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4.1 Nutrition and Cell Chemistry • Metabolism – The sum total of all chemical reactions that occur in a cell • Catabolic reactions (catabolism) – Energy-releasing metabolic reactions • Anabolic reactions (anabolism) – Energy-requiring metabolic reactions • Most knowledge of microbial metabolism is based on study of laboratory cultures © 2012 Pearson Education, Inc. 4.1 Nutrition and Cell Chemistry • Nutrients – Supply of monomers (or precursors of) required by cells for growth • Macronutrients – Nutrients required in large amounts • Micronutrients – Nutrients required in trace amount © 2012 Pearson Education, Inc. 4.1 Nutrition and Cell Chemistry • Carbon – – – – – Required by all cells Typical bacterial cell ~50% carbon (by dry weight) Major element in all classes of macromolecules Heterotrophs use organic carbon Autotrophs use inorganic carbon © 2012 Pearson Education, Inc. 4.1 Nutrition and Cell Chemistry • Nitrogen – Typical bacterial cell ~12% nitrogen (by dry weight) – Key element in proteins, nucleic acids, and many more cell constituents © 2012 Pearson Education, Inc. 4.1 Nutrition and Cell Chemistry • Other Macronutrients – Phosphorus (P) • Synthesis of nucleic acids and phospholipids – Sulfur (S) • Sulfur-containing amino acids (cysteine and methionine) • Vitamins (e.g., thiamine, biotin, lipoic acid) and coenzyme A – Potassium (K) • Required by enzymes for activity © 2012 Pearson Education, Inc. 4.1 Nutrition and Cell Chemistry • Other Macronutrients (cont’d) – Magnesium (Mg) • Stabilizes ribosomes, membranes, and nucleic acids • Also required for many enzymes – Calcium (Ca) • Helps stabilize cell walls in microbes • Plays key role in heat stability of endospores – Sodium (Na) • Required by some microbes (e.g., marine microbes) © 2012 Pearson Education, Inc. 4.1 Nutrition and Cell Chemistry • Iron – Key component of cytochromes and FeS proteins involved in electron transport – Under anoxic conditions, generally ferrous (Fe2+) form; soluble – Under oxic conditions: generally ferric (Fe3+) form; exists as insoluble minerals – Cells produce siderophores (iron-binding agents) to obtain iron from insoluble mineral form (Figure 4.2) © 2012 Pearson Education, Inc. 4.2 Culture Media • Culture Media – Nutrient solutions used to grow microbes in the laboratory • Two broad classes – Defined media: precise chemical composition is known – Complex media: composed of digests of chemically undefined substances (e.g., yeast and meat extracts) © 2012 Pearson Education, Inc. 4.2 Culture Media • Selective Media – Contains compounds that selectively inhibit growth of some microbes but not others • Differential Media – Contains an indicator, usually a dye, that detects particular chemical reactions occurring during growth © 2012 Pearson Education, Inc. 4.5 Catalysis and Enzymes • Enzymes – – – – – Biological catalysts Typically proteins (some RNAs) Highly specific Generally larger than substrate Typically rely on weak bonds • Examples: hydrogen bonds, van der Waals forces, hydrophobic interactions – Active site: region of enzyme that binds substrate © 2012 Pearson Education, Inc. Figure 4.7 Substrate Products Active site Free lysozyme © 2012 Pearson Education, Inc. Enzyme-substrate complex Free lysozyme III. Oxidation–Reduction and EnergyRich Compounds • 4.6 Electron Donors and Electron Acceptors • 4.7 Energy-Rich Compounds and Energy Storage © 2012 Pearson Education, Inc. 4.6 Electron Donors and Electron Acceptors • Energy from oxidation–reduction (redox) reactions is used in synthesis of energy-rich compounds (e.g., ATP) • Redox reactions occur in pairs (two half reactions; Figure 4.8) • Electron donor: the substance oxidized in a redox reaction • Electron acceptor: the substance reduced in a redox reaction © 2012 Pearson Education, Inc. Essentials of Catabolism • • • • • 4.8 Glycolysis 4.9 Respiration and Electron Carriers 4.10 The Proton Motive Force 4.11 The Citric Acid Cycle 4.12 Catabolic Diversity © 2012 Pearson Education, Inc. 4.8 Glycolysis • Two reaction series are linked to energy conservation in chemoorganotrophs: fermentation and respiration (Figure 