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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.
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

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

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

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

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

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

Oxidation

The loss of electrons from an atom or chemical


compound.
Results in the generation of energy.

Reduction

The gain of electrons


Requires energy

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 = 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)

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

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

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

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

Electron

Transport and
Oxidative Phosphorylation

Mitochondrial Electron Transport (Figure 9.13)

Three differences between


prokaryotes and eukaryotes

#1: Prokaryotes use different electron carriers


(cytochromes vary)

#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)

#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

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

Oxidative

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

The

chemiosmotic hypothesis of
oxidative phosphorylation (Peter
Mitchell)
Membrane-bound

carriers transfer
electrons to oxygen across a chain
Cytochromes,

flavoprotein, quinones, nonheme iron containing proteins


Coupling of electron transport to oxidative
phosphorylation

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

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)

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

Inhibitors of ATP synthesis fall into two main


categories:

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)

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

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

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

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

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

The

Breakdown of Glucose
to Pyruvate

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

ATP

the expenditure of two molecules of

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

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.)

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

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

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.

Fermentation

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

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

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

#1:

Homolactic acid fermentation

Pyruvate

#2:

lactic acid

Alcoholic fermentation

Pyruvate

acetaldehyde ethanol

#3: Propionic acid fermentation


acetic acid, OAA, malate,
fumarate, succinate, propionate

Pyruvate

#4: Butylene glycol fermentation


(butanediol)
acetolactic acid acetoin
2,3 butylene glycol (2,3 butanediol)

Pyruvate

#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

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

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

Oxygen = Terminal Electron Acceptor


Glucose CO2
38 ATP

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 OXPHOS still used)

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

2 NO3- + 12e- + 12H+ N2 + 6H2O


Examples:

Escherichia
Enterobacter
Bacillus
Pseudomonas
Micrococcus
Rhizobium

SO42- sulfate reducers

TEA = sulfate

which is reduced to sulfide

SO42- + 8e- + 8H+ S2- + 4H2O


Examples:

Desulfovibrio
Desulfotomaculum

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

Metals can also be reduced


Elemental sulfur (So)
Ferric iron (Fe3+)

The

Tricarboxylic Acid
Cycle-a series of reactions

Acetyl-CoA

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

Three molecules of NADH and one


molecule of FADH2 are produced per acetylCoA, 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

Catabolism

of Carbohydrates
and Intracellular Reserve
Polymers

proceeds

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

Lipid

Catabolism

Lipases
Degrade

acids

lipids to glycerol + free fatty

Fatty

acid degradation proceeds by


the beta -oxidation pathway, which
produces acetyl-CoA, which can enter
the TCA cycle

Glycerol degradation proceeds via the


Embden-Meyerhof pathway (glycolysis)
entering as glyceraldehyde 3-phosphate

Protein

and Amino Acid


Catabolism

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

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.

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

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

Lipid biosynthesis

Two lipids predominate in bacteria in the lipid


bilayer:
Phosphatidyl glycerol
Phosphatidylethanolamine

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)

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

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.