Microbial Metabolism
Chapter 5
Why microbial metabolism is
important
• How do cells gain energy to form cell structures?
• How do pathogens acquire energy and nutrients
at the expense of a patients health?
• How does grape juice turn into wine?
Metabolism
• Metabolism – the sum of all of the ________
___________within a living organism
– Biosynthetic
– Energy harvesting processes
– 1000s of chemical reactions and control
mechanisms
• The chemical reactions that occur in a cell are
referred to _______________________
Metabolic Pathways
• Metabolic pathway (Biochemical pathway):
Series of chemical reactions required to
breakdown or build a cellular component
– ____________(“food”)
– Intermediates (“partially digested food”)
– ____________(“by-product”)
– ____________are involved
Enzymes Role in Metabolic Pathways
• Enzyme facilitate each step of a metabolic
pathway
– Made of _______________
– Act as a Biological _____________
• _________________________of a chemical reaction
Enzyme 1
Enzyme 2
Enzyme 3
Enzyme + Substrate -----------------------------------------------> Enzyme + End Product
Energy
Activation energy
without enzyme
Activation energy
with enzyme
Products
Progress of reaction
Enzymes Role in Metabolic Pathways
• Naming Enzymes – many are named by adding
“ase”
• Enzymes are ________________
– Lipases
– Proteases
Lipids
Proteins
Enzyme Specificity can be explained by
a lock and key theory called the
____________________
Key (____________)
_______________: substrate binding
site
Lock (______________: protein)
Many protein enzymes are complete on their own, others have
protein and non protein components.
Induced fit model
____________________
What happens to the enzyme once products have been made?
Apoenzyme Components
Apoenzymes:
1. Enzyme - protein portion
2. Cofactor - non-protein
• ______________- inorganic ions (iron, magnesium, or zinc)
• ______________- organic vitamins which cannot be synthesized by
certain organisms
Inorganic cofactor
Active site
Coenzyme
(organic
cofactor)
Apoenzyme (protein)
Coenzymes
• E. coli can synthesize or make its own vitamins
and convert them to coenzymes
• Humans and other animals must consume
vitamins from external sources
– E. coli synthesizes vitamin K which we can absorb
Factors that Influence Enzymatic
Activity
• A cells ability to survive in extreme
temperatures or pH is due to their enzymes
• Enzymes are influenced by environmental
factors
– Temperature, pH, and substrate concentrations
– Have optimal activity ranges
Temperature
• Denaturation of an Active Protein
pH
Many enzymes work
best at neutral pH.
Acetic acid pH 3.0 can
act as a preservative
Substrate concentration
When the substrate
concentration (enzyme
food) gets to high the
enzymatic activity levels off,
since all enzymes are
working at their maximum
rate.
Without enzymes…
• Energy yielding reactions could occur but rates
would be extremely slow
Allosteric Enzymes
• Cells can rapidly regulate or control the
activity of key enzymes
– They have an _____________that is ________
from the active site
– Molecules bind and _____________________
which prevents them from working
Feedback inhibition
• When ______________of a biosynthetic
pathway can act as an ______________of the
______________in that pathway.
– Allows for controlling its own synthesis or building
– Example: E. coli, the presence of the amino acid
isoleucine allosterically inhibits the first enzyme in
the pathway. Prevents the synthesis of isoleucine
when available. Once depleted E. coli can resume
production
Substrate
Pathway
shuts down
Pathway
operates
Enzyme 1
Bound
end-product
(allosteric
inhibitor)
Allosteric
site
Feedback
inhibition
Intermediate A
Enzyme 2
Intermediate B
End-product
Enzyme Inhibition
Enzymes can be inhibited by a variety of
compounds other than regulatory molecules such
as allosteric regulators
Inhibitors can effect enzymatic activity
1. Competitive Inhibitors
2. Noncompetitive Inhibitors
Why is enzyme inhibition important?
