Microbial Metabolism
L2300
Metabolism
• Pathogenic bacteria exhibit heterotrophic metabolism.
• Heterotrophs require preformed organic compounds (sugars,
amino acids) for growth.
• Bacterial transport systems involve CM-associated binding, or
transport proteins for CHO & AAs.
• Energy frequently required to concentrate substrates inside cell
• Transport - inducible for nutrients that are catabolized
• Glucose – exception  transport constitutive
• Phosphotransferase systems frequently used for sugar transport
• Videos
– 1. Facilitated Diffusion
– 2. Active Transport by Group Translocation
– 3. Proton Pump
Fig. 6.1
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
CATABOLISM
ANABOLISM
Energy source
(glucose)
Cell structures
(cell wall, membrane,
ribosomes, surface
structures)
Energy
Macromolecules
(proteins, nucleic acids,
polysaccharides, lipids)
Energy
Subunits
(amino acids,
nucleotides, sugars,
fatty acids)
Energy
Precursor
metabolites
Waste products
(acids, carbon
dioxide)
Catabolic processes harvest
the energy released during the
breakdown of compounds and
use it to make ATP. The
processes also produce
precursor metabolites used in
biosynthesis.
Nutrients
(source of nitrogen,
sulfur, etc.)
Anabolic processes
(biosynthesis) synthesize and
assemble subunits of macromolecules that make up the cell
structures. The processes use
the ATP and precursor metabolites
produced in catabolism.
Figure 6.8
Videos
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1. Glycolysis
http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::512::342::/sites/dl/free/0073375314/927345/
How_Glycolysis_Works.swf::How_Glycolysis_Works
2. Krebs Cycle
http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::512::342::/sites/dl/free/0073375314/927345/
How_the_Krebs_Cycle_Works.swf::How_the_Krebs_Cycle_Works
3. Proton Pump
http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::512::342::/sites/dl/free/0073375314/927345/
Proton_Pump.swf::Proton_Pump
4. ETC & Formation of ATP x2 / Nester 6th ARIS – Chapter 16
http://www.youtube.com/watch?v=6W-7FG9KlpA
Superoxide
• What enzyme produces superoxide (O2-) from
oxygen (O2)?
• Oxidase = a group of enzymes (now termed
oxidoreductases) that bring about oxidation by
the addition of oxygen to a metabolite or by the
removal of hydrogen or of one or more e-.
• O2 acts as an acceptor (of H or of electrons)
• Removal of hydrogen = dehydrogenases
Superoxide
• NADPH oxidase
• Nicotinamide adenine dinucleotide phosphate
oxidase
• Neutrophil / Macrophage cell membrane
• Superoxide is generated by both prokaryotes &
eukaryotes and is an important product of the
metabolic burst of neutrophil leukocytes.
• A very active oxygen species, it can cause
substantial damage.
Peroxide
• What enzyme converts superoxide free radical
(O2-) to peroxide (H2O2) and oxygen (O2)?
• 2O2-+ 2H+  H2O2 + O2
• Dismutase = enzyme catalyzing the reaction of
2 identical molecules to produce 2 molecules in
differing states of oxidation (e.g., superoxide
dismutase).
Superoxide dismutase
•
•
•
•
•
•
Present in:
Obligate aerobes
Facultative anaerobes
Aerotolerant anaerobes
Microaerophiles
Pathogens - Bacteroides
Peroxide
• What enzyme degrades peroxide (H2O2) into
H2O and oxygen (O2), and thereby protects
bacteria?
• 2 H2O2  H2O + O2
• Hydrogen peroxide is toxic to cells
Catalase
• Catalase 
• 2 H2O2  H2O + O2
• Hydrogen peroxide can also be oxidized by a
peroxidase enzyme
• Peroxidase =
• H2O2 + NADH + H+  2 H2O + NAD+
Hypochlorite
• What enzyme forms hypochlorite (HOCl-)
from peroxide (H2O2) and chloride (Cl-)?
