Chapter 3
Microbial Metabolism
I. Laboratory Culture of Microorganisms
• 3.1 Cell Chemistry and Nutrition
• 3.2 Culture Media
• 3.3 Laboratory Culture
3.1 Cell Chemistry and Nutrition
• Nutrients
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Supply of monomers (or precursors of) required by cells for
growth
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Macronutrients
• Nutrients required in large amounts
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Micronutrients
• Nutrients required in trace amounts
3.1 Cell Chemistry and Nutrition
• Carbon
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Required by ALL cells
Typical bacterial cell is ~50% carbon (by dry weight)
Major element in ALL classes of macromolecules
Heterotrophs use organic carbon
Autotrophs use carbon dioxide (CO2)
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3.1 Cell Chemistry and Nutrition
• Nitrogen
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Typical bacterial cell is ~13% nitrogen (by dry weight)
Key element in proteins, nucleic acids, and many more cell
constituents
3.1 Cell Chemistry and Nutrition
• Other macronutrients
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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
3.1 Cell Chemistry and Nutrition
• Other macronutrients (cont'd)
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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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3.1 Cell Chemistry and Nutrition
• Iron
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Key component of cytochromes and FeS proteins involved
in electron transport
Growth factors
• Organic compounds required in small amounts by certain
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organisms
• Examples: vitamins, amino acids, purines, pyrimidines
Vitamins
• Most commonly required growth factors
• Most function as coenyzmes
3.2 Media and Laboratory Culture
• Culture media
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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)
3.2 Media and Laboratory Culture
• Enriched media
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Contain complex media plus additional nutrients
Selective media
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Contain compounds that selectively inhibit growth of some
microbes but not others
Differential media
• Contain an indicator, usually a dye, that detects particular
chemical reactions occurring during growth
3.2 Media and Laboratory Culture
• 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
3.2 Media and Laboratory Culture
• Pure culture: culture containing only a single kind of
microbe
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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
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(agar or gelatin)
When grown on solid media, cells form isolated masses
(colonies)
3.2 Media and Laboratory Culture
• Microbes are everywhere
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Sterilization of media is critical
Aseptic technique should be followed (Figure 3.3)
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3.2 Media and Laboratory Culture
• Pure culture technique
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Streak plate (Figure 3.4)
Pour plate
Spread plate
II. Energetics, Enzymes and Redox
• 3.3 Energy Classes of Microorganisms
• 3.4 Bioenergetics
• 3.5 Catalysis and Enzymes
• 3.6 Electron Donors and Electron Acceptors
• 3.7 Energy-Rich Compounds
3.3 Energy Classes of Microorganisms
• Metabolism
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The sum total of all of the chemical reactions that occur in a
cell
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Catabolic reactions (catabolism)
• Energy-releasing metabolic reactions
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Microorganisms grouped into energy classes (Figure 3.5)
• Chemorganotrophs
• Chemolithotrophs
• Phototrophs
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Heterotrophs
Autotrophs
3.4 Bioenergetics
• Energy is defined in units of kilojoules (kJ), a measure of
heat energy
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In any chemical reaction, some energy is lost as heat
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The change in free energy during a reaction is referred to
as ΔG0′
Free energy (G): energy released that is available to do
work
3.4 Bioenergetics
• Reactions with a negative ΔG0′ release free energy
(exergonic)
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Reactions with a positive ΔG0′ require energy
(endergonic)
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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)
3.4 Bioenergetics
• For the reaction A + B  C + D,
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ΔG0′ = Gf0 [C+D] - Gf0[A+B]
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ΔG0′ is not always a good estimate of actual free-energy
changes
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ΔG: free energy that occurs under actual conditions
• ΔG = ΔG0′ + RT in K
• where R and T are physical constants and K is the
equilibrium constant for the reaction in question
3.5 Catalysis and Enzymes
• Free-energy calculations do not provide information on
reaction rates
