (p)ppGpp regulates RNA polymerase (RNAP). We propose that GMK is an ancestral (p)ppGpp target and RNAP evolved more recently as a direct target in Proteobacteria.

  • Holwerda EK, Thorne PG, Olson DG, Amador-Noguez D, Engle NL, Tschaplinski TJ, van Dijken JP, Lynd LR (2014) The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading. Biotechnol Biofuels 7(1):155 (PMC4207885) · Pubmed

    Clostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum. Using a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel) initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids - predominantly L-valine and L-alanine - were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso- and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis. C. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.

  • Baeza J, Dowell JA, Smallegan MJ, Fan J, Amador-Noguez D, Khan Z, Denu JM (2014) Stoichiometry of site-specific lysine acetylation in an entire proteome. J. Biol. Chem. 289(31):21326-38 (PMC4118097) · Pubmed

    Acetylation of lysine ϵ-amino groups influences many cellular processes and has been mapped to thousands of sites across many organisms. Stoichiometric information of acetylation is essential to accurately interpret biological significance. Here, we developed and employed a novel method for directly quantifying stoichiometry of site-specific acetylation in the entire proteome of Escherichia coli. By coupling isotopic labeling and a novel pairing algorithm, our approach performs an in silico enrichment of acetyl peptides, circumventing the need for immunoenrichment. We investigated the function of the sole NAD(+)-dependent protein deacetylase, CobB, on both site-specific and global acetylation. We quantified 2206 peptides from 899 proteins and observed a wide distribution of acetyl stoichiometry, ranging from less than 1% up to 98%. Bioinformatic analysis revealed that metabolic enzymes, which either utilize or generate acetyl-CoA, and proteins involved in transcriptional and translational processes displayed the highest degree of acetylation. Loss of CobB led to increased global acetylation at low stoichiometry sites and induced site-specific changes at high stoichiometry sites, and biochemical analysis revealed altered acetyl-CoA metabolism. Thus, this study demonstrates that sirtuin deacetylase deficiency leads to both site-specific and global changes in protein acetylation stoichiometry, affecting central metabolism.

  • Tepper N, Noor E, Amador-Noguez D, Haraldsdóttir HS, Milo R, Rabinowitz J, Liebermeister W, Shlomi T (2013) Steady-state metabolite concentrations reflect a balance between maximizing enzyme efficiency and minimizing total metabolite load. PLoS ONE 8(9):e75370 (PMC3784570) · Pubmed

    Steady-state metabolite concentrations in a microorganism typically span several orders of magnitude. The underlying principles governing these concentrations remain poorly understood. Here, we hypothesize that observed variation can be explained in terms of a compromise between factors that favor minimizing metabolite pool sizes (e.g. limited solvent capacity) and the need to effectively utilize existing enzymes. The latter requires adequate thermodynamic driving force in metabolic reactions so that forward flux substantially exceeds reverse flux. To test this hypothesis, we developed a method, metabolic tug-of-war (mTOW), which computes steady-state metabolite concentrations in microorganisms on a genome-scale. mTOW is shown to explain up to 55% of the observed variation in measured metabolite concentrations in E. coli and C. acetobutylicum across various growth media. Our approach, based strictly on first thermodynamic principles, is the first method that successfully predicts high-throughput metabolite concentration data in bacteria across conditions.

  • Xu YF, Amador-Noguez D, Reaves ML, Feng XJ, Rabinowitz JD (2012) Ultrasensitive regulation of anapleurosis via allosteric activation of PEP carboxylase. Nat. Chem. Biol. 8(6):562-8 (PMC3433955) · Pubmed

    Anapleurosis is the filling of the tricarboxylic acid cycle with four-carbon units. The common substrate for both anapleurosis and glucose phosphorylation in bacteria is the terminal glycolytic metabolite phosphoenolpyruvate (PEP). Here we show that Escherichia coli quickly and almost completely turns off PEP consumption upon glucose removal. The resulting buildup of PEP is used to quickly import glucose if it becomes available again. The switch-like termination of anapleurosis results from depletion of fructose-1,6-bisphosphate (FBP), an ultrasensitive allosteric activator of PEP carboxylase. E. coli expressing an FBP-insensitive point mutant of PEP carboxylase grow normally when glucose is steadily available. However, they fail to build up PEP upon glucose removal, grow poorly when glucose availability oscillates and suffer from futile cycling at the PEP node on gluconeogenic substrates. Thus, bacterial central carbon metabolism is intrinsically programmed with ultrasensitive allosteric regulation to enable rapid adaptation to changing environmental conditions.

