Bacterial Metabolism and Energy Generation

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• Bacterial Metabolism and Energy Generation
•  

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• An Overview of Metabolism

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• Metabolism the sum of all chemical reactions
  occurring within a cell simultaneously. Involves
  degradation and biosynthesis of complex
  molecules.
• Catabolism-the breakdown of larger, more complex
  molecules into smaller, simpler ones, during
  which energy is released, trapped, and made
  available for work
• Anabolism-the synthesis of complex molecules from
  simpler ones during which energy is added as input

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• Multi-stage process of catabolism
• Stage 1-breakdown of large molecules
  (polysaccharides, lipids, proteins) into their
  component constituents with the release of little
  (if any) energy

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• Stage 2-degradation of the products of stage 1
  aerobically or anaerobically to even simpler
  molecules with the production of some ATP, NADH,
  and/or FADH2

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• Stage 3-complete aerobic oxidation of stage 2
  products with the production of ATP, NADH, and
  FADH2 the latter two molecules are processed by
  electron transport to yield much of the ATP
  produced

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• Metabolic efficiency is maintained by the use of
  a few common catabolic pathways, each degrading
  many nutrients
• Microorganisms are catabolically diverse, but are
  anabolically quite uniform
• Amphibolic pathways function both catabolically
  and anabolically, and sometimes employ separate
  enzymes to catalyze the forward and reverse
  reactions this separation enables independent
  regulation of the forward and reverse reactions

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Definitions

• Oxidation
• Reduction
• Redox reactions
• Standard Reduction potential
• Oxidative phosphorylation
• Chemiosmosis
• Electron transport chain

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Oxidation

• The loss of electrons from an atom or chemical
  compound.
• Results in the generation of energy.

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Reduction

• The gain of electrons
• Requires energy

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Redox reactions

• Reactions involving the transfer of electrons
  from a donor to an acceptor
• Redox couple
• Reducing agent reductant donor
• Oxidizing agent oxidant acceptor
• Oxidant ne- (number of electrons transferred)?
  Reductant

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Standard Reduction Potential (Equilibrium
Constant Eo at pH 7.0)

• Measures the tendency of the reductant to lose
  electrons.
• Redox couples with more negative Eo values will
  donate elecrron to redox couples with more
  positive Eo values, releasing free enegy (G)
• Energy is required to reverse the process (e.g.
  photosynthesis)

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• The larger the difference in Eo values between
  electron donors and electron acceptors, the
  greater the free energy that is generated.
• Therefore, the more negative the reduction
  potential, the better electron donor it is, and
  the more numerous the potential acceptors.

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Oxidative phosphorylation

• A metabolic sequence of reactions occurring
  within a membrane in which an electrons
  transferred from a reduced coenzyme by a series
  of electron carriers, establishing an
  electrochemical gradient across the membrane that
  drives the formation of ATP from ADP and
  inorganic phosphate by chemiosmosis.
• Powered by redox reactions
• Aerobic respiration uses O2 as TEA
• Anaerobic respiration uses SO42-, NO3-, CO2 as
  TEA (not as efficient as using O2 as TEA)

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Chemiosmosis

• The generation of ATP by the movement of hydrogen
  ions into pores in the cytoplasmic membrane that
  are associated with the ATPase system.

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Electron Transport Chain

• A series of oxidation-reduction reactions in
  which electrons are transported from a substrate
  through a series of intermediate electron
  carriers to a final acceptor, establishing an
  electrochemical gradient across a membrane that
  results in the formation of ATP.
• Electrochemical gradient proton motive force
• Examples of donors coenzymes NADH, NADPH, and
  FADH2 --gt often referred to as reducing power

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• Electron Transport and Oxidative Phosphorylation

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• Mitochondrial Electron Transport (Figure 9.13)

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Three differences between prokaryotes and
eukaryotes

• 1 Prokaryotes use different electron carriers
  (cytochromes vary)

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• 2 The bacterial electron transport chain may
  be extensively branched with several terminal
  oxidases
• Electrons may enter at several different points
  and use different TEAs
• Often dependent on growing conditions of bacteria
• Different cytochromes used depending on O2 status
  (log vs stationary)