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Activation energy: energy required to bring all molecules
in a chemical reaction into the reactive state (Figure 3.6)
• Catalysis is usually required to breach activation energy
barrier
3.5 Catalysis and Enzymes
• Catalyst: substance that
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Lowers the activation energy of a reaction
Increases reaction rate
Does not affect energetics or equilibrium of a reaction
3.5 Catalysis and Enzymes
• Enzymes
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Biological catalysts
Typically proteins (some RNAs)
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Highly specific
Generally larger than substrate
Typically rely on weak bonds
• Examples: hydrogen bonds, van der Waals forces, hydrophobic
interactions
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Active site: region of enzyme that binds substrate
3.5 Catalysis and Enzymes
• Enzymes (cont'd)
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Increase the rate of chemical reactions by 108 to 1020 times
the spontaneous rate
Enzyme catalysis: E + S  E — S  E + P (Figure 3.7)
Catalysis dependent on
• Substrate binding
• Position of substrate relative to catalytically active amino acids
in active site
3.5 Catalysis and Enzymes
• Many enzymes contain small nonprotein molecules that
participate in catalysis but are not substrates
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Prosthetic groups
• Bind tightly to enzymes
• Usually bind covalently and permanently (e.g., heme group
in cytochromes)
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Coenzymes
• Loosely bound to enzymes
• Most are derivatives of vitamins (e.g., NAD+/NADH)
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3.6 Electron Donors and Electron Acceptors
• Energy from oxidation–reduction (redox) reactions is
used in synthesis of energy-rich compounds (e.g., ATP)
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Redox reactions occur in pairs (two half reactions; Figure
3.8)
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Electron donor: the substance oxidized in a redox
reaction
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Electron acceptor: the substance reduced in a redox
reaction
3.6 Electron Donors and Electron Acceptors
• Reduction potential (E0′): tendency to donate electrons
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Expressed as volts (V)
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Substances can be either electron donors or electron
acceptors under different circumstances (redox couple)
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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′
3.6 Electron Donors and Electron Acceptors
• The redox tower represents the range of possible
reduction potentials (Figure 3.9)
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The reduced substance at the top of the tower donates
electrons
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The oxidized substance at the bottom of the tower
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accepts electrons
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The farther the electrons "drop," the greater the amount
of energy released
3.6 Electron Donors and Electron Acceptors
• Redox reactions usually involve reactions between
intermediates (carriers)
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Electron carriers are divided into two classes
• Prosthetic groups (attached to enzymes)
• Coenzymes (diffusible)
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Examples: NAD+, NADP (Figure 3.10)
3.6 Electron Donors and Electron Acceptors
• NAD+ and NADH facilitate redox reactions without being
consumed; they are recycled (Figure 3.11)
3.7 Energy-Rich Compounds
• Chemical energy released in redox reactions is primarily
stored in certain phosphorylated compounds (Figure
3.12)
• ATP; the prime energy currency
• Phosphoenolpyruvate
• Glucose 6-phosphate
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Chemical energy also stored in coenzyme A
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3.7 Energy-Rich Compounds
• Long-term energy storage involves insoluble polymers
that can be oxidized to generate ATP
• Examples in prokaryotes
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• Glycogen
• Poly-β-hydroxybutyrate and other polyhydroxyalkanoates
• Elemental sulfur
Examples in eukaryotes
• Starch
• Lipids (simple fats)
III. Fermentation and Respiration
• 3.8 Glycolysis
• 3.9 Fermentative Diversity and the Respiratory Option
• 3.10 Respiration: Electron Carriers
• 3.11 Respiration: The Proton Motive Force
• 3.12 Respiration: Citric Acid and Glyoxylate Cycle
• 3.13 Catabolic Diversity
3.8 Glycolysis
• Two reaction series are linked to energy conservation in
chemoorganotrophs: fermentation and respiration
(Figure 3.13)
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Differ in mechanism of ATP synthesis
• Fermentation: substrate-level phosphorylation; ATP is
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directly synthesized from an energy-rich intermediate
Respiration: oxidative phosphorylation; ATP is produced
from proton motive force formed by transport of electrons
3.8 Glycolysis
• Fermented substance is both an electron donor and an
electron acceptor
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Glycolysis (Embden–Meyerhof pathway): a common