  • Amador-Noguez D, Brasg IA, Feng XJ, Roquet N, Rabinowitz JD (2011) Metabolome remodeling during the acidogenic-solventogenic transition in Clostridium acetobutylicum. Appl. Environ. Microbiol. 77(22):7984-97 (PMC3209008) · Pubmed

    The fermentation carried out by the biofuel producer Clostridium acetobutylicum is characterized by two distinct phases. Acidogenesis occurs during exponential growth and involves the rapid production of acids (acetate and butyrate). Solventogenesis initiates as cell growth slows down and involves the production of solvents (butanol, acetone, and ethanol). Using metabolomics, isotope tracers, and quantitative flux modeling, we have mapped the metabolic changes associated with the acidogenic-solventogenic transition. We observed a remarkably ordered series of metabolite concentration changes, involving almost all of the 114 measured metabolites, as the fermentation progresses from acidogenesis to solventogenesis. The intracellular levels of highly abundant amino acids and upper glycolytic intermediates decrease sharply during this transition. NAD(P)H and nucleotide triphosphates levels also decrease during solventogenesis, while low-energy nucleotides accumulate. These changes in metabolite concentrations are accompanied by large changes in intracellular metabolic fluxes. During solventogenesis, carbon flux into amino acids, as well as flux from pyruvate (the last metabolite in glycolysis) into oxaloacetate, decreases by more than 10-fold. This redirects carbon into acetyl coenzyme A, which cascades into solventogenesis. In addition, the electron-consuming reductive tricarboxylic acid (TCA) cycle is shutdown, while the electron-producing oxidative (clockwise) right side of the TCA cycle remains active. Thus, the solventogenic transition involves global remodeling of metabolism to redirect resources (carbon and reducing power) from biomass production into solvent production.

  • Vander Heiden MG, Locasale JW, Swanson KD, Sharfi H, Heffron GJ, Amador-Noguez D, Christofk HR, Wagner G, Rabinowitz JD, Asara JM, Cantley LC (2010) Evidence for an alternative glycolytic pathway in rapidly proliferating cells. Science 329(5998):1492-9 (PMC3030121) · Pubmed

    Proliferating cells, including cancer cells, require altered metabolism to efficiently incorporate nutrients such as glucose into biomass. The M2 isoform of pyruvate kinase (PKM2) promotes the metabolism of glucose by aerobic glycolysis and contributes to anabolic metabolism. Paradoxically, decreased pyruvate kinase enzyme activity accompanies the expression of PKM2 in rapidly dividing cancer cells and tissues. We demonstrate that phosphoenolpyruvate (PEP), the substrate for pyruvate kinase in cells, can act as a phosphate donor in mammalian cells because PEP participates in the phosphorylation of the glycolytic enzyme phosphoglycerate mutase (PGAM1) in PKM2-expressing cells. We used mass spectrometry to show that the phosphate from PEP is transferred to the catalytic histidine (His11) on human PGAM1. This reaction occurred at physiological concentrations of PEP and produced pyruvate in the absence of PKM2 activity. The presence of histidine-phosphorylated PGAM1 correlated with the expression of PKM2 in cancer cell lines and tumor tissues. Thus, decreased pyruvate kinase activity in PKM2-expressing cells allows PEP-dependent histidine phosphorylation of PGAM1 and may provide an alternate glycolytic pathway that decouples adenosine triphosphate production from PEP-mediated phosphotransfer, allowing for the high rate of glycolysis to support the anabolic metabolism observed in many proliferating cells.

  • Amador-Noguez D, Feng XJ, Fan J, Roquet N, Rabitz H, Rabinowitz JD (2010) Systems-level metabolic flux profiling elucidates a complete, bifurcated tricarboxylic acid cycle in Clostridium acetobutylicum. J. Bacteriol. 192(17):4452-61 (PMC2937365) · Pubmed

    Obligatory anaerobic bacteria are major contributors to the overall metabolism of soil and the human gut. The metabolic pathways of these bacteria remain, however, poorly understood. Using isotope tracers, mass spectrometry, and quantitative flux modeling, here we directly map the metabolic pathways of Clostridium acetobutylicum, a soil bacterium whose major fermentation products include the biofuels butanol and hydrogen. While genome annotation suggests the absence of most tricarboxylic acid (TCA) cycle enzymes, our results demonstrate that this bacterium has a complete, albeit bifurcated, TCA cycle; oxaloacetate flows to succinate both through citrate/alpha-ketoglutarate and via malate/fumarate. Our investigations also yielded insights into the pathways utilized for glucose catabolism and amino acid biosynthesis and revealed that the organism's one-carbon metabolism is distinct from that of model microbes, involving reversible pyruvate decarboxylation and the use of pyruvate as the one-carbon donor for biosynthetic reactions. This study represents the first in vivo characterization of the TCA cycle and central metabolism of C. acetobutylicum. Our results establish a role for the full TCA cycle in an obligatory anaerobic organism and demonstrate the importance of complementing genome annotation with isotope tracer studies for determining the metabolic pathways of diverse microbes.