Competitive Inhibitors
•
• Generally this occurs since the inhibitor has a
________________________as the substrate
Example
• Sulfanilamide (Sulfa Drugs)
– Antibiotic
– competes for the active site on bacterial enzymes that
converts PABA into Folic Acid
•
•
•
•
Has a similar chemical structure to PABA
Prevents PABA from binding to active site
Folic Acid - required for the synthesis of DNA and RNA
No Folic acid= no DNA RNA synthesis= no cell replication
– Selective toxicity: Doesn’t affect human cells since we
can not synthesize Folic acid
Non-competitive Inhibitors
•
Figure 5.11 Feedback inhibition
Substrate
Pathway
shuts down
Pathway
operates
Enzyme 1
Bound
end-product
(allosteric
inhibitor)
Allosteric
site
Feedback
inhibition
Intermediate A
Enzyme 2
Intermediate B
End-product
Enzyme 3
WHO CARES?
• What are the 3 methods we will use in this
class to identify unknown bacteria?
WHO CARES?????
• When laboratory personnel perform
biochemical tests to identify unknown
microorganisms we are testing for the
production of an __________by that
organism.
Scenario
• You observe Gram positive coccus using the
microscope. You are having a difficult time
determining arrangement (which is common)
– You see a mixture of chains: indicating
Streptococcus
– You also see clumps: indicating Staphylococcus
• What biochemical test/tests can you perform
to further differentiate between the two?
Biochemical Testing
• Every bacterial species produce different
enzymes which allow them to produce
different metabolic by-products.
• Identify and differentiate bacteria.
Streptococcus pyogenes
Staphylococcus aureus
•
Example of a biochemical test
– Test used to demonstrate the ability of a bacterium to
produce the enzyme catalase
– Simplest test used to differentiate between
Staphylococcus and Streptococcus
– Streptococcus are catalase negative (obligate
fermenter)
– Staphylococcus are catalase positive
Streptococcus pyogenes
Staphylococcus aureus
Catalase test
• The catalase test is simply performed by
placing a few drops of H2O2 (hydrogen
peroxide) on bacterial growth on an agar
media.
• A positive test is indicated by the formation of
bubbles (H2O2 + catalase -> H2O + O2 (bubbles)
Streptococcus pyogenes
Staphylococcus aureus
Basics of Metabolism
Two components of metabolism
• 1. __________________( Catabolic )
– _____________of complex organic molecules into
simpler compounds
–
• 2. _________________( Anabolic )
– the _____________of complex organic molecules
from simpler ones
–
Energy
• Energy: the capacity to do work
– 2 forms
• Potential: stored energy
• Kinetic: energy of motion
– Example: Rock on top of a hill?
– Rock tumbling down a hill?
Role of ATP
• Adenosine triphosphate (ATP)
– Energy currency of a cell
– Donor of free energy
– 3 phosphates
– Energy is stored in the phosphate bonds
• Adenosine diphosphate (ADP)
– Acceptor of free energy
– 2 phosphates
Metabolism
Energy lost
as heat
Energy lost
as heat
Energy
stored
Energy
used
ANABOLISM
Precursor
molecules
Larger building
blocks
Macromolecules
Nutrients
Energy storage
(carbohydrates,
lipids, etc.)
Cellular
processes
(cell growth,
cell division,
etc.)
Cellular structures
(membranes,
ribosomes, etc.)
Role of ATP
• Cells constantly generate and use ATP
– Power biosynthetic reactions
• 2 processes used by heterotrophic bacteria to
form ATP
– Substrate-level phosphorylation
– Oxidative phosphorylation
How cells make ATP (part 1)
• Substrate-level phosphorylation
–
• Ex. Glycolosis
– Only a small amount of ATP is made
How cells make ATP (part 2)
• Oxidative phosphorylation
–
Chemical Energy Production
• Energy Source or electron donor
• Compound is broken down by a cell to release
energy
• Example: Glucose
• 1.
– refers to the ________of electrons
• 2.
