1. Nutrition, Culture, and Metabolism of Microorganisms
Chapter 4
Nutrition, Culture, and Metabolism of Microorganisms

2. I. Nutrition, Culture, and Metabolism of Microorganisms
4.1 Nutrition and Cell Chemistry
4.2 Culture Media
4.3 Laboratory Culture
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3. 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
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4. 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
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5. 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
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6. 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
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7. 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
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8. 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)
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9. 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)
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10. Hydroxamate group (Ferric) Hydroxamate Cell wall Ferric hydroxamate
Figure 4.2
Hydroxamate group
(Ferric)
Hydroxamate
Cell wall
Ferric hydroxamate
Cytoplasmic membrane
Figure 4.2 Mechanism of hydroxamate siderophores.
Ferric hydroxamate
Reduction
Hydroxamate
(Ferrous)
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11. 4.1 Nutrition and Cell Chemistry
Growth Factors
Organic compounds required in small amounts by certain organisms
Examples: vitamins, amino acids, purines, pyrimidines
Vitamins
Most commonly required growth factors
Most function as coenyzmes
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12. 4.2 Culture Media Culture Media Two broad classes
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)
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13. 4.2 Culture Media Selective Media Differential 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
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14. 4.2 Culture Media
For successful cultivation of a microbe, it is important to know the nutritional requirements and supply them in proper form and proportions in a culture medium
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15. 4.3 Laboratory Culture
Pure culture: culture containing only a single kind of microbe
Contaminants: unwanted organisms in a culture
Cells can be grown in liquid or solid culture media
Solid media are prepared by addition of a gelling agent (agar or gelatin)
When grown on solid media, cells form isolated masses (colonies)
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16. 4.3 Laboratory Culture Microbes are everywhere
Sterilization of media is critical
Aseptic technique should be followed (Figure 4.4)
Animation: Aseptic Transfer and the Streak Plate Method
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17. Figure 4.4
Figure 4.4 Aseptic transfer.
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18. 4.3 Laboratory Culture Pure culture technique
Streak plate (Figure 4.5)
Pour plate
Spread plate
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19. Isolated colonies at end of streak
Figure 4.5
Isolated colonies at end of streak
Confluent growth at beginning of streak
Figure 4.5 Making a streak plate to obtain pure cultures.
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20. II. Energetics and Enzymes
4.4 Bioenergetics
4.5 Catalysis and Enzymes
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21. 4.4 Bioenergetics
Energy is defined in units of kilojoules (kJ), a measure of heat energy
In any chemical reaction, some energy is lost as heat
Free energy (G): energy released that is available to do work
The change in free energy during a reaction is referred to as G0′
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22. 4.4 Bioenergetics
Reactions with a negative G0′ release free energy (exergonic)
Reactions with a positive G0′ require energy (endergonic)
To calculate free-energy yield of a reaction, we need to know the free energy of formation (Gf0; the energy released or required during formation of a given molecule from the elements)
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23. 4.4 Bioenergetics For the reaction A + B C + D,
G0′ = Gf0 [C+D] - Gf0[A+B]
G0′ not always a good estimate of actual free-energy changes
G: free energy that occurs under actual conditions
G = G0′ + RT ln K
where R and T are physical constants and K is the equilibrium constant for the reaction in question
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24. 4.5 Catalysis and Enzymes
Free-energy calculations do not provide information on reaction rates
Activation energy: energy required to bring all molecules in a chemical reaction into the reactive state (Figure 4.6)
A catalysis is usually required to breach activation energy barrier
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25. Substrates (A + B) Products (C + D) Activation energy— no enzyme
Figure 4.6
Activation energy— no enzyme
Substrates (A + B)
Activation energy with enzyme
Free energy
 G0 = Gf0(C + D)  Gf0(A + B)
Figure 4.6 Activation energy and catalysis.