  • Lu W, Clasquin MF, Melamud E, Amador-Noguez D, Caudy AA, Rabinowitz JD (2010) Metabolomic analysis via reversed-phase ion-pairing liquid chromatography coupled to a stand alone orbitrap mass spectrometer. Anal. Chem. 82(8):3212-21 (PMC2863137) · Pubmed

    We present a liquid chromatography-mass spectrometry (LC-MS) method that capitalizes on the mass-resolving power of the orbitrap to enable sensitive and specific measurement of known and unanticipated metabolites in parallel, with a focus on water-soluble species involved in core metabolism. The reversed phase LC method, with a cycle time 25 min, involves a water-methanol gradient on a C18 column with tributylamine as the ion pairing agent. The MS portion involves full scans from 85 to 1000 m/z at 1 Hz and 100,000 resolution in negative ion mode on a stand alone orbitrap ("Exactive"). The median limit of detection, across 80 metabolite standards, was 5 ng/mL with the linear range typically >or=100-fold. For both standards and a cellular extract from Saccharomyces cerevisiae (Baker's yeast), the median inter-run relative standard deviation in peak intensity was 8%. In yeast exact, we detected 137 known compounds, whose (13)C-labeling patterns could also be tracked to probe metabolic flux. In yeast engineered to lack a gene of unknown function (YKL215C), we observed accumulation of an ion of m/z 128.0351, which we subsequently confirmed to be oxoproline, resulting in annotation of YKL215C as an oxoprolinase. These examples demonstrate the suitability of the present method for quantitative metabolomics, fluxomics, and discovery metabolite profiling.

  • Reddy AK, Amador-Noguez D, Darlington GJ, Scholz BA, Michael LH, Hartley CJ, Entman ML, Taffet GE (2007) Cardiac function in young and old Little mice. J. Gerontol. A Biol. Sci. Med. Sci. 62(12):1319-25 (PMC2771398) · Pubmed

    We studied cardiac function in young and old, wild-type (WT), and longer-living Little mice using cardiac flow velocities, echocardiographic measurements, and left ventricular (LV) pressure (P) to determine if enhanced reserves were in part responsible for longevity in these mice. Resting/baseline cardiac function, as measured by velocities, LV dimensions, +dP/dt(max), and -dP/dt(max), was significantly lower in young Little mice versus young WT mice. Fractional shortening (FS) increased significantly, and neither +dP/dt(max) nor -dP/dt(max) declined with age in Little mice. In contrast, old WT mice had no change in FS but had significantly lower +dP/dt(max) and -dP/dt(max) versus young WT mice. Significant decreases were observed in the velocity indices of old Little mice versus old WT mice, but other parameters were unchanged. The magnitude of dobutamine stress response remained unchanged with age in Little mice, while that in WT mice decreased. These data suggest that while resting cardiac function in Little mice versus WT mice is lower at young age, it is relatively unaltered with aging. Additionally, cardiac function in response to stress was maintained with age in Little mice but not in their WT counterparts. Thus, some mouse models of increased longevity may not be associated with enhanced reserves.

  • Amador-Noguez D, Dean A, Huang W, Setchell K, Moore D, Darlington G (2007) Alterations in xenobiotic metabolism in the long-lived Little mice. Aging Cell 6(4):453-70 (PMC2859448) · Pubmed