– the _________of electrons
Chemical Energy Production
• Oxidation and Reduction Reactions
– Electron transfer from an electron _______to an
electron ____________
– Reactions always occur simultaneously
– Cells use ___________________to carry electrons
(often in H atoms)
Reduction
Oxidized
donor
Electron
donor
Electron
acceptor
Oxidation
Reduced
acceptor
Chemical Energy Production
Electron carriers
• Oxidized form
– NAD+
– FAD
•
Reduced form
NADH
FADH2
Chemical Energy Production
Terminal Electron Acceptors
• Electrons are transferred to a molecule such
as oxygen which functions as a terminal eacceptor.
– Aerobic Respiration
Carbohydrate Catabolism
• Microorganisms oxidize carbohydrates as their
primary source of energy for anabolic
reactions
• Glucose - most common energy source
• Energy obtained from Glucose by:
–
–
Key metabolic pathways
• Gradually oxidize glucose completely to
carbon dioxide
– Glycolysis
– Transition reaction
– Krebs cycle
– Electron Transport system
Oxidation of Glucose
Chemical Equation
• C6H12O6 + 6 O2 -------> 6 CO2 + 6 H2O
•
38 ADP + 38 P
38 ATP
• Oxygen is the terminal e- acceptor
Glycolysis
• Oxidation of Glucose (_________) into ___ molecules of
____________ (___carbon)
• Investment phase
–
• Pay off phase
– Generates __________
– Net gain ____________
• Overall End Products of Glycolysis:
– ___ pyruvate
– ___ NADH (reducing power used in electron transport system)
– ___ ATP
Cellular Respiration
• Includes
– Synthesis of Acetyl-CoA (transition reaction)
– Krebs cycle
– ETS
• This can be aerobic (with oxygen)
• Or anaerobic (without oxygen)
– Varying amounts of ATP are produced depending
on e- acceptor
Cellular Respiration
Transition Reaction (synthesis of Acetyl-CoA)
• Connects Glycolysis to Krebs Cycle
• Input from glycolysis
– _____________
• End Products:
–
–
–
Cellular Respiration
Krebs Cycle
• The cyclical process needs to “turn” twice for
1 molecule of glucose
• Input from transition step
–
• Products:
–
–
–
–
Cellular Respiration
Electron Transport System
• Input
– _______________from glycolysis, transition, and TCA cycle
• Electron carriers _______and _______(“Reducing
power”) ____________________________________
_____________________________
• The electron energy is used to ____________
_______________from the _______________
– Forms the _______________________
Electron Transport System
• When the electron carriers transfer electrons
the electron carriers are recycled
• Aerobic respiration ultimately passes e- to
oxygen the terminal e- acceptor
• Anaerobic respiration uses a molecule other
than oxygen
Electron Transport System creates the
Proton Motive Force
• The lipid bilayer is not permeable to H+ atoms
• Positively charged protons build up and are
concentrated immediately outside the cell
membrane
Figure 5.18 One possible arrangement of an electron transport chain
Bacterium
Mitochondrion
Intermembrane
space
Matrix
Exterior
Cytoplasmic
membrane
Cytoplasm
Exterior of prokaryote
or intermembrane space
of mitochondrion
FMN
Ubiquinone
Cyt b
Phospholipid
membrane
NADH
from glycolysis,
Krebs cycle,
pentose phosphate
pathway, and
Entner-Doudoroff
pathway
Cyt c
Cyt a3
Cyt a
Cyt c2
FADH2
from
Krebs cycle
Cytoplasm of prokaryote
or matrix of mitochondrion
ATP synthase
Proton Motive Force continued….
• Separation of charged ions creates an
_____________________across the
membrane
–This gradient has ________________
• Energy is harvested when protons flow
through protein channels called _____
____________________
• ATPases phosphorylate ADP to generate
ATP
How 34 ATP from E.T.S. ?
3 ATP for each NADH
2 ATP for each FADH2
• FADH2
• NADH
• Glycolysis
• T. R.
• Krebs Cycle
2
2
6
• Glycolysis
• T.R.
• Krebs Cycle
0
0
2
• Total
10
• Total
2
• 10 x 3 = 30 ATP
• 2 x 2 = 4 ATP
Total ATP production for the complete
oxidation of 1 molecule of glucose in
Aerobic Respiration
•
•
•
•
Glycolysis
Transition Reaction
Krebs Cycle
E.T.S.
