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 3Enzyme 2

Enzyme + Substrate -----------------------------------------------> Enzyme + End Product

Ener

gy

Progress of reaction

Products

Activation energywithout enzyme

Activation energywith enzyme

• Naming Enzymes – many are named by adding “ase”

• Enzymes are ________________– Lipases Lipids– Proteases Proteins

Enzymes Role in Metabolic Pathways

Enzyme Specificity can be explained by a lock and key theory called the

____________________

Lock (______________: protein)

Key (____________)

_______________: substrate binding site

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 ComponentsApoenzymes: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(organiccofactor)

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

pHMany enzymes work best at neutral pH.

Acetic acid pH 3.0 can act as a preservative

Substrate concentrationWhen 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

Feedbackinhibition

Boundend-product(allostericinhibitor)

Substrate

Enzyme 1

Allostericsite

Intermediate A

Intermediate B

Enzyme 2

End-product

Pathwayshuts down

Pathwayoperates

Enzyme Inhibition

Inhibitors can effect enzymatic activity

1. Competitive Inhibitors

2. Noncompetitive Inhibitors

Why is enzyme inhibition important?

Enzymes can be inhibited by a variety of compounds other than regulatory molecules such as allosteric regulators

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

Feedbackinhibition

Boundend-product(allostericinhibitor)

Substrate

Enzyme 1

Allostericsite

Intermediate A

Intermediate B

Enzyme 2

Enzyme 3

End-product

Pathwayshuts down

Pathwayoperates

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 lostas heat

Nutrients

Energystored

Energyused

Energy lostas heat

Precursormolecules

Larger buildingblocks

Energy storage(carbohydrates,lipids, etc.)

Macromolecules

Cellularprocesses(cell growth,cell division, etc.)

Cellular structures(membranes,ribosomes, etc.)

ANABOLISM

CATABOL ISM

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)

Electrondonor

Electronacceptor

Oxidation

ReductionOxidizeddonor

Reduced acceptor

Chemical Energy ProductionElectron carriers

• Oxidized form Reduced form– NAD+ NADH– FAD FADH2

•

Chemical Energy ProductionTerminal Electron Acceptors

• Electrons are transferred to a molecule such as oxygen which functions as a terminal e- acceptor. – 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 GlucoseChemical 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 RespirationTransition Reaction (synthesis of Acetyl-CoA)

• Connects Glycolysis to Krebs Cycle• Input from glycolysis

– _____________

• End Products:– – –

Cellular RespirationKrebs 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

Exterior

Cytoplasmicmembrane

Cytoplasm

Intermembranespace

Matrix

Mitochondrion

Exterior of prokaryoteor intermembrane spaceof mitochondrion

FMN

Phospholipidmembrane

NADHfrom glycolysis,Krebs cycle,pentose phosphatepathway, andEntner-Doudoroffpathway

FADH2

from Krebs cycle

Ubiquinone

Cytoplasm of prokaryoteor matrix of mitochondrion

Cyt c2

Cyt b

Cyt c Cyt aCyt a3

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 NADH2 ATP for each FADH2

• NADH

• Glycolysis 2• T. R. 2• Krebs Cycle 6

• Total 10

• 10 x 3 = 30 ATP

• FADH2

• Glycolysis 0• T.R. 0• Krebs Cycle 2

• Total 2

• 2 x 2 = 4 ATP

Total ATP production for the complete oxidation of 1 molecule of glucose in

Aerobic Respiration ATP•Glycolysis 2•Transition Reaction 0•Krebs Cycle 2•E.T.S. 34

•Total 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=endscreen&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 themGlucose

Pyruvic acid

Organisms

Fermentation

Fermentationproducts

CO2, propionic acid Lactic acid CO2, ethanol Acetone, isopropanol

Swiss cheese Cheddar cheese,yogurt, soy sauce

Wine, beer Nail polis remover,rubbing alcohol

PropionibacteriumAspergillusLactobacillusStreptococcus

Saccharomyces Clostridium

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