    Our previous microarray expression analysis of the long-lived Little mice (Ghrhr(lit/lit)) showed a concerted up-regulation of xenobiotic detoxification genes. Here, we show that this up-regulation is associated with a potent increase in resistance against the adverse effects of a variety of xenobiotics, including the hepatotoxins acetaminophen and bromobenzene and the paralyzing agent zoxazolamine. The classic xenobiotic receptors Car (Constitutive Androstane Receptor) and Pxr (Pregnane X Receptor) are considered key regulators of xenobiotic metabolism. Using double and triple knockout/mutant mouse models we found, however, that Car and Pxr are not required for the up-regulation of xenobiotic genes in Little mice. Our results suggest instead that bile acids and the primary bile acid receptor Fxr (farnesoid X receptor) are likely mediators of the up-regulation of xenobiotic detoxification genes in Little mice. Bile acid levels are considerably elevated in the bile, serum, and liver of Little mice. We found that treatment of wild-type animals with cholic acid, one of the major bile acids elevated in Little mice, mimics in large part the up-regulation of xenobiotic detoxification genes observed in Little mice. Additionally, the loss of Fxr had a major effect on the expression of the xenobiotic detoxification genes up-regulated in Little mice. A large fraction of these genes lost or decreased their high expression levels in double mutant mice for Fxr and Ghrhr. The alterations in xenobiotic metabolism in Little mice constitute a form of increased stress resistance and may contribute to the extended longevity of these mice.

  • Amador-Noguez D, Zimmerman J, Venable S, Darlington G (2005) Gender-specific alterations in gene expression and loss of liver sexual dimorphism in the long-lived Ames dwarf mice. Biochem. Biophys. Res. Commun. 332(4):1086-100 · Pubmed

    Genetic mutations that increase lifespan in mice frequently involve alterations in the growth hormone/insulin-like growth factor-I signaling pathway. Although several of the effects of GH on gene expression are known to be sex-dependent, an understanding of the gender-specific vs. gender-independent effects of lifespan-extending mutations of the GH/IGF-I axis is currently lacking. The Ames dwarf mice (prop1(df/df)) are GH, prolactin and thyroid-stimulating hormone deficient and exhibit an increase in mean lifespan of 49% in males and 68% in females. We used oligonucleotide arrays containing over 14,000 genes to study the gender-specific vs. gender-independent effects of the prop1(df) mutation in liver of male and female Ames mice. We identified 381 gender-independent and 110 gender-specific alterations in gene expression produced by the Prop1(df/df) genotype. The gender-specific alterations corresponded to genes with a strong sexual dimorphism in wild-type mice and produced an almost complete loss of sex-specific gene expression in the liver of Ames dwarf mice: out of 123 genes that showed sexual dimorphism in wild-type mice only six maintained a gender difference in mutant mice. However, the Prop1(df/df) genotype did not introduce new sexually dimorphic patterns of gene expression in Ames dwarf mice that were not present in the wild-type animals. The gender-specific alterations accounted for a large fraction of the most significant changes in gene expression in male and female Ames mice livers and affected several metabolic processes, particularly fatty acid metabolism, steroid hormone metabolism, and xenobiotic metabolism.

  • Amador-Noguez D, Yagi K, Venable S, Darlington G (2004) Gene expression profile of long-lived Ames dwarf mice and Little mice. Aging Cell 3(6):423-41 · Pubmed

    Ames dwarf mice (Prop1df/df) and Little mice (Ghrhrlit/lit) are used as models of delayed aging and show significant increases in lifespan (50% and 25%, respectively) when compared with their wild-type siblings. To gain further insight into the molecular basis for the extended longevity of these mice, we used oligonucleotide microarrays to measure levels of expression of over 14 000 RNA transcripts in liver during normal aging at 3, 6, 12 and 24 months. We found that the Prop1df/df and Ghrhrlit/lit genotypes produce dramatic alterations in gene expression, which are predominantly maintained at all ages. We found 1125 genes to be significantly affected by the Prop1df/df genotype and 1152 genes were significantly affected by the Ghrhrlit/lit genotype; 547 genes were present in both gene lists and showed parallel changes in gene expression, suggesting common mechanisms for the extended longevity in these mutants. Some of the functional gene classes most affected in these mutants included: amino acid metabolism, TCA cycle, mitochondrial electron transport, fatty acid, cholesterol and steroid metabolism, xenobiotic metabolism and oxidant metabolism. We found that the Prop1df/df genotype, and to a minor extent the Ghrhrlit/lit genotype, also produced complex alterations in age-dependent changes in gene expression as compared with wild-type mice. In some cases these alterations reflected a partial delay or deceleration of age-related changes in gene expression as seen in wild-type mice but they also introduced age-related changes that are unique for each of these mutants and not present in wild-type mice.

  • Rito-Palomares M, Nuñez L, Amador-Noguez D (2001) Practical application of aqueous two-phase systems for the development of a prototype process for c-phycocyanin recovery from Spirulina maxima J. Chemical Technology and Biotechnology 76(12):1273-80 · DOI

    No abstract available.