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• 3 Electron transport in bacteria occurs in the
  cytoplasmic membrane
• In eukaryotic cells this occurs on the inner
  membranes of mitochondria
• But the mechanism of Ox-Phos is remarkably
  similar
• Did mitochondria arise from bacteria?
  Endosymbiont theory

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• Electrons from NADH and FADH2 are transported in
  a series of redox reactions to a terminal
  electron acceptor
• Reduced coenzymes (NADH and FADH2) generated in
  glycolysis and TCA cycles must be reoxidized

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• Oxidative Phosphorylation
• Some of the energy liberated during electron
  transport is used to drive the synthesis of ATP
  in a process called oxidative phosphorylation

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• The chemiosmotic hypothesis of oxidative
  phosphorylation (Peter Mitchell)
• Membrane-bound carriers transfer electrons to
  oxygen across a chain
• Cytochromes, flavoprotein, quinones, non-heme
  iron containing proteins
• Coupling of electron transport to oxidative
  phosphorylation

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• Postulates that the energy released during
  electron transport is used to establish a proton
  gradient (proton motive force due to different
  charge distributions)
• As electrochemical potential is formed (PMF)
  protons are attracted back into the cell through
  a proton channel
• F1F0 adenosine triphosphatase (F1F0 ATPase) in
  proton channel releases energy as protons enter

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• Blockers
• inhibit the flow of electrons through the system
• Examples Cyanide or Azide (block electron
  transport between cyt a and O2
• Examples Piericidin (competes with Coenzyme Q)
  and Antimycin A (blocks electron transport
  between cyt b and cyt c)

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• This proton-motive force is then used to drive
  ATP synthesis
• Energy is converted to chemical energy by
  phosphorylation of ADP to ATP
• High energy phosphate bonds used for biosynthetic
  pathways
• Net result
• 1 NADP or NADPH 3 ATP
• 1 FADH2 2 ATP

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Inhibition of aerobic synthesis of ATP

• Inhibitors of ATP synthesis fall into two main
  categories

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• Uncouplers
• allow electron flow, but disconnect it from
  oxidative phosphorylation (inhibit ATP synthesis,
  not electron transport)
• Uncouple electron transport from Ox-Phos ? The
  energy from electron transport is lost as heat,
  not ATP
• Examples Valinomycin, Dinitrophenol (DNP)

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• The Yield of ATP in Glycolysis and Aerobic
  Respiration
• The yield of ATP in glycolysis and aerobic
  respiration varies with each organism, but has a
  theoretical maximum of 38 molecules of ATP per
  molecule of glucose catabolized
• Anaerobic organisms using glycolysis can only
  produce two molecules of ATP per molecule of
  glucose catabolized

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Pasteur Effect

• Switch from fermentation (anaerobic) to aerobic
  respiration when oxygen is available
• Much more efficient
• More ATP using oxygen as TEA
• Sugar catabolism dramatically decreases

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Distinction between Respiration and Fermentation

• Respiration
• Electrons from oxidative process enter the
  electron transport system, protons are generated
  and energy is generated through OX-PHOS
• Electrons are passed to an inorganic acceptor
• Electron transport is used
• OX-PHOS is used

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• Fermentation
• Electrons and protons from oxidized substrates
  are transferred directly to another organic
  compound (organic acceptor) in the pathway
• No electron transport
• No OX-PHOS
• Substrate-level phosphorylation used instead
• The oxidation of a phosphorylated compound
  resuluting in the direct formation of a
  high-energy phosphate bond
• Transferred to ADP to form ATP

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• Example of fermentation
• Glycolysis
• Glucose ? Pyruvate ? Lactic Acid or Ethanol
• The final electron acceptor pyruvate (or a
  product of pyruvate such as acetaldehyde
  intermediate)
• Summary In fermentation, organic compounds are
  the electron donors AND acceptors with some
  energy generated

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• The Breakdown of Glucose to Pyruvate

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• The glycolytic (Embden-Meyerhof) pathway is the
  most common pathway and is divided into two
  parts
• The 6-carbon sugar stage involves the
  phosphorylation of glucose twice to yield
  fructose 1,6-bisphosphate
• requires the expenditure of two molecules of ATP

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• The 3-carbon sugar stage cleaves fructose
  1,6-bisphosphate into two 3-carbon molecules,
  which are each processed to pyruvate
• two molecules of ATP are produced by
  substrate-level phosphorylation from each of the
  3-carbon molecules for a net yield of two
  molecules of ATP
• 2 molecules of NADH are also produced per glucose
  molecule

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• 1 Glucose ? 2 Pyruvate
• 2 ATP used
• 4 ATP generated
• Net yield 2 ATP
• 2 NAD used
• 2 NADH generated
• Net yield 2 NADH (THIS MUST BE OXIDIZED TO KEEP
  THIS PATHWAY RUNNING.)