• Hypochlorite = bleach
Respiratory burst
O2-dependent MPO-dependent reactions
H2O2 +
-
Cl-
2OCl + H2O
myeloperoxidase
OCl- + H2O
1O
2
Toxic compounds: hypochlorous acid OCl-, singlet oxygen 1O2
+ Cl- + H2O
Myeloperoxidase
• Myeloperoxidase
• Hypochlorite is 50x more potent than
peroxide at killing bacteria
• Neutrophils  green pus!
Energy
• ATP derived from controlled breakdown of various organic
substrates
• CHO, lipids, proteins
• Catabolism = substrate breakdown + conversion into usable energy
• Metabolic process – hydrolysis of large macromolecules in external
cell environment by enzymes
• Monosaccharides, short peptides, FA’s  CM  cytoplasm
• Glucose, Fructose, Galactose
• Metabolites converted  common universal intermediate =
pyruvate
• Pyruvic acid – carbons channeled toward 
– 1. E production or
– 2. Synthesis of new CHO, AA’s, lipids, or NA’s
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Fig. 6.10
GLUCOSE
2
Pentose phosphate
pathway
Starts the oxidation of glucose
1
Glycolysis
Oxidizes glucose to pyruvate
Yields
~ ~
+
Reducing
power
ATP
by substrate-level
phosphorylation
Yields
Reducing
power
Biosynthesis
5
Acids, alcohols, and gases
Pyruvate
Pyruvate
Fermentation
Reduces pyruvate
or a derivative
3a Transition step
CO2
CO2
Yields
Reducing
power
AcetylCoA
AcetylCoA
X2
CO2
CO2
3b
TCA cycle
Incorporates an acetyl
group and releases CO2
(TCA cycles twice)
Yields
~
ATP
by substrate-level
phosphorylation
~
+
Reducing
power
4
Respiration
Uses the electron transport
chain to convert reducing
power to proton motive force
Yields
~ ~
ATP
by oxidative
phosphorylation
Table 6.2
Metabolism of Glucose
• Instead of releasing all the molecule’s E as heat
(burning), bacteria break down glucose in
discrete steps allow E capture in usable form
• Efficiency
• 1. Aerobic respiration – complete conversion of
glucose to 6 CO2 + H2O
• 2. Anaerobic respiration
• 3. Glycolysis – 3C end products
• 4. Fermentation – 2C & 3C end products
Embden-Meyerhof-Parnas Pathway
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•
•
•
•
Glycolysis
Primary pathway used to convert glucose  pyruvate
Energy produced in two different forms:
1. Chemical – SLP (substrate level phosphorylation)
2. Electrochemical – NADH  converted to ATP
through series of oxidation reactions
• Absence of O2, SLP = primary means of E production
Niacin - Vitamin B3
• There are 2 co-enzyme forms of niacin:
– 1. Nicotinamide adenine dinucleotide (NAD)
– 2. Nicotinamide adenine dinucleotide phosphate
(NADP)
• Both play an important role in energy transfer reactions
in the metabolism of glucose, fat and alcohol.
• A precursor of coenzymes NAD & NADP, which are
needed in many metabolic processes.
Riboflavin – B2
• Riboflavin is involved in release of energy in:
– 1. The electron transport chain (ETC)
– 2. The citric acid cycle (CAC)
– 3. Catabolism of fatty acids (beta oxidation)
• A precursor of cofactors FAD & FMN
– Needed for flavoprotein enzyme reactions, including
activation of other vitamins.
Cofactor / Coenzyme
• Cofactor = a non-protein chemical compound or
metallic ion that is required for a protein's
biological activity.
• Cofactors can be subclassified as either:
– 1. Inorganic ions
– 2. Coenzymes = complex organic molecules
• Coenzymes - mostly derived from vitamins &
other essential organic nutrients.
