M122 Microbiology MIDTERM REVIEW

Tutors:Suleiman Saroia

(M, W 10-11am Nat Sci 2108)Amy Vanmali

(M,W 11-12am Nat Sci 2108)

The Basics Microbiology is the study of

microorganisms

A pathogen is a microbe that causes disease. Normal flora don't cause disease and therefore

are non-pathogenic E. Coli in our intestines S. Aureus on our skin HOWEVER: in immunocompromised people

(i.e. AIDS) non-pathogenic microbes can become pathogenic when unregulated!

Germ Theory - the theory that diseases are caused by germs (microbes)

Earth’s life support:agriculture, energy and

environmentlegumes

ruminants

Impact on Humans:

Important people and Discoveries of/regarding Microorganisms

• Antony von Leeuwenhoek- The first to accurately describe microoraganisms (discovered bacteria/protozoa)•Robert Hooke -First to publish description about

microorganism(discovered fruiting mold)

•Luis Pasteur - Proved biogenesis for microorganisms.•Franscesco Redi- Proved biogenesis for large organisms.•Robert Koch- Was the first to establish relation between

bacteria and disease (Germ Theory)

•Carl Woese - Used rRNA sequencing which led to discovery of archaea.

Spontaneous Generation vs. Biogenesis

Definition of Biogenesis: Life arises from pre-existing life

PROOF: Large organisms: Redi “maggot

experiment” Microorganisms: Pasteur “Swan

Neck Flask”

Definition of Spontaneous Generation: Life arises spontaneously from non-living

matter

Redi's Maggot Experiment

Conclusion of Redi’s Maggot Experiment

• Conclusion:• - Rotting meat does not spontaneously

produce maggots.• - Maggots form only when flies are

present, i.e. flies carry eggs of maggots.

No Spontaneous Generation of Microorganism !

Robert KochDemonstrated the Role of Bacteria

in Causing Diseases via“Koch’s Postulates”

1.The microbe must only be present in infected organism, not in a healthy animals blood.

2. Isolate the microbe, and grow it in pure culture .

3. Take isolated microbe and inject into a healthy organism(should develop same signs and symptoms)

4. Re-isolate the microbe from healthy organism and should be same as the original microbe.

Observepathogenicorganism

Culturepathogenicorganism

Injectpathogenicorganism

Recoverpathogenicorganism

Importance of Koch’s Work

•Developed pure culture methods:

- growth on solid media- isolation of pure cultures

(like in micro lab …Word.)

•Scientific method to prove causation of diseaseEX)*(established the link between bacillus anthracis (bacteria) anthrax (disease).

What is a cell?Basic unit of all cellular life

-organisms can be cellular or acellular-Cells are enclosed by a sort of barrier (cell wall or cell membrane)

-Cellular cells include to main types:- prokaryotes (bacteria, archae)-eukaryotes (fungi, protists, algae)

… almost everything is cellular except VIRUSES ARE ACELLULAR

Basic Properties of Cells

• 6 Basic Properties of Cells:• 1) Metabolism (anabolism/catabolism)• 2) Reproduction (grow)• 3) Locomotion (movement)• 4) Differentiation• 5) Communication• 6) Evolve

Prokaryotes vs. Eukaryotes

Prokaryotes- 70S ribosomes

(30s+50s)- NO nucleus- NO membrane bound

organelles

Note: prokaryotic cells are typically smaller than eukaryotic cells.

Eukaryotes- 80S ribosomes

(40s+60s)- Nucleus- membrane-bound

organelles (i.e. mitochondria, golgi, ER, etc.)

Prokaryotes (Archae vs. Bacteria)Archae- cell wall composition is

more complex- seen as having both

bacterial/eukaryote characteristics!

- live in extreme environments (*Streptomyces thermoautotrophicus – thermophile)

*REMEMBER: Archae was discovered by Carl Woese who compared rRNA sequences

Bacteria- have peptidoglycan in

cell wall composition- simple shapes like

round, rod, etc.