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• The pentose phosphate (hexose monophosphate)
  pathway uses a different set of reactions to
  produce a variety of 3-, 4-, 5-, 6-, and 7-carbon
  sugar phosphates
• These phosphates can be used to produce ATP and
  NADPH, as well as to provide the carbon skeletons
  for the synthesis of amino acids, nucleic acids,
  and other macromolecules

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• The NADPH can be used to provide electrons for
  biosynthetic processes or can be converted to
  NADH to yield additional ATP through the electron
  transport chain

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• The Entner-Doudoroff pathway can also be used to
  produce pyruvate with a lower yield of ATP, but
  is accompanied by the production of NADPH as well
  as NADH.

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• Fermentation

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• In the absence of oxygen, NADH is not usually
  oxidized by the electron transport chain because
  no external electron acceptor is available
• However, NADH must still be oxidized to replenish
  the supply of NAD for use in glycolysis

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• Fermentations are reactions that regenerate NAD
  from NADH in the absence of oxygen
• Fermentations involve pyruvate or pyruvate
  derivatives as electron acceptors
• Fermentations may or may not produce additional
  ATP for the cell

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Six Common Pathways of Fermentation

• Homoloactic acid
• Alcoholoic
• Propionic acid
• Butylene glycol (Butanediol)
• Mixed acid fermentation
• Butyric acid, butanol, acetone

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• 1 Homolactic acid fermentation
• Pyruvate ? lactic acid
• 2 Alcoholic fermentation
• Pyruvate ? acetaldehyde ? ethanol

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• 3 Propionic acid fermentation
• Pyruvate ? acetic acid, OAA, malate, fumarate,
  succinate, propionate
• 4 Butylene glycol fermentation (butanediol)
• Pyruvate ? acetolactic acid ? acetoin ? 2,3
  butylene glycol (2,3 butanediol)

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• 5 Mixed acid fermentation
• Pyruvate ? Lactate, formate, acetate, ethanol,
  succinate
• 6 Butyric acid, butanol, acetone fermentation
• Pyruvate ? acetyl CoA, adetate, ethanol
• Pyruvate ? acetyl CoA, acetone, isopropanol
• Pyruvate ? acetyl CoA, butyryl CoA, butanol,
  butyric acid

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Mixed Acid versus Butanediol Fermenters 3 Tests
to Divide Groups

• Voges-Proskauer Test
• Detection of acetoin (intermediary metabolite)
• Methyl red test
• Mixed acid fermenter produces a lot of acid
• pH is 4.4
• Red color produced for MAF only
• CO2/H2 ratios
• Mixed acid 11
• Butanediol 51

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Respiration

• Use of electron transport chain passing electrons
  to an inorganic terminal electron acceptor (TEA)
• Energy generated through OX-PHOS
• More energy efficient than fermentation

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Aerobic Respiration

• Oxygen Terminal Electron Acceptor
• Glucose ? CO2
• 38 ATP

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Anaerobic Respiration

• Uses inorganic molecules other than oxygen as
  terminal electron acceptors this produces
  additional ATP for the cell, but not usually as
  much as is produced by aerobic respiration
• Used mainly by anaerobes but many facultative
  anaerobes may use anaerobic respiration (electron
  transport and OX-PHOS still used)

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• Non-oxygen Terminal Electron Acceptor
• Three major types
• NO3- ? nitrate reducers
• Facultative anaerobes
• TEA nitrate
• Results in denitrification (loss of nitrates
  froom the soil agricultural dilamma)
• Advantageous when removing nitrates from sewage

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• 2 NO3- 12e- 12H ? N2 6H2O
• Examples
• Escherichia
• Enterobacter
• Bacillus
• Pseudomonas
• Micrococcus
• Rhizobium

