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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
    amadornoguez@wisc.edu

Start and Promotion Dates

  • Assistant Professor: 2013
  • Associate Professor: 2020

Education

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.

Teaching

Microbiology 526: Physiology of Microorganisms

Lab Personnel

Picture of Buasakdi
Chavin Buasakdi
Grad Student
buasakdi@wisc.edu
Picture of Callaghan
Melanie Callaghan
Postdoc
callaghan2@wisc.edu
Picture of Getahun
Fit Getahun
Research Intern
getahun@wisc.edu
Picture of Jacobson
Tyler Jacobson
Postdoc
tbjacobson@wisc.edu
Picture of Khana
Daven Khana
Grad Student
khana@wisc.edu
Picture of Lucas
Lauren Lucas
Grad Student
llucas3@wisc.edu
Picture of Rivera Vazquez
Julio Rivera Vazquez
Grad Student
riveravazque@wisc.edu
Picture of Stevenson
David Stevenson
Sr Research Specialist
Lab Manager
dmstevenson@wisc.edu
Picture of Williams
Jack Williams
Research Intern
jlwilliams28@wisc.edu

Research Papers

  • Trujillo EA, Hebert AS, Rivera Vazquez JC, Brademan DR, Tatli M, Amador-Noguez D, Meyer JG, Coon JJ (2022) Rapid Targeted Quantitation of Protein Overexpression with Direct Infusion Shotgun Proteome Analysis (DISPA-PRM). Analytical chemistry : · Pubmed · DOI

    No abstract available.

  • Foster C, Boorla VS, Dash S, Gopalakrishnan S, Jacobson TB, Olson DG, Amador-Noguez D, Lynd LR, Maranas CD (2022) Assessing the impact of substrate-level enzyme regulations limiting ethanol titer in Clostridium thermocellum using a core kinetic model. Metabolic engineering 69:286-301 · Pubmed · DOI

    No abstract available.

  • Khana DB, Callaghan MM, Amador-Noguez D (2022) Novel computational and experimental approaches for investigating the thermodynamics of metabolic networks. Current opinion in microbiology 66:21-31 · Pubmed · DOI

    No abstract available.

  • Martien JI, Trujillo EA, Jacobson TB, Tatli M, Hebert AS, Stevenson DM, Coon JJ, Amador-Noguez D (2021) Metabolic Remodeling during Nitrogen Fixation in Zymomonas mobilis. mSystems 6((6)):e0098721 PMC8594446 · Pubmed · DOI

    No abstract available.

  • Hromada S, Qian Y, Jacobson TB, Clark RL, Watson L, Safdar N, Amador-Noguez D, Venturelli OS (2021) Negative interactions determine Clostridioides difficile growth in synthetic human gut communities. Molecular systems biology 17((10)):e10355 PMC8543057 · Pubmed · DOI

    No abstract available.

  • 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 Zymomonas mobilis ZM4. Frontiers in microbiology 10:2270 PMC6777484 · Pubmed · DOI

    No abstract available.

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

    No abstract available.

  • 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

    No abstract available.

  • 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

    No abstract available.

  • 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

    No abstract available.

  • 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[ a ]Pyrene by Aspergillus Species. mBio 10((3)): PMC6538779 · Pubmed · DOI

    No abstract available.

  • 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

    No abstract available.

  • 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 Caldicellulosiruptor bescii utilizes two enzymes to oxidize glyceraldehyde 3-phosphate during glycolysis. The Journal of biological chemistry 294((25)):9995-10005 PMC6597818 · Pubmed · DOI

    No abstract available.

  • 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

    No abstract available.

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

    No abstract available.

  • 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

    No abstract available.

  • 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. Applied and environmental microbiology 84((24)): PMC6275354 · Pubmed · DOI

    No abstract available.

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

    No abstract available.

  • 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

    No abstract available.

  • 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 Listeria -Stimulated Immunity. Journal of immunology (Baltimore, Md. : 1950) 200((11)):3729-3738 PMC5964023 · Pubmed · DOI

    No abstract available.

  • 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 C. thermocellum by ethanol. Biotechnology for biofuels 10:276 PMC5708176 · Pubmed · DOI

    No abstract available.

  • 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. Nature microbiology 2((12)):1624-1634 PMC5705400 · Pubmed · DOI

    No abstract available.

  • 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

    No abstract available.

  • 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. Metabolic engineering 41:182-191 · Pubmed · DOI

    No abstract available.

  • 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. Metabolic engineering 39:169-180 · Pubmed · DOI

    No abstract available.

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

    No abstract available.

  • 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.

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    No abstract available.