Eukaryotes(Protozoa, Fungi, Algae)

Protozoa Fungi Plants• Single-celled single/multi celled single/multi celled

• No cell walls have cell walls have cell walls

• Motile No photosyn. pigments Photosynthesis

• Aquatic environments

• Part of food chain

• Some are pathogens

In soils, oceans, lakes

Some produce toxins

Acellular microorganisms:

VirusesCharacteristics:

A Major class of microorganism

Not cellular

Made of nucleic acid + protein

Obligate intracellular parasites

An organism is found to live in a lake which is found to have 5M NaCl. This is known to be a salinity in which most microorganisms cannot survive. This microorganism is most likely:a. bacteriab. archaec. protistd. fungie. all of the above

An organism is found to live in a lake which is found to have 5M NaCl. This is known to be a salinity in which most microorganisms cannot survive. This microorganism is most likely:a. bacteriab. archaec. protistd. fungie. all of the above

An organism is found to have peptidoglycan cell wall composition. Which of the following statements is true?

a. It is bacteria, and has 70S ribosomesb. It is bacteria, and has 80S ribosomesc. It is archae, and has 70S ribosomesd. It is archae, and has 80S ribosomese. none of the above are true statements

An organism is found to have peptidoglycan cell wall composition. Which of the following statements is true?

a. It is bacteria, and has 70S ribosomesb. It is bacteria, and has 80S ribosomesc. It is archae, and has 70S ribosomesd. It is archae, and has 80S ribosomese. none of the above are true statements

Lecture 2Prokaryotes

Typical Eukaryotic CellTypical Prokaryotic Cell

Membrane-enclosed Nucleus

Mitochondria & Chloroplasts

“Open”NucleoidPlasma Membrane

Prokaryotic Cell Sizes

• Typical Sizes:–Width - 1-2 m–Length - 2-10 m long

• Exceptions:–“Nanobacteria” - less than 0.2 m–A few large bacteria - up to 750 m

Sizes of Bacteria and Viruses

Cyanobacterium

Smallest bacteria are

about the size of the largest

viruses

Thiomargarita namibiensis (white sphere) is comparable in size to the head of a fruit fly.PRETTY BIG

Epulopiscium fishelsoni

Comparable to the sizeof a printed hyphen

Prokaryotic Cell Shapes

Bacillus

Hypha e.g. Streptomyces (fungus-like bacteria)

Function Supports & protects the cells.

Stalke.g. Caulobacter

Contains cytoplasmic material that is devoid of ribosomes and DNA.

Function Maybe in nutrient absorption.

Spherical (Cocci) Bacteria:Diplo – pairs

Staphylo – grape-like clustersStrepto – chain

Tetrads – groups of 4

Sarcinae – groups of 8

Spiral Bacteria:Vibrio, Spirilla,

Spirochete

• Vibrios - curved rods

• Spirilla - 2 or more twists

• Spirochetes - corkscrew shaped

Prokaryotic Structure:Plasma or Cell Membrane

Fimbra/Pilus

Plasma MembraneComposed of:

•Phospholipid bilayer asymmetric polar & nonpolar end•Membrane proteins=integral & peripheral protein)•Hopanoids - embedded in bilayer

– Sterol-like (similar to cholesterol) which stabilize membrane

–Stabilize membrane

AmphipathicAmphipathic

Structure of Plasma Membrane:“Fluid Mosaic Model of Bacterial Membrane Structure”

Floating in a lipid bilayer

Loosely associated with the inner membrane surface

Hydrophilic ends of the membrane phospholipids

Hydrophobic fatty acid chains

Plasma Membrane

Functions• Separates cell • Selectively permeable• Location of metabolic

reactions• Responds to

surroundings • Chemotaxis

Components• Phospholipids• Hopanoids• Integral Proteins• Peripheral Proteins

Internal Membrane System

Mesosomes: • invaginations of plasma membrane • Artifacts of chemical fixation

Complex in-foldings of plasma membrane:• Usually in photosynthetic or other prokaryotes

with high respiratory activity• Vesicles or tubular membranes

Cytoplasm

• 70 % water• Thick / Elastic• Has ribosomes and

inclusion bodies• Highly organized

with respect to protein location

Inclusion Bodies• Structure: made of organic or

inorganic materials• Function: nutrient & energy storage;

others• Examples:

– poly--hydroxybutyrate: phosphate storage. Plastics!