Products (C + D)
Progress of the reaction
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26. 4.5 Catalysis and Enzymes Catalyst: substance that
Lowers the activation energy of a reaction
Increases reaction rate
Does not affect energetics or equilibrium of a reaction
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27. 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
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28. 4.5 Catalysis and Enzymes Enzymes (cont’d)
Increase the rate of chemical reactions by 108 to 1020 times the spontaneous rate
Enzyme catalysis: E + S E  S E + P (Figure 4.7)
Catalysis dependent on
Substrate binding
Position of substrate relative to catalytically active amino acids in active site
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29. Substrate Products Free lysozyme Enzyme-substrate complex
Figure 4.7
Substrate
Products
Active site
Figure 4.7 The catalytic cycle of an enzyme.
Free lysozyme
Enzyme-substrate complex
Free lysozyme
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30. 4.5 Catalysis and Enzymes
Many enzymes contain small nonprotein molecules that participate in catalysis but are not substrates
Prosthetic groups
Bind tightly to enzymes
Usually bind covalently and permanently (e.g., heme group in cytochromes)
Coenzymes
Loosely bound to enzymes
Most are derivatives of vitamins (e.g., NAD+/NADH)
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31. III. Oxidation–Reduction and Energy-Rich Compounds
4.6 Electron Donors and Electron Acceptors
4.7 Energy-Rich Compounds and Energy Storage
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32. 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
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33. Electron- donating half reaction Electron- accepting half reaction
Figure 4.8
Electron donor
Electron acceptor
Electron- donating half reaction
Electron- accepting half reaction
Formation of water
Net reaction
Figure 4.8 Example of an oxidation–reduction reaction.
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34. 4.6 Electron Donors and Electron Acceptors
Reduction potential (E0′): tendency to donate electrons
Expressed as volts (V)
Substances can be either electron donors or acceptors under different circumstances (redox couple)
Reduced substance of a redox couple with a more negative E0′ donates electrons to the oxidized substance of a redox couple with a more positive E0′
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35. 4.6 Electron Donors and Electron Acceptors
The redox tower represents the range of possible reduction potentials (Figure 4.9)
The reduced substance at the top of the tower donates electrons
The oxidized substance at the bottom of the tower accepts electrons
The farther the electrons “drop,” the greater the amount of energy released
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36. Figure 4.9 Redox couple E0 (V) 0.0 (1) H2  fumarate succinate
0.60
0.50
0.40
0.30
(1)
0.20
0.10
0.0
+0.10
(2)
+0.20
+0.30
+0.40
+0.50
Figure 4.9 The redox tower.
+0.60
+0.70
(3)
+0.80
+0.90
(1) H2  fumarate
succinate
G0  86 kJ
(2) H2  NO3
NO2  H2O
G0  163 kJ
(3) H2  O2 1
H2O
G0  237 kJ 2
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37. 4.6 Electron Donors and Electron Acceptors
Redox reactions usually involve reactions between intermediates (carriers)
Electron carriers are divided into two classes
Prosthetic groups (attached to enzymes)
Coenzymes (diffusible)
Examples: NAD+, NADP (Figure 4.10 )
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38. NAD NADH  H Nicotinamide NAD/ NADH E0  0.32V Adenine Figure 4.10
Figure 4.10 The oxidation–reduction coenzyme nicotinamide adenine dinucleotide (NAD+).
NAD/ NADH E0  0.32V
Adenine
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39. 4.6 Electron Donors and Electron Acceptors
NAD+ and NADH facilitate redox reactions without being consumed; they are recycled (Figure 4.11)
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40. Figure 4.11 NAD reduction Enzyme– substrate complex NAD binding site
Enzyme I reacts with electron donor and oxidized form of coenzyme, NAD
Enzyme– substrate complex
NAD binding site
Active site
Enzyme I
NAD
Electron donor (substrate)
NADH
Electron donor oxidized (product)
Electron acceptor reduced (product)
Figure 4.11 NAD+/NADH cycling.