4.13) • Differ in mechanism of ATP synthesis – Fermentation: substrate-level phosphorylation; ATP directly synthesized from an energy-rich intermediate – Respiration: oxidative phosphorylation; ATP produced from proton motive force formed by transport of electrons © 2012 Pearson Education, Inc. 4.8 Glycolysis • Fermented substance is both an electron donor and an electron acceptor • Glycolysis (Embden-Meyerhof pathway): a common pathway for catabolism of glucose (Figure 4.14) – Anaerobic process – Three stages © 2012 Pearson Education, Inc. Figure 4.14 Stage I Glucose Stage II Pyruvate 2 lactate Stage III 2 Pyruvate 2 ethanol 2 CO2 Intermediates 1,3-Bisphosphoglycerate Enzymes Phosphoglycerokinase Glucose 6-P 3-P-Glycerate Hexokinase Phosphoglyceromutase Fructose 6-P 2-P-Glycerate Isomerase Enolase Fructose 1,6-P Phosphoenolpyruvate Phosphofructokinase Pyruvate kinase Dihydroxyacetone-P Aldolase Lactate dehydrogenase Glyceraldehyde-3-P Triosephosphate isomerase Pyruvate decarboxylase Glyceraldehyde-3-P dehydrogenase Alcohol dehydrogenase Energetics Yeast Lactic acid bacteria © 2012 Pearson Education, Inc. 4.8 Glycolysis • Glycolysis – Glucose consumed – Two ATPs produced – Fermentation products generated • Some harnessed by humans for consumption © 2012 Pearson Education, Inc. 4.9 Respiration and Electron Carriers • Aerobic Respiration – Oxidation using O2 as the terminal electron acceptor – Higher ATP yield than fermentations • ATP produced at the expense of the proton motive force, which is generated by electron transport © 2012 Pearson Education, Inc. 4.9 Respiration and Electron Carriers • Electron Transport Systems – Membrane associated – Mediate transfer of electrons – Conserve some of the energy released during transfer and use it to synthesize ATP – Many oxidation–reduction enzymes are involved in electron transport (e.g., NADH dehydrogenases, flavoproteins, iron–sulfur proteins, cytochromes) © 2012 Pearson Education, Inc. 4.9 Respiration and Electron Carriers • NADH dehydrogenases: proteins bound to inside surface of cytoplasmic membrane; active site binds NADH and accepts 2 electrons and 2 protons that are passed to flavoproteins • Flavoproteins: contains flavin prosthetic group (e.g., FMN, FAD) that accepts 2 electrons and 2 protons but only donates the electrons to the next protein in the chain (Figure 4.15) © 2012 Pearson Education, Inc. Figure 4.15 Isoalloxazine ring Ribitol Oxidized Reduced © 2012 Pearson Education, Inc. 4.9 Respiration and Electron Carriers • Cytochromes – Proteins that contain heme prosthetic groups (Figure 4.16) – Accept and donate a single electron via the iron atom in heme © 2012 Pearson Education, Inc. Figure 4.16 Porphyrin ring Pyrrole Heme (a porphyrin) Protein Histidine-N N-Histidine Cysteine-S S-Cysteine Amino acid Amino acid Cytochrome © 2012 Pearson Education, Inc. 4.9 Respiration and Electron Carriers • Iron–Sulfur Proteins – Contain clusters of iron and sulfur (Figure 4.17) • Example: ferredoxin – Reduction potentials vary depending on number and position of Fe and S atoms – Carry electrons © 2012 Pearson Education, Inc. Figure 4.17 Cysteine Cysteine Cysteine Cysteine Cysteine Cysteine Cysteine Cysteine © 2012 Pearson Education, Inc. 4.9 Respiration and Electron Carriers • Quinones – Hydrophobic non-protein-containing molecules that participate in electron transport (Figure 4.18) – Accept electrons and protons but pass along electrons only © 2012 Pearson Education, Inc. 4.10 The Proton Motive Force • Electron transport system oriented in cytoplasmic membrane so that electrons are separated from protons (Figure 4.19) • Electron carriers arranged in membrane in order of their reduction potential • The final carrier in the chain donates the electrons and protons to the terminal electron acceptor © 2012 Pearson Education, Inc. Figure 4.19 E0(V) 0.22 ENVIRONMENT 0.0 Complex II Q cycle Fumarate 0.1 CYTOPLASM 0.36 0.39 E0(V) © 2012 Pearson Education, Inc. Succinate 4.10 The Proton Motive Force • During electron transfer, several protons are released on outside of the membrane – Protons originate from NADH and the dissociation of water • Results in generation of pH