• Alcaligenes eutrophus

– gas vacuoles: provide buoyancy• Cyanobacterium

– magnetosome: iron containing, orientation in magnetic field

• Magnetospirillum magnetotacticum

Ribosomes/Nucleoid

Ribosomes• ribosomal RNA

(rRNA) + protein• Site of protein

synthesis• 70S

Nucleoid• Irregularly shaped

region, containing bacterial chromosome

Plasmids

• Extra-chromosomal DNA• Small, circular “mini-

chromosomes”• Function:

– Extra info: NOT required for growth

– May provide antibiotic resistance

• Transferrable between bacteria via conjugation

Cell Wall

•Gives shape / support

•Protects from osmosis lysis and toxins

•Made of peptidoglycan (murein)

Peptidoglycan

Formed by identical subunits

Alternating NAG/NAM form sugar chains

Peptidoglycan

The peptidoglycan “subunit” of Escherichia coli

N-acetyl-muramic acid (NAM)

N-acetyl-glucosamine (NAG)

Tetrapeptide side chain composed of alternating D- and L-amino acids

(these D-amino acids are not found in proteins)

Peptidoglycan Cross-Links

Diaminopimelic acidDirect cross-linking

Gram NEGATIVE = DIRECT

Peptide interbridgeGram POSITIVE=PEPTIDE

INTERBRIDGE

Gram Staining

1.) Crystal violet for 1 min

2.) Iodine for 3 min

3.) Add alcohol (decolorizer)

4.) Safranin for 1-2 min

All cells purple

All cells purple

G+ = purple, G- = colorless

G+ = purple, G- = red/pink

Gram +

•No outer membrane•Peptidoglycan layer is thick•Has Teichoic Acid•Periplasmic Space is thin•Sensitive to Penicillin•Flagella has two rings (inner and outer)

Gram -

•Has outer membrane•Peptidoglycan is thin•Has LPS•Penicillin resistant•Periplasmic space is thick•Flagella has 4 rings (LPSM)

Outer Membrane for G-

•Another barrier for transport•Protects from antibiotics and digestive enzymes•Has porin proteins•More permeable than inner membrane•Is strengthened by Braun’s lipoprotein

Periplasmic Space

•Thin in G+ bacteria•Contains exoenzymes

•Thick in G- bacteria•Peptidoglycan synthesis•Nutrient acquisition •Modifies toxins

Teichoic Acid and LPS

•Both are antigens that give negative charges•Techoic (G +) •LPS (G -)

•LPS stabilizes membrane (contains anO-side chain recognized by antibodies, core polysaccharide, lipid A)•LPS can act as an endotoxin

Capsule + Slim Layer

•Gives adherence•Helps resists dessication•Helps resists phagocytosis•Improves motility

Fimbrae and Pili

•Only in G-•Hair like•Attachment, invasion and nutrient uptake•Pili may be used to transfer DNA

Flagella

•Helps cell move•CCW = run•CW = tumble

•Chemotaxis-Go toward

nutrients-Avoids waste

Monotrichous

Lophotrichous

Amphitrichous

Peritrichous

Endospores

•Dormant structure inside cell•Sporulation = formation under stress•Germination = spore turning into vegetative cell

Activation Germination Outgrowth

Lecture 3Microbial Growth and Nutrition

Growth basically constrained by nutrients present and environmental limitations of the organism

Requirements for Growth: Physical Factors:

Temperature, pH, osmotic pressure, oxygen concentration

Chemical Factors: Macro-elements (e.g. C, N)

Trace-elements (e.g. Mn, Cu)Organic growth factors (e.g. amino acids)

Definition of Microbial Growth:Increase in cell number by raw material or defined

nutrients

What kind of effects do physical factors have?

TemperatureDisplays “Cardinal Growth temperatures” (min, optimum, max)

Psychrophiles (cold-loving) and thermophiles (hot-loving) have specific adaptations that allow their growth at their respective temperature ranges

Relative to the graph, what would a thermophile plot look like?

Psychrophiles (withstand extreme cold)• Proteins work well at lower

temperatures• Unsaturation of bilayer (increase

fluidity) to prevent “freezing”

Thermophiles (Withstand heat)• Proteins are stabilized to prevent

denaturation (more H-bonds,prolines, chaperones)

• DNA is stabilized (histones)• Membrane is saturated (lower “melting”

point) to maintain stable)

UNDERSTANDING THIS GRAPH AND THE FACT THAT YOU HAVE ADAPTATIONS ALLOWING YOU TO FILL A NICHE CUTS DOWN ON MEMORIZING ALL THIS :)

Temperature (cont…)Mesophiles: “moderate” temperaturesInclude human pathogensMakes sense because our body is 37C which is quite moderate….