NADH binding site
Active site
Enzyme II
Electron acceptor (substrate)
NADH oxidation
Enzyme– substrate complex
Enzyme II reacts with electron acceptor and reduced form of coenzyme, NADH
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41. 4.7 Energy-Rich Compounds and Energy Storage
Chemical energy released in redox reactions is primarily stored in certain phosphorylated compounds (Figure 4.12)
ATP; the prime energy currency
Phosphoenolpyruvate
Glucose 6-phosphate
Chemical energy also stored in coenzyme A
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42. Adenosine triphosphate (ATP) (ATP) Glucose 6-phosphate
Figure 4.12
Ester bond
Anhydride bonds
Ester bond
Anhydride bond
Phosphoenolpyruvate
Adenosine triphosphate (ATP) (ATP)
Glucose 6-phosphate
Anhydride bond
Thioester bond
Figure 4.12 Phosphate bonds in compounds that conserve energy in bacterial metabolism.
Acetyl
Coenzyme A
Acetyl-CoA
Acetyl phosphate
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43. 4.7 Energy-Rich Compounds and Energy Storage
Long-term energy storage involves insoluble polymers that can be oxidized to generate ATP
Examples in prokaryotes
Glycogen
Poly--hydroxybutyrate and other polyhydroxyalkanoates
Elemental sulfur
Examples in eukaryotes
Starch
Lipids (simple fats)
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44. IV. 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
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45. 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
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46. Substrate-level phosphorylation
Figure 4.13
Energy-rich intermediates
Intermediates
Substrate-level phosphorylation
Energized membrane
Figure 4.13 Energy conservation in fermentation and respiration.
Less energized membrane
Oxidative phosphorylation
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47. 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
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48. Figure 4.14 Figure 4.14 Embden–Meyerhof–Parnas pathway (glycolysis).
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
Figure 4.14 Embden–Meyerhof–Parnas pathway (glycolysis).
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
Energetics
Glyceraldehyde-3-P dehydrogenase
Alcohol dehydrogenase
Yeast
Lactic acid bacteria
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49. 4.8 Glycolysis Glycolysis Glucose consumed Two ATPs produced
Fermentation products generated
Some harnessed by humans for consumption
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50. 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
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51. 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)
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52. 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)
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53. Oxidized Reduced Isoalloxazine ring Ribitol Figure 4.15
Figure 4.15 Flavin mononucleotide (FMN), a hydrogen atom carrier.
Ribitol
Oxidized
Reduced
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54. 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
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55. Figure 4.16 Porphyrin ring Pyrrole Heme (a porphyrin) Protein
Histidine-N
N-Histidine
Figure 4.16 Cytochrome and its structure.
Cysteine-S
S-Cysteine
Amino acid
Amino acid
Cytochrome
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56. 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
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57. Cysteine Cysteine Cysteine Cysteine Cysteine Cysteine Cysteine
Figure 4.17
Cysteine
Cysteine
Cysteine
Cysteine
Cysteine
Cysteine
Figure 4.17 Arrangement of the iron–sulfur centers of nonheme iron–sulfur proteins.
Cysteine
Cysteine
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58. 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
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59. Oxidized Reduced Figure 4.18
Figure 4.18 Structure of oxidized and reduced forms of coenzyme Q, a quinone.
Reduced
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60. 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
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61. Figure 4.19
E0(V)
Complex I
0.22
0.0
Complex II
Q cycle
Succinate
Fumarate
ENVIRONMENT
0.1
CYTOPLASM
Complex III
Figure 4.19 Generation of the proton motive force during aerobic respiration.
Complex IV
0.36
0.39
E0(V)
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62. 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
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63. 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
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64. 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
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65. 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
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66.   F1 F1 In In b2 b2 c a a Fo Fo c12 Out Out           
Figure 4.20        F1 F1   In In b2 b2     c a a
Figure 4.20 Structure and function of ATP synthase (ATPase) in Escherichia coli. Fo
Membrane Fo
c12
Out
Out
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67. 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)
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68. Figure 4.21a Pyruvate (three carbons) C2 Acetyl-CoA C4 C5 C6
Oxalacetate2
Citrate3
Aconitate3
Malate2
Isocitrate3
Fumarate2
Figure 4.21 The citric acid cycle.
Succinate2
-Ketoglutarate2
Succinyl-CoA
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69. Energetics Balance Sheet for Aerobic Respiration
Figure 4.21b
Energetics Balance Sheet for Aerobic Respiration
Figure 4.21 The citric acid cycle.