gradient and an electrochemical potential across the membrane (the proton motive force) – The inside becomes electrically negative and alkaline – The outside becomes electrically positive and acidic © 2012 Pearson Education, Inc. 4.10 The Proton Motive Force • Complex I (NADH:quinone oxidoreductase) – NADH donates e to FAD – FADH donates e to quinone • Complex II (succinate dehydrogenase complex) – Bypasses Complex I – Feeds e and H+ from FADH directly to quinone pool © 2012 Pearson Education, Inc. 4.10 The Proton Motive Force • Complex III (cytochrome bc1 complex) – Transfers e from quinones to cytochrome c – Cytochrome c shuttles e to cytochromes a and a3 • Complex IV (cytochromes a and a3) – Terminal oxidase; reduces O2 to H2O © 2012 Pearson Education, Inc. 4.10 The Proton Motive Force • ATP synthase (ATPase): complex that converts proton motive force into ATP; two components (Figure 4.20) – F1: multiprotein extramembrane complex, faces cytoplasm – Fo: proton-conducting intramembrane channel – Reversible; dissipates proton motive force © 2012 Pearson Education, Inc. Figure 4.20 F1 F1 In In b2 c a Membrane c12 © 2012 Pearson Education, Inc. b2 Fo a Fo Out Out 4.11 The Citric Acid Cycle • Citric acid cycle (CAC): pathway through which pyruvate is completely oxidized to CO2 (Figure 4.21a) – Initial steps (glucose to pyruvate) same as glycolysis – Per glucose molecule, 6 CO2 molecules released and NADH and FADH generated – Plays a key role in catabolism and biosynthesis • Energetics advantage to aerobic respiration (Figure 4.21b) © 2012 Pearson Education, Inc. Figure 4.21a Pyruvate (three carbons) C2 C4 Acetyl-CoA C5 C6 Oxalacetate2 Citrate3 Aconitate3 Malate2 Isocitrate3 Fumarate2 Succinate2 -Ketoglutarate2 Succinyl-CoA © 2012 Pearson Education, Inc. Figure 4.21b Energetics Balance Sheet for Aerobic Respiration © 2012 Pearson Education, Inc. 4.11 The Citric Acid Cycle • The citric acid cycle generates many compounds available for biosynthetic purposes -Ketoglutarate and oxalacetate (OAA): precursors of several amino acids; OAA also converted to phosphoenolpyruvate, a precursor of glucose – Succinyl-CoA: required for synthesis of cytochromes, chlorophyll, and other tetrapyrrole compounds – Acetyl-CoA: necessary for fatty acid biosynthesis © 2012 Pearson Education, Inc. 4.12 Catabolic Diversity • Microorganisms demonstrate a wide range of mechanisms for generating energy (Figure 4.22) – – – – – Fermentation Aerobic respiration Anaerobic respiration Chemolithotrophy Phototrophy © 2012 Pearson Education, Inc. Figure 4.22 Fermentation Carbon flow Organic compound Carbon flow in respirations Electron transport/ generation of pmf Biosynthesis Aerobic respiration Chemotrophs Electron acceptors Anaerobic respiration Chemoorganotrophy Electron transport/ generation of pmf Electron acceptors Biosynthesis Aerobic respiration Anaerobic respiration Chemolithotrophy Light Photoheterotrophy Phototrophs Organic compound Photoautotrophy Electron transport e donor Generation of pmf and reducing power Biosynthesis Phototrophy © 2012 Pearson Education, Inc. Biosynthesis 4.12 Catabolic Diversity • Anaerobic Respiration – The use of electron acceptors other than oxygen • Examples include nitrate (NO3), ferric iron (Fe3+), sulfate (SO42), carbonate (CO32), certain organic compounds – Less energy released compared to aerobic respiration – Dependent on electron transport, generation of a proton motive force, and ATPase activity © 2012 Pearson Education, Inc. 4.12 Catabolic Diversity • Chemolithotrophy – Uses inorganic chemicals as electron donors • Examples include hydrogen sulfide (H2S), hydrogen gas (H2), ferrous iron (Fe2+), ammonia (NH3) – Typically aerobic – Begins with oxidation of inorganic electron donor – Uses electron transport chain and proton motive force – Autotrophic; uses CO2 as carbon source © 2012 Pearson Education, Inc. 4.12 Catabolic Diversity • Phototrophy: uses light as energy source – Photophosphorylation: light-mediated ATP synthesis – Photoautotrophs: use ATP for assimilation of CO2 for biosynthesis – Photoheterotrophs: use ATP for assimilation of organic carbon for biosynthesis © 2012 Pearson Education, Inc.