• Non-covalent bond
Fermentation
• Fermentation – pyruvic acid converted to end products
• Organic molecules (not O2) used as e- acceptors to
recycle NADH
• Absence of O2, SLP = primary means of E production
• ATP formation not coupled to e- transfer
• Specific metabolic end products synthesized  aid in ID
of bacterial species
Tricarboxylic Acid Cycle (TCA)
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Krebs Cycle , Citric Acid Cycle
+O2  pyruvate  completely oxidized
Controlled burning of pyruvate to H2O + CO2 using TCA
Transition step = oxidative decarboxylation (release of
CO2)
TCA allows generation of >> E from glucose vs.
glycolysis
GTP (ATP equivalent) – SLP
NADH / FADH2  ETC  ATP
Aerobic = Oxygen
Anaerobic = Nitrate, sulfate, CO2, ferric iron
Functions of TCA
• TCA – C’s derived from lipids (acetyl CoA) shunted
towards E production or biosynthesis
• Deaminated AA’s  alpha ketoglutarate, oxaloacetate
• Functions:
• 1. Most efficient mechanism for generation of ATP
• 2. Final common pathway for complete oxidation of AA’s,
FA’s, and CHO.
• 3. Supplies key intermediates for synthesis of AA’s,
lipids, purines & pyrimidines
Pentose Phosphate Pathway
• Hexose monophosphate shunt (HMP)
• Alternative pathway to breakdown glucose
• Pentose shunt = A pathway of hexose oxidation in which G6P
undergoes two successive oxidations by NADP, the final one being
an oxidative decarboxylation to form a pentose phosphate.
• Functions
• 1. Provide nucleotide (NA) precursors
– Ribose-5-phosphate = precursor in nucleotide synthesis
• 2. Provide reducing power (NADPH) for biosynthesis
– NADP is a coenzyme in lipid and nucleic acid synthesis.
• 3. Amino acid precursor metabolite
– Erythrose 4-phosphate
Respiration
• Respiration uses reducing power (NADH) generated in
glycolysis, the transition step, and the TCA cycle to
synthesize ATP.
• Oxidative phosphorylation = mechanism of ATP
synthesis
• Requires:
• 1. ETC (electron transport chain) generates PMF (proton
motive force)
– CM with cytochrome enzymes, lipid cofactors, & coupling factors
• 2. ATP synthase (enzyme) uses E of PMF to drive
synthesis of ATP
Obligate Aerobes
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+Superoxide dismutase (SOD)
+Catalase
+Peroxidase
Glycolysis
Transition step
TCA or Krebs Cyle or Citric Acid Cycle
Respiration – Aerobic (Oxygen)
G+: Nocardia, Bacillus, Mycobacterium
G-: Neisseria, Pseudomonas, Bordetella, Legionella,
Brucella
• Acid-fast: Mycobacterium, Nocardia (weakly acid-fast)
Obligate Anaerobes
• Killed by O2- (superoxide anion)
• Lack superoxide dismutase (SOD) & catalase
• Require another substance as a H+/e- acceptor during
generation of metabolic E
• Glycolysis
• Fermentation
• Respiration – Anaerobic (Nitrate, Sulfate)
• G+: Clostridium, Actinomyces
• G-: Bacteroides
Facultative Anaerobes
• Shift from fermentative to respiratory metabolism in presence of
oxygen
• Most pathogenic bacteria are facultative anaerobes
• +Superoxide dismutase (SOD)
• +Catalase
• Glycolysis
• Transition step
• TCA
• Fermentation
• Respiration – Aerobic (+/- Anaerobic)
• Enterics (Enterobacteriaceae) – Escherichia coli
• Listeria, Staphylococcus
Microaerophiles
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+Superoxide dismutase (SOD)
(-)Catalase
Glycolysis
Fermentation
Campylobacter , Helicobacter
Aerotolerant Anaerobes
• Resemble facultative anaerobes, but have fermentative
metabolism both +/- oxygen environment
• Obligate fermenters
• +Superoxide dismutase (SOD)
• (-)Catalase
• Glycolysis
• Fermentation
• Lactic acid bacteria – Streptococcus, Lactobacillus,
Enterococcus