pathway for catabolism of glucose (Figure 3.14)
• Anaerobic process
• Three stages
3.8 Glycolysis
• Glycolysis
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Glucose is consumed
Two ATPs are produced
Fermentation products are generated
• Some harnessed by humans for consumption
3.9 Fermentative Diversity and the Respiratory
Option
• Fermentations classified by products formed
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Ethanol
Lactic acid
Propionic acid
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Mixed acids
Butyric acid
Butanol
3.9 Fermentative Diversity and the Respiratory
Option
• Fermentations classified by substrate fermented
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Usually NOT glucose
Amino acids
Purines and pyrimidines
Aromatic compounds
3.9 Fermentative Diversity and the Respiratory
Option
• Saccharomyces cerevisiae can carry out fermentation or
respiration
• Carries out the one most beneficial
• Respiration generates more ATP
• Fermentation occurs when conditions are anoxic
3.10 Respiration: Electron Carriers
• Aerobic respiration
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Oxidation using O2 as the terminal electron acceptor
Higher ATP yield than fermentations
• ATP is produced at the expense of the proton motive force,
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which is generated by electron transport
3.10 Respiration: Electron Carriers
• Electron transport systems
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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)
3.10 Respiration: 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
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Flavoproteins: contains flavin prosthetic group (e.g.,
FMN, FAD) that accepts 2 electrons and 2 protons but
donates the electrons only to the next protein in the
chain (Figure 3.16)
3.10 Respiration: Electron Carriers
• Cytochromes
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Proteins that contain heme prosthetic groups (Figure 3.17)
Accept and donate a single electron via the iron atom in
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heme
3.10 Respiration: Electron Carriers
• Iron–sulfur proteins
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Contain clusters of iron and sulfur (Figure 3.18)
• Example: ferredoxin
Reduction potentials vary depending on number and
position of Fe and S atoms
Carry electrons
3.10 Respiration: Electron Carriers
• Quinones
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Hydrophobic non-protein-containing molecules that
participate in electron transport (Figure 3.19)
Accept electrons and protons but pass along electrons only
3.11 Respiration: The Proton Motive Force
• Electron transport system oriented in cytoplasmic
membrane so that electrons are separated from protons
(Figure 3.20)
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Electron carriers arranged in membrane in order of their
reduction potential
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The final carrier in the chain donates the electrons and
protons to the terminal electron acceptor
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3.11 Respiration: 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
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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
3.11 Respiration: The Proton Motive Force
• Complex I (NADH:quinone oxidoreductase)
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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
3.11 Respiration: The Proton Motive Force
• Complex III (cytochrome bc1 complex)
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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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3.11 Respiration: The Proton Motive Force
• ATP synthase (ATPase): complex that converts proton
motive force into ATP; two components (Figure 3.21)
• F1: multiprotein extramembrane complex; faces cytoplasm
• Fo: proton-conducting intramembrane channel
• Reversible; dissipates proton motive force
3.12 Respiration: Citric Acid and Glyoxylate
Cycle
• Citric acid cycle (CAC): pathway through which pyruvate
is completely oxidized to CO2 (Figure 3.22a)
• Initial steps (glucose to pyruvate) same as glycolysis
• Per glucose molecule, 6 CO2 molecules released and
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NADH and FADH generated
Plays a key role in catabolism AND biosynthesis
Energetics advantage to aerobic respiration (Figure
3.22b)
3.12 Respiration: Citric Acid and Glyoxylate
Cycle
• The citric acid cycle generates many compounds
available for biosynthetic purposes
• - α-Ketoglutarate and oxaloacetate (OAA): precursors of
several amino acids; OAA also converted to
phosphoenolpyruvate, a precursor
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of glucose
Succinyl-CoA: required for synthesis of cytochromes,
chlorophyll, and other tetrapyrrole compounds
Acetyl-CoA: necessary for fatty acid biosynthesis
3.12 Respiration: Citric Acid and Glyoxylate
Cycle
• Organic acids can be metabolized as electron donors
and carbon sources by many microbes
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C4-C6 citric acid cycle intermediates (e.g., citrate,
malate, fumarate, and succinate) are common natural
plant and fermentation products and can be readily