Physical Factors: pHNeutrophiles pH 5.5-8.0Acidophiles (<pH 5.5)Alkalophiles (>pH 5.5)

Like most organisms, all of the above are near neutral (~7.4 pH)..however clearly they live in different pH Environments. How is this done?

• H+ pumps (proton pumps) to maintain internal pH

• Chaperonins made for protection(“acid shock proteins” )

• Waste products affecting environmental pH

Acidithiobacillus ferrooxidans

Physical Factors: Osmotic PressureOsmosis: Water diffuses from a region of high water concentration to a region of lower water concentration (high [solute]).

Hypotonic soln.: osmotic lysis (movement of water in) Hypertonic soln.: plasmolysis (movement of waterout)Isotonic: no net movement (but there still is movement…)*Adaptation of osmotolerant organisms: uptake of compatible solutes

Hypotonic Hypertonic

Extreme Halophiles

• Extreme halophiles = require salt

• Have high intracellular [K+] or [Na+]

• Found in:–Marine environments–Great Salt Lake, Utah

Physical Factors: O2 Concentration-O2 can come in very reactive forms which are toxic to all living things ex) H2O2, O2 radical

Mechanisms:• Superoxide dismutase

–Removes superoxide radicals–2 O2

-• + 2 H+ H2O2 + O2

• Catalase/Peroxidase–Remove hydrogen peroxide

–Catalase reaction: 2 H2O2 2 H2O + O2

–Peroxidase reaction: H2O2 + 2 H+ 2 H2O

“Making it make sense”- Obligate aerobe NEEDS O2. So clearly it has the mechanisms to tolerate and uses it as part of its metabolism- Facultative clearly prefers. So it has the mechanisms to tolerate and likely benefits from aerobic respiration- Aerotolerant has the mechanims but likely does not use it in metabolism- Strict anaerobe likely lacks the mechanisms and therefore cannot tolerate O2- Microaerophile is more tricky. It probably has the mechanisms to survive, and likely relies on O2 as part of its

metabolism but in limiting quantitiesOf course the above things I stated are likely not the type of depth questions on the test, but may help to understand the material.

needsoxygen

prefersoxygen

ignoresoxygen

hates oxyge

n

2 – 10%oxygen

Common Nutrient Requirements

• Macroelements (macronutrients)• CHOPKINS Ca Fe Mg

• Trace elements (micronutrients)• NiMo ‘CuMnZn’ Co

– (NiMo ‘CuMnZn’ when its Cold)

Special Nutrient Requirements• Organic growth factors (

– Factors NEEDED for growth that body cannot synthesize

Classes of Growth Factors

• Amino acids

• Purines and Pyrimidines

• Vitamins (cofactors for enzymes)

Uptake of Nutrients by the Cell

• Some nutrients enter through:-passive diffusion (no energy)

• H2O, O2, CO2 small molecules

• Most nutrients enter by:– facilitated diffusion– active transport– group translocation

Ex: of facilitated diffusion (permeases) Ex: of active transport (ABC

transporters)

Binary Fission in Bacteria

Bacterial Growth Curve in a Closed System

Bacterial growth in a CLOSED systemLag phase: adjustment to new media, preparation before growth

Exponential phase: maximal growth, constant growth rate (“doubling..doubling..and again..and again”) LINEAR relationship between (log (# of cells) and time

Stationary phase: death rate = growth rate. Maximal population size. Competition for resources peaks population size.

Death phase: nutrient depletion/waste accumulation results in death of population (like the log phase it is constant..”halving..halving..”)

Exponential Phase Parameters

Number of generations (n) (number of “doublings” during a period of time)Mean growth rate constant (k) (generations per hour)Generation doubling time (g) (time required to double population size)

How can you measure bacterial growth?