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70. 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
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71. 4.12 Catabolic Diversity
Microorganisms demonstrate a wide range of mechanisms for generating energy (Figure 4.22)
Fermentation
Aerobic respiration
Anaerobic respiration
Chemolithotrophy
Phototrophy
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72. Generation of pmf and reducing power
Figure 4.22
Fermentation
Carbon flow
Organic compound
Carbon flow in respirations
Electron transport/ generation of pmf
Biosynthesis
Aerobic respiration
Electron acceptors
Anaerobic respiration
Chemoorganotrophy
Chemotrophs
Electron transport/ generation of pmf
Biosynthesis
Electron acceptors
Aerobic respiration
Anaerobic respiration
Chemolithotrophy
Light
Photoheterotrophy
Photoautotrophy
Figure 4.22 Catabolic diversity.
Organic compound
e donor
Electron transport
Phototrophs
Generation of pmf and reducing power
Biosynthesis
Biosynthesis
Phototrophy
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73. 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
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74. 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
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75. 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
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76. V. Essentials of Anabolism
4.13 Biosynthesis of Sugars and Polysaccharides
4.14 Biosynthesis of Amino Acids and Nucleotides
4.15 Biosynthesis of Fatty Acids and Lipids
4.16 Regulating the Activity of Biosynthetic Enzymes
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77. 4.13 Biosynthesis of Sugars and Polysaccharides
Prokaryotic polysaccharides are synthesized from activated glucose (Figure 4.23a)
Adenosine diphosphoglucose (ADPG)
Precursor for glycogen biosynthesis
Uridine diphosphoglucose (UDPG)
Precursor of some glucose derivatives needed for biosynthesis of important polysaccharides
Examples: N-acetylglucosamine, N-acetylmuramic acid
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78. Uridine diphosphoglucose (UDPG)
Figure 4.23a
Glucose
Figure 4.23 Sugar metabolism.
Uridine diphosphoglucose (UDPG)
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79. 4.13 Biosynthesis of Sugars and Polysaccharides
Gluconeogenesis (Figure 4.23b)
Synthesis of glucose from phosphoenolpyruvate
Phosphoenolpyruvate can be synthesized from oxalacetate
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80. Phosphoenolpyruvate  CO2
Figure 4.23b
C2, C3, C4, C5, Compounds
Citric acid cycle
Oxalacetate
Phosphoenolpyruvate  CO2
Figure 4.23 Sugar metabolism.
Reversal of glycolysis
Glucose-6-P
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81. 4.13 Biosynthesis of Sugars and Polysaccharides
Pentoses are formed by the removal of one carbon atom from a hexose (Figure 4.23c)
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82. Deoxyribonucleotides DNA
Figure 4.23c
Glucose-6-P
Ribulose-5-P  CO2
Ribose-5-P
Ribonucleotides
Ribonucleotides
Figure 4.23 Sugar metabolism.
NADPH
Ribonucleotide reductase
RNA
Deoxyribonucleotides
DNA
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83. 4.14 Biosynthesis of Amino Acids and Nucleotides
Biosynthesis of amino acids and nucleotides often involves long, multistep pathways
Amino acid biosynthesis
Carbon skeletons come from intermediates of glycolysis or citric acid cycle (Figure 4.24)
Ammonia is incorporated by glutamine dehydrogenase or glutamine synthetase (Figure 4.25)
Amino group transferred by transaminase and synthase
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84. Glutamate family Citric acid cycle Aspartate family Alanine family
Figure 4.24
Glutamate family
Proline Glutamine Arginine
-Ketoglutarate
Citric acid cycle
Aspartate family
Asparagine Lysine Methionine Threonine Isoleucine
Oxalacetate
Alanine family
Pyruvate
Valine Leucine
Glycolysis
Figure 4.24 Amino acid families.
Serine family
3-Phosphoglycerate
Glycine Cysteine
Phospho- enolpyruvate
Aromatic family
Phenylalanine Tyrosine Tryptophan
Chorismate
Erythrose-4-P
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85. Glutamate dehydrogenase
Figure 4.25
Glutamate dehydrogenase
Glutamine synthetase
Transaminase
Figure 4.25 Ammonia incorporation in bacteria.