• Total
ATP
2
0
2
34
38 ATP
The Epic Journey of Glucose in Bacteria!
By: Michael Pressler
This story begins with a 6 carbon molecule of glucose, he will become
completely oxidized into CO2 while releasing ATP by substrate level
phosphorylation and gaining reducing power in the form of electron
carriers (NADH and FADH2).
The story does not end with out a climatic event…. When the
“powered” electron carriers give their electrons to a protein in the cell
membrane a bucket brigade of electron transportation occurs by
passing electrons through a series of proteins called the electron
transport chain. The electron transport chain develops the “force”
(the proton motive force that is…) and pumps hydrogen ions out of
the cell membrane. By utilizing the Terminal electron accepting
abilities of Oxygen, Oxygen and hydrogen combine to form water! The
hero comes in the form of ATP synthase, as she allows Hydrogen back
into the cell, ATP is formed by oxidative phosphorylation!
• Metabolism video clip
Respiration
• 2 types
–
–
Aerobic Respiration
• Oxygen (O2) is the final electron acceptor is
aerobic respiration.
• Aerobic bacteria will generally use O2 when it
is available due to the large amount of energy
produced compared to using other electron
acceptors.
Anaerobic Respiration
• Anaerobic respiration occurs when an
inorganic molecule other than oxygen is used
for the electron acceptor.
– Less efficient (Less ATP is generated) form of
respiration than aerobic
– nitrate, sulfate, and carbonate.
• Facultative anaerobes
– Can use oxygen or other inorganic molecules as
electron acceptors
Anaerobic respiration
• Methanogens
• Archaea that generate ATP by oxidizing hydrogen
gas
– Release methane
– http://www.youtube.com/watch?NR=1&feature=e
ndscreen&v=mL57IO-MzJA
– http://www.youtube.com/watch?v=seZotwOEI5k
General Fermentation
Characteristics
•
• Does not inhibit growth
• Does not use Oxygen as electron acceptor
• During fermentative metabolism, _________
______________ act as electron acceptors.
• Facultative anaerobes
– Some of the Facultative anaerobes we use are obligate
fermenters
– Can use oxygen or other inorganic molecules as electron
acceptors
Fermentation
• Sometimes cells cannot completely oxidize glucose
by cellular respiration
– Fermenters go through glycolysis to form pyruvic
acid (3 carbon), 2 ATP, and NADH
– Pyruvic acid is oxidized to acetylaldehyde (2
carbon) with CO2 as a by product.
– NADH donate electrons and Hydrogen to
acetylaldehyde to form ethanol and NAD+
Figure 5.22 Representative fermentation products and the organisms that produce them
Glucose
Pyruvic acid
Organisms
Propionibacterium
Aspergillus
Lactobacillus
Streptococcus
Saccharomyces
Clostridium
Fermentation
CO2, propionic acid
Lactic acid
CO2, ethanol
Acetone, isopropanol
Wine, beer
Nail polis remover,
rubbing alcohol
Fermentation
products
Swiss cheese
Cheddar cheese,
yogurt, soy sauce
Scenario
• You observe an unknown organism growing in
throughout an FT tube indicating the
unknown organism is a facultative anaerobe.
• How can you determine if this organism is an
obligate fermenter or if this organism is
capable of switching between aerobic and
anaerobic fermentation?
– Oxidase test
– Catalase test
Differentiating between types of
metabolism: aerobic, anaerobic, or
fermentation
• 2 biochemical tests used
•
Catalase Test
• Catalase is usually only present in _________
__________________
• Facultative anaerobes can be catalase positive
– Obligate fermenters are usually catalase negative
• Obligate anaerobes lack catalase and
superoxide dismutase
– Therefore catalase negative
Oxidase test
• The oxidase test is used to _______________
_____________________
• In aerobic respiration, ____________(electron
transport proteins) carry the electrons to O2
• This test identifies the enzyme cytochrome
oxidase which is associated with cytochrome c
in the ETS
• Purple is positive after adding the oxidase
reagent
– Must be read within 2 minutes