Direct countsCounting chambers

• Caveat: can’t distinguish living cells from dead

Plate countsSerial dilution, filtrationIndirectTurbidimetric measures

• More light scattered = more dense amount of microbes

Media compositions that help identify microbes

Differential “differentiates” between microbesEx: Blood agar (streptococcus) we can see secretions of hemolysins Selective promotes/inhibits growth of an organism over anotherEx: MacConkey Agar: Dye or LactoseEnrichment is used in isolation of particular organismsEx: use media without fixed nitrogen source to isolate nitrogen fixing bacteria

Lecture 4. How can we control Microbes? Sterilize-kills everything!~spores,

viruses, etc.. Disinfection-only kills pathogens (i.e.

disease causing microbes Sanitation-lets just make it safe Antisepsis- kill/inhibit pathogens that

are tissue related

Is our method killing or inhibiting microbes? Bacteriocidal methods- cause microbe death

by taking away the microbes ability to reproduce

Bacteriostatic methods- halts/inhibits/does not kill microbes-microbes reproduction is still possible.

Random things to know about Microbe Death It is exponential just like growth Microbes are only dead if they can no

longer reproduce Vegetative (normal cell) vs. endospore-

endospore is always hardest to kill

Factors affecting Antimicrobials Population size-more=more work Population composition-spores=more

work Duration of exposure-longer

duration=more effective Concentration or intensity of agent-more

of antimicrobials=better job of killing Temperature-high temp=more effective Local environment-organic matter-turkey

stuffing vs NaCl solution?

What areas do our antimicrobials attack in the microbe? Membrane-leakage of cellular

contents/nutrients, and entry of toxins Denature DNA and RNA Both will stop microbe

reproduction=Death

How can we kill Microbes?

Physical methods and Chemical methods 1st Physical Method= Heat

Bacteriocidal, Rules: 60-70˚=kills most microbes

120˚ and higher=sterilization (spores)

Dry heat vs. Moist heatDry heat:

1) Hot even 2) Direct Flame

Moist Heat

Boiling- Good for vegetative cells, not spores Autoclave- uses steam under pressure

Sterilizes/kills spores at 121˚

• Pasteurization Kills only pathogens, mainly used for food.

How can we measure our method’s effectiveness? Thermal death time (TDT)- time it takes to

kill all microbes at a certain temperature Decimal Reduction Time (D):

Time required to kill 90% of microbes in sample at a specific temp

Z Value-increase in temperature required to reduce D value to 1/10

Next Physical Method: Filtration

Filtration- the problem with heat is that is kills vitamins, amino acids, andtiobiotics..How can we sterilize those things then?

We filter

Low Temperature/Freezing

Low temperature MethodsBacteriostatic-ice crystals are indirect

killingRefrigeration and Freezing

-Lack of liquid water

Next physical method: Radiation

Weaker Radiation: Non-Ionizing Radiation It is UV light that only penetrates surfaces

Example- Would not penetrate bacteria culture, only the surface. Good for metal surfaces, cabinets, not good for glass or water

Stronger Radiation: Ionizing Radiation Uses X/gamma rays-deeply penetrates all surfaces generates e-, OH-, and H● Better for spores

Deinococcus radiodurans

Bacteria that is super resistant to RadiationWas found still living in spoiled meat after

shooting canned food with gamma raysHas multiple copies of DNA and DNA repair

mechanisms

Last Physical Methods

Dessication-Drying, used in foods Osmotic Pressure-

High concentration of sugars/salts

Chemical methods of Control:

List of chemicals in lecturesPhenol, Alcohol, Halogens, Heavy Metals,

Detergents, Aldehydes, Sterilizing Gases

How can we measure how good are chemical methods are? Phenol Coefficient-phenol is the standard test agent you

compare all other agents with. How good is your agent compared to phenol?

Lecture example-Max activity of Agent A: 1/450Max activity of Phenol: 1/90

-PC = 450 / 90 = 5 PC > 1 Agent is more effective than phenol

Dilution Tests-can agent A kill as many microbes as agent B at a lower concentration?

Random Stuff from Lec. 4

To sterilize a space craft use a dry heat baking method. It kills microbes that might survive in high pressure/space environment.

Mars might have methane on it produced by microbes?

Lecture 5.Different types of Microbes Know these associations: 1)Chemo=Chemical 2) Organo=organic 3) Litho=Inorganic

4)Photo=Light 5) Auto=inorganic 6) Hetero=organic Order of naming= Energy Electron Carbon• Chemoorganotrophic heterotrophy= Chemical

Energy Source, Organic Electron Source, and Organic Carbon Source

• Use this pattern for all types of microbes.

What are those sources?