catabolized through the citric acid cycle alone
3.12 Respiration: Citric Acid and Glyoxylate
Cycle
• Glyoxylate cycle
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Catabolism of C2-C3 organic acids typically involves
production of oxaloacetate through the glyoxylate cycle
(Figure 3.23)
A variation of the citric acid cycle
Glyoxylate is a key intermediate
3.13 Catabolic Diversity
• Microorganisms demonstrate a wide range of
mechanisms for generating energy (Figure 3.24)
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Fermentation
Aerobic respiration
Anaerobic respiration
Chemolithotrophy
Phototrophy
3.13 Catabolic Diversity
• Anaerobic respiration
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The use of electron acceptors other than oxygen
• Examples include nitrate (NO3–), ferric iron (Fe3+), sulfate (SO42–
), carbonate (CO32–), certain organic compounds
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Less energy released compared to aerobic respiration
Dependent on electron transport, generation of a proton
motive force, and ATPase activity
3.13 Catabolic Diversity
• Chemolithotrophy
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Uses inorganic chemicals as electron donors
• Examples include hydrogen sulfide (H2S), hydrogen gas (H2),
ferrous iron (Fe2+), ammonia (NH3)
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Typically aerobic
Begins with oxidation of inorganic electron donor
Uses electron transport chain and proton motive force
Autotrophic; uses CO2 as carbon source
3.13 Catabolic Diversity
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Phototrophy: uses light as energy source
• Photophosphorylation: light-mediated ATP synthesis
• Photoautotrophs: use ATP for assimilation of CO2 for
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biosynthesis
Photoheterotrophs: use ATP for assimilation of organic
carbon for biosynthesis
IV. Biosyntheses
• 3.14 Sugars and Polysaccharides
• 3.15 Amino Acids and Nucleotides
• 3.16 Fatty Acids and Lipids
• 3.17 Nitrogen Fixation
3.14 Sugars and Polysaccharides
• Prokaryotic polysaccharides are synthesized from
activated glucose (Figure 3.25a)
• Adenosine diphosphoglucose (ADPG)
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• Precursor for glycogen biosynthesis
Uridine diphosphoglucose (UDPG)
• Precursor of some glucose derivatives needed for biosynthesis
of important polysaccharides
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Examples: N-acetylglucosamine, N-acetylmuramic acid
3.14 Sugars and Polysaccharides
• Gluconeogenesis (Figure 3.25b)
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Synthesis of glucose from phosphoenolpyruvate
• Phosphoenolpyruvate can be synthesized from oxaloacetate
3.14 Sugars and Polysaccharides
• Pentoses are formed by the removal of one carbon atom
from a hexose (Figure 3.25c)
3.14 Sugars and Polysaccharides
• Pentoses are required for the synthesis of nucleic acids
• If pentoses are not readily available from the
environment, organisms must synthesize them
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The major pathway for pentose production is the pentose
phosphate pathway (Figure 3.26)
3.15 Amino Acids and Nucleotides
• Biosynthesis of amino acids and nucleotides often
involves long, multistep pathways
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Amino acid biosynthesis
• Carbon skeletons come from intermediates of glycolysis or
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citric acid cycle (Figure 3.27)
Ammonia is incorporated by glutamine dehydrogenase or
glutamine synthetase (Figure 3.28)
Amino group transferred by transaminase and synthase
(Figure 3.28)
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3.15 Amino Acids and Nucleotides
• Purine and pyrimidine biosyntheses are complex
• Purines (Figure 3.29a and b)
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Adenine and guanine
Inosinic acid intermediate
Pyrimidines (Figure 3.29c and d)
• Thymine, cytosine, and uracil
• Orotic acid intermediate
3.16 Fatty Acids and Lipids
• Fatty acids are biosynthesized two carbons at a time
(Figure 3.30)
• Acyl carrier protein (ACP) involved
• ACP holds the growing fatty acid as it is being synthesized
3.16 Fatty Acids and Lipids
• In Bacteria and Eukarya, the final assembly of lipids
involves addition of fatty acids to glycerol
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In Archaea, lipids contain phytanyl side chains instead of
fatty acids
• Phytanyl biosynthesis is distinct from that of fatty acids
3.17 Nitrogen Fixation
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Only certain prokaryotes can fix nitrogen
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Reaction is catalyzed by nitrogenase
• Sensitive to the presence of oxygen
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A wide variety of nitrogenases use different metal
cofactors (Figure 3.31)
Some nitrogen fixers are free-living, and others are
symbiotic
3.17 Nitrogen Fixation
• Electron flow in nitrogen fixation (Figure 3.33)
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electron donor  dinitrogenase reductase  dinitrogenase
 N2
Ammonia is the final product
3.17 Nitrogen Fixation
• Nitrogenase activity can be assayed using the acetylene
reduction assay (Figure 3.34)
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