Glutamate synthase
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86. 4.14 Biosynthesis of Amino Acids and Nucleotides
Purine and pyrimidine biosyntheses are complex
Purines (Figure 4.26)
Adenine and guanine
Inosinic acid intermediate
Pyrimidines
Thymine, cytosine, and uracil
Orotic acid intermediate
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87. Purine skeleton Inosinic acid Orotic acid Uridylate
Figure 4.26
Amino group of aspartate
CO2
Glycine
Formyl group (from folic acid)
Amide nitrogen of glutamine
Ribose-5-P
Purine skeleton
Inosinic acid
Purine biosynthesis
Aspartic acid
NH3
Figure 4.26 Composition of purines and pyrimidines.
CO2
Orotic acid
Uridylate
Pyrimidine biosynthesis
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88. 4.15 Biosynthesis of Fatty Acids and Lipids
Fatty acids are biosynthesized two carbons at a time (Figure 4.27)
Acyl carrier protein (ACP) involved
ACP holds the growing fatty acid as it is being synthesized
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89. Figure 4.27
Figure 4.27 The biosynthesis of the C16 fatty acid palmitate.
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90. 4.15 Biosynthesis of Fatty Acids and Lipids
In Bacteria and Eukarya, the final assembly of lipids involves addition of fatty acids to glycerol
In Archaea, lipids contain phytanyl side chains instead of fatty acids
Phytanyl biosynthesis is distinct from that of fatty acids
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91. 4.16 Regulating the Activity of Biosynthetic Enzymes
Two major modes of enzyme regulation
Amount
Regulation at the gene level
Activity
Temporary inactivation of the protein through changes in enzyme structure
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92. 4.16 Regulating the Activity of Biosynthetic Enzymes
Feedback Inhibition: mechanism for turning off the reactions in a biosynthetic pathway (Figure 4.28)
End product of the pathway binds to the first enzyme in the pathway, thus inhibiting its activity
The inhibited enzyme is an allosteric enzyme (Figure 4.29)
Two binding sites: active and allosteric
Reversible reaction
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93. Starting substrate Enzyme A Enzyme B Enzyme C Enzyme D End product
Figure 4.28
Starting substrate
The allosteric enzyme
Enzyme A
Intermediate I
Enzyme B
Intermediate II
Feedback inhibition
Enzyme C
Figure 4.28 Feedback inhibition of enzyme activity.
Intermediate III
Enzyme D
End product
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94. Active site Allosteric site
Figure 4.29
End product (allosteric effector)
Active site
Allosteric site
Enzyme
Substrate
INHIBITION: Substrate cannot bind; enzyme reaction inhibited
ACTIVITY: Enzyme reaction proceeds
Figure 4.29 The mechanism of allosteric inhibition by the end product of a pathway.
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95. 4.16 Regulating the Activity of Biosynthetic Enzymes
Some pathways controlled by feedback inhibition use isoenzymes
Isoenzymes
Different enzymes that catalyze the same reaction but are subject to different regulatory controls (Figure 4.30)
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96. DAHP synthases (isoenzymes 1, 2, 3)
Figure 4.30
Phosphoenol- pyruvate
Erythrose 4-phosphate
Initial substrates 
DAHP synthases (isoenzymes 1, 2, 3)
DAHP
Chorismate
Figure 4.30 Isoenzymes and feedback inhibition.
Tyrosine
Tryptophan
Final products
Phenylalanine
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97. 4.16 Regulating the Activity of Biosynthetic Enzymes
Biosynthetic enzymes can also be regulated by covalent modifications
Regulation involves a small molecule attached to or removed from the protein (Figure 4.31)
Results in conformational change that inhibits activity
Common modifiers include adenosine monophosphate (AMP), adenosine diphosphate (ADP), inorganic phosphate (PO42), methyl groups (CH3)
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98. Glutamine concentration
Figure 4.31
100
Enzyme activity
Glutamine concentration
Relative GS activity
Glutamine
Glutamine 50 GS
GS-AMP6
GS-AMP12
Figure 4.31 Regulation of glutamine synthetase by covalent modification. 3 6 9 12
AMP
AMP groups added
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