Example of chemical sources of ENERGY: - organic: glucose inorganic: H2S

Examples of electron sources- organic: glucose Litho/Inorganic:

Fe2+, H2

Examples of carbon sources- organic: Glucose inorganic:

CO2

Continued…

• Chemoorganotrophic heterotrophy• -pathogenic microbes

• Chemolithotrophic Autotrophy• -Nitrifying Bacteria, S and Fe oxidizing

bacteria• Photolithotrophic Autotrophy

• -Cyanobacteria, algae• Photoorganotrophic Heterotrophy

• -Non-sulfur bacteria

Metabolism What are the nutrients microbes need in

large quantities? ~ The macro nutrientsCHOPKINS CaFe Mg

No iodine~

What are the nutrients microbes require in small amounts?~ The micro nutrientsMNMCCZ- you decide on knowing the

enzymes they’re associated with

Metabolism…

Metabolism=all chemical reactions in an organism Catabolism-break down large molecules to small

molecules-releases/gains energy Anabolism- put together small molecules to male big

molecules-requires energy How is energy made?

Use sun (photo) or chemical compounds (chemo). For chemical compounds, you oxidize the reduced chemical source of energy (glucose) so that electrons from glucose can be stored in a reduced compound like NADH. Then electronsE.T.C.ATP/Energy

Glucose(reduced) + NAD+(oxidized) Glucose(oxidized) +NADH(reduced)

Overview

Aerobic Respiration- Electron Donor (inorganic or organic)gets stripped of electrons (oxidized) and Oxygen (exogenous**) accepts these electrons at end of ETC.

Anaerobic Respiration Same thing as above except*** Oxygen does not accept

the final electrons, but rather nitrate, sulfate, CO2, or fumurate (exogenous**)

Fermentation-completely different. It is anaerobic (don’t need O2) but its purpose is to regain certain molecules (NAD+)

Stages of Catabolism

1. All the big moleculessmall moleculesProteins, fats, carbsmonomers or most broken down form

NO energy gained from this process! 2. All the monomersAcetyl-CoA

Glycolysis stage, getting ready for Krebs Cycle We have gained ATP, NADH, and FADH2!

3. Krebs Cycle, ETC. By the end of 3, all electron/energy sources are oxidized and we gain CO2, ATP, NADH and FADH2!

Catabolic Stages 1-3

Gycolysis

Goal: take monosacharides like glucose and oxidize them. In the process we produce the reduced compounds NADH (stored energy), and ATP (energy).

Occurs in cytosol Strictly Catabolic. Is Anaerobic-doesn’t require O2 but O2 doesn’t

harm the process. Starts with glucose (6 C), ends with pyruvate (3

C)

Points to remember about glycolysis Prep. Phase-we put in 2 ATP.

This happens at step 1: glucoseG-6-P and at step 3: F-6-P F-1-6-BP.

Remember we put in 2 ATP before we break into 2 G-3-P’s.

Pay off phase- we gain back 2 NET ATP. These occur after we have broken into 2 G-3-P molecules and gain back 2 ATP’s per pyruvate (we end with

two). - They occur at 1,3 BPG3-P-G, and at

PhosphoenolpyruvatePyruvate.- - There is also one reduction (per G-3-P) of NAD+NADH

More Glycolysis..

Substrate-level Phosphorylation- take a high energy organic substrate molecule that has a Phosphate, and exergonically break it down by transferring the phosphate to ADP which gives ATP.

glucose + 2ADP + 2Pi + 2NAD+

2 pyruvate + 2ATP + 2NADH + 2H+

Pentose-Phosphate Pathway

Both Anabolic and Catabolic Goal: Lets start like glycolysis and

continue like glycolysis if we need ATP (the catabolic part), or if we don’t need ATP lets make amino acids or nucleotides (the anabolic part). How do we do that?

Pentose Phosphate pathway continued… So we start with G-6-P, just like the

starting of glycolysis. BUT** Instead all we do is go from G-6-P to Ribulose-5-P and produce 2NADPH’s along the way. (oxidative phase)

The important part: Ribulose-5-P=precursor for nucleotides (anabolic)

Continued..

Then, R-5-P undergoes transaldolase nad transketolase reactions that produce 3-7 carbon sugars. Some of them are things like F-6-P and F1-6-BP like in

glycolysis. Other random 3-7 carbons are made thatAmino Acids

(Anabolic). ~NADPH=electron source The sugars from glycolysis made from Pentose pathway

can all convert back to the orignal G-6-P and go into glycolysis (catabolic).

