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Bacterial Metabolism and Energy Generation. An Overview of Metabolism. Metabolism – the sum of all chemical reactions occurring within a cell simultaneously. Involves degradation and biosynthesis of complex molecules.

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slide3
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
slide4
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
slide5
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
slide6
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
slide7
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
definitions
Definitions
  • Oxidation
  • Reduction
  • Redox reactions
  • Standard Reduction potential
  • Oxidative phosphorylation
  • Chemiosmosis
  • Electron transport chain
oxidation
Oxidation
  • The loss of electrons from an atom or chemical compound.
  • Results in the generation of energy.
reduction
Reduction
  • The gain of electrons
  • Requires energy
redox reactions
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
standard reduction potential equilibrium constant e o at ph 7 0
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)
slide13
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.
oxidative phosphorylation
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)
chemiosmosis
Chemiosmosis
  • The generation of ATP by the movement of hydrogen ions into pores in the cytoplasmic membrane that are associated with the ATPase system.
electron transport chain
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 --> often referred to as “reducing power”
three differences between prokaryotes and eukaryotes
Three differences between prokaryotes and eukaryotes
  • #1: Prokaryotes use different electron carriers (cytochromes vary)
slide20
#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)
slide21
#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
slide22
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
slide23
Oxidative Phosphorylation
  • Some of the energy liberated during electron transport is used to drive the synthesis of ATP in a process called oxidative phosphorylation
slide24
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
slide25
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
slide26
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)
slide27
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
inhibition of aerobic synthesis of atp
Inhibition of aerobic synthesis of ATP
  • Inhibitors of ATP synthesis fall into two main categories:
slide29
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)
slide30
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
pasteur effect
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
distinction between respiration and fermentation
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
slide33
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
slide34
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
slide36
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
slide37
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
slide38
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.)
slide39
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
slide40
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
slide41
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.
slide43
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
slide44
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
six common pathways of fermentation
Six Common Pathways of Fermentation
  • Homoloactic acid
  • Alcoholoic
  • Propionic acid
  • Butylene glycol (Butanediol)
  • Mixed acid fermentation
  • Butyric acid, butanol, acetone
slide46
#1: Homolactic acid fermentation
    • Pyruvate  lactic acid
  • #2: Alcoholic fermentation
    • Pyruvate  acetaldehyde  ethanol
slide47
#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)
slide48
#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
mixed acid versus butanediol fermenters 3 tests to divide groups
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 ~1:1
    • Butanediol ~5:1
respiration
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
aerobic respiration
Aerobic Respiration
  • Oxygen = Terminal Electron Acceptor
  • Glucose  CO2
  • 38 ATP
anaerobic respiration
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)
slide53
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
slide54
2 NO3- + 12e- + 12H+  N2 + 6H2O
  • Examples:
    • Escherichia
    • Enterobacter
    • Bacillus
    • Pseudomonas
    • Micrococcus
    • Rhizobium
slide55
SO42-  sulfate reducers
    • TEA = sulfate
      • which is reduced to sulfide
    • SO42- + 8e- + 8H+  S2- + 4H2O
    • Examples:
      • Desulfovibrio
      • Desulfotomaculum
slide56
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
slide57
Metals can also be reduced
    • Elemental sulfur (So)
    • Ferric iron (Fe3+)
slide59
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
slide60
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
slide62
proceeds by either hydrolysis or
  • phosphorolysis to produce molecules that can enter the common catabolic pathways already discussed
slide64
Lipases
    • Degrade lipids to glycerol + free fatty acids
slide65
Fatty acid degradation proceeds by the beta -oxidation pathway, which produces acetyl-CoA, which can enter the TCA cycle
slide66
Glycerol degradation proceeds via the Embden-Meyerhof pathway (glycolysis) entering as glyceraldehyde 3-phosphate
slide68
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
anabolism
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.
slide70
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
slide71
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 (NAG:UDP intermediate)
      • Gluconeogenesis also used
        • Synthesis of glucose from a non-carbohydrate precursor
slide72
Lipid biosynthesis
    • Two lipids predominate in bacteria in the lipid bilayer:
      • Phosphatidyl glycerol
      • Phosphatidylethanolamine
slide73
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)
slide74
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
slide75
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 Kreb’s cycle
  • Once made  protein synthesis on ribosomes can ensue.