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• SO42- ? sulfate reducers
• TEA sulfate
• which is reduced to sulfide
• SO42- 8e- 8H ? S2- 4H2O
• Examples
• Desulfovibrio
• Desulfotomaculum

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• CO2 ? methane bacteria
• TEA CO2 which is reduced to methane
• Habitat rumen of cud-chewing animals, black mud
  of ponds and composts and sewage tanks
• CO2 8e- 8H ? CH4 2H2O

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• Metals can also be reduced
• Elemental sulfur (So)
• Ferric iron (Fe3)

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• The Tricarboxylic Acid Cycle-a series of reactions

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• Acetyl-CoA (produced by decarboxylation of
  pyruvate) reacts with oxaloacetate to produce a
  6-carbon molecule
• Subsequently, two molecules of carbon dioxide are
  released, regenerating the oxaloacetate
• ATP is produced by substrate-level phosphorylation

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• Three molecules of NADH and one molecule of FADH2
  are produced per acetyl-CoA, and can be further
  processed to produce more ATP
• Even those organisms that lack the complete TCA
  cycle usually have most of the cycle enzymes
  because one of the TCA cycle's major functions is
  to provide carbon skeletons for use in
  biosynthesis

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• Catabolism of Carbohydrates and Intracellular
  Reserve Polymers

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• proceeds by either hydrolysis or
• phosphorolysis to produce molecules that can
  enter the common catabolic pathways already
  discussed

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• Lipid Catabolism

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• Lipases
• Degrade lipids to glycerol free fatty acids

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• Fatty acid degradation proceeds by the beta
  -oxidation pathway, which produces acetyl-CoA,
  which can enter the TCA cycle

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• Glycerol degradation proceeds via the
  Embden-Meyerhof pathway (glycolysis) entering as
  glyceraldehyde 3-phosphate

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• Protein and Amino Acid Catabolism

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• Proteins are degraded by secreted proteases to
  their component amino acids, which are
  transported into the cell and catabolized
• The amino group is removed by deamination or
  transamination
• The resulting organic acids are converted to
  pyruvate, acetyl-CoA, or a TCA-cycle intermediate

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Anabolism

• Building up structural and functional components
  of a cell using energy and building blocks (small
  molecular intermediates)
• Includes synthesis of nucleic acids (DNA RNA),
  cell wall (lipid bilayer PTG) and proteins.

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• Two important features
• Biosynthetic pathways of most cellular components
  is different from degradative pathways at key
  regulatory steps (regulated by endproducts
  ATP NAD
• Biosynthetic pathways are often induced by
  combining reactants to form activated
  intermediates

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• Polysaccharide biosynthesis
• Some monosaccharides are derived from metabolic
  pathway intermediates
• React with nucleoside triphosphates to form
  activated intermediates (or adenosine or uridine
  diphosphate)
• Examples in bacteria
• Capsular polysaccharides
• Outer membrane LPS
• Glycogen
• PTG (NAGUDP intermediate)
• Gluconeogenesis also used
• Synthesis of glucose from a non-carbohydrate
  precursor

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• Lipid biosynthesis
• Two lipids predominate in bacteria in the lipid
  bilayer
• Phosphatidyl glycerol
• Phosphatidylethanolamine

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• Glycerol phosphate backbone plus two fatty acids
  (12-18 carbons in length)
• Glycerol derived from dihydroxyacetone phosphate
  (Embden-Meyerhoff)
• Fatty acids added from fatty acyl-CoA
  intermediates (FAs built up 2 carbons at a time
  using acetyl CoA)

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• Amino acid and protein biosynthesis
• Autotrophs
• Capable of synthesizing all 20 amino acids
  starting with CO2 (can survive in completely
  inorganic environment)
• Majority of prototrophs
• Similarly do not require added amino acids
• Some bacteria (e.g. Neisseria and Streptococci)
  require preformed amino acids
• Auxotrophs
• Mutant strains that DO require growth factors and
  sometimes amino acids

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• Amino acids are synthesized by complex pathways
  often involving multiple unique enzymes
• Histidine synthesis requires 9 enzymes
• Synthesis begins with metabolic pathway
  intermediates of glycolysis or Krebs cycle
• Once made ? protein synthesis on ribosomes can
  ensue.