The Entner-Doudoroff Pathway Yield per glucose molecule:

1 ATP1 NADPH1 NADH

Just a combination of both pathways. Ends with pyruvate

Summary

Pathways Endproducts Glycolysis Pyruvate

-catabolic

Pentose phosphate Various sugars: 3-7 C -catabolic/anabolic

Entner-Doudoroff Pyruvate-catabolic/anabolic

PyruvateAcetyl CoA End of Stage 2 Pyruvate + CoA + NAD Acetyl-CoA + NADH2 + CO2

Pyruvate Dehydrogenase All aerobic microbes

• Pyruvate + CoA + 2 Fd Acetyl-CoA + 2 FdH + CO2 Pyruvate:Ferredoxin-Oxidoreductase

-All anaerobic glucose metabolism-Clostridium during anaerobic glucose metabolism

• Pyruvate + CoA Acetyl-CoA + Formate Pyruvate Formate Lyase E. coli during anaerobic glucose metabolism

Maint point:pyruvate is oxidized to Acetyl Coa

Fermentation

This is an anaerobic process. Purpose: we take pyruvate (endogenous

electron receptor) and reduce it but in the process we oxidize NADHNAD+. This is good because we need NAD+ in order for glycolysis to run. So when fermentation occurs, it keeps glycolysis running without any O2 needed.

Small amount of ATP forming, but still good under no O2 conditions.

Pyruvate Acetaldehyde + CO2

Pyruvate Decarboxylase

Alcoholic Fermentation

Acetaldehyde + NADH Ethanol + NAD+

Alcohol Dehydrogenase

Homolactic fermentation:Pyruvate + NADH Lactate + NAD+

Lactate DehydrogenaseExample organism: Lactic acid bacteriaCommercial uses: YogurtAlso - involved in tooth decay

Lactic Acid Fermentation

Formic Acid Fermentations“useful in identification in members of the Enterobacteriaceae”

Mixed acid fermentation

FormsFormic acid (CO2 + H2)EthanolAcetic acidLactic acidSuccinic acid

Ex: E. coli

Formic Acid Fermentations“useful in identification in members of the Enterobacteriaceae”

2,3 Butanediol Fermentation

Forms 2,3 butanediolEthanol

Ex: Enterobacter

Using Fermentation Endproducts to Identify Bacteria

Methyl Red test: Measures acidity of fermentation endproducts

Mixed acid - produces acidic endproducts2,3 butanediol - mostly neutral

endproductsPos. methyl red - acid endproducts (red)Neg. methyl red - neutral endproducts

(yellow)

Fermentations of amino acids

Stickland reaction:

oxidation of one amino acid with use of second amino acid as electron acceptor (gets reduced)

TCA Cycle/Krebs Cycle

Acetyl-Coa from stage 2TCA cycle Stage 3 A 4 carbon intermediate oxalloacetate picks up

A-CoA to make a 6 Carbon molecule. Redox reactions happen, decarboxylations

happen, and one substrate Phosphorylation occurs and we end back up with oxalloacetate.

4 C6C5C4C Our products=3 NADH, 2 CO2, 1FADH2, and 1

GTP (ATP).

TCA continued…

This is aerobic mainly, but remember we could have anaerobic respiration.

Note***- all carbons from glucose are lost to CO2 by the end of TCA. GlucosepyruvateA-CoaTCAall lost as CO2.

All are oxidations. Catabolic and Anabolic

TCA intermediates used for Amino Acid synthesis. Occurs in Mitochondria In Eukaryotes. What

about prokaryotes?

Electron Transport and Oxidative Phosphorylation

Only 4 ATP molecules synthesized directly from oxidation of glucose to CO2Most ATP made when NADH and FADH2

(formed as glucose degraded) are oxidized in electron transport chain (ETC)

2 ATP

2 ATP

Electron Transport Chain

So we have made 3NADH and 1FADH2, and 2 NADH from glycolysis. Why?

Continued..

Electron flow goes from from carriers with more negative E0 (NADH) to carriers with more positive E0 (O2)

NADH, and FADH2 get oxidized by cytochrome enzymes, meaning the cytochromes get reduced.

Coenzyme Q & Cytochrome c connect the 4 complexes and carry the electrons between complexes which reduces each cytochrome on the way,when they pass through the last cytochrome, the electrons are given to oxygen to form water.

Summary:Electron transport chain…

electrons transferred energy released

released energy used to make ATP by oxidative phosphorylation

3 ATP molecules made per NADH using oxygen as acceptor

2 ATP molecules per FADH2

The Pasteur Effect

Facultative microbes have both fermentation and aerobic respiration

With O2 - Consumes less glucose

Without O2 - Consumes more glucose

Anaerobic Respiration

ETS uses electron acceptor other than O2

Ex: Nitrate (NO3-)

Sulfate (SO42-)

Carbon dioxide (CO2)

Where do these bugs live??Swamps, soil sediments, intestinal tracts

The Electron Transport Chain of E. coli with high O2 concentrations

The Electron Transport Chain of E. coli with NO3

-

Not as much H+ as aerobic resp. but still better than fermentation

The Electron Transport Chain of Pseudomonas stutzeri with NO3

-

Anaerobic Respiration

Sufate reduction (Sulfide)SO4

2- H2S

MethanogensCO2 CH4

Chemolithotrophs

Specifically talking about the Electrons*** for ETC obtained from inorganic molecules

Ex: H2, ammonia (NH4+), H2S, CO, Iron

However, even with inorganic Electron sources, Carbon sources may still be inorganic (autotrophic) or organic (heterotrophic)

Chemolithotrophs

Hydrogen oxidizing bacteria:

H2 2H+ + 2e-

Hydrogenase (Ni and/or Fe)

AlcaligenesStreptomyces thermoautotrophicus

Chemolithotrophs

Nitrifying bacteria:Oxidize ammonia to nitrite or Nitrate (Nitrification).

NH4+ + 1 1/2 O2 NO2

- + H2O + 2H+

Nitrosomonas

NO2- + 1/2 O2 NO3

-

Nitrobacter

Chemolithotrophs

CO oxidizing bacteria:

CO CO2

Streptomyces thermoautotrophicus

Oligotropha carboxidovorans

Carboxydothermus hydrogenoformans

Fe2+ oxidation by Acidithiobacillus ferrooxidans

(Cu)

Photosynthesis in Eukaryotes and Cyanobacteria

Have chlorophyll and other pigments

Use 2 photosystems (noncyclic) Electron source: H2O Produce ATP, NADPH Produce O2 (oxygenic

photosynthesis)

Photosynthesis in Green and Purple Bacteria or Prokaryotes Have bacteriochlorophyll Use 1 photosystem: Cyclic Electron source: H2S, S, H2 Produce ATP Do not produce O2

(anoxygenic photosynthesis)

More principles…

Catabolic and anabolic pathways are not identical, despite sharing many enzymes

permits independent regulation

Carbon Assimilation (Carbon fixation)

CO2 is needed which is incorporated via Calvin-Benson cycle- process that makes organic carbon.

Energy source:photoautotroph - lightchemolithotrophs - inorganic chemicals

Product:organic carbon needed by heterotrophs

Carbon fixation..How it works

Stage 1. Carboxylation phase RuBPcarboxylate it by Rubisscoforms two 3-PGA’s.

Stage 2. Reduction phase Take two 3-PGA two G3P’s by using 2ATP’s (1

per 3PGA) and gaining 2 NADPH (1 per 3-PGA) Stage 3. Regeneration- take 1 G3Pback to

RuBP by using one ATP and*** the 2nd G3P produces carbon molecules like glucose/fructose (need 6 cycles)

Incorporation of 1 CO2 into organic material costs 3 ATPs and 2 NADPHs

Nitrogen Assimilation

Nitrogen needed for protein, nucleic acid, and coenzyme synthesis

Incorporated in Prokaryotes fromAmmoniaNitrateAtmospheric N2 (Nitrogen fixation)

How does Ammonia as a good nitrogen source for proteins, DNA etc.. Prokaryotes do this to make amino acids. They take NH4+alpha KG+

Glut.DHGlutamatetake NH4 and transfer Alpha keto acid to make amino acid by transaminase.

The NH4 we start with gets oxidized/reduced? (Hint: NADPH is used as an electron source)

This is Ammonia Assimilation.

How does Nitrate helps give useful Nitrogen to the Prokaryote? The useful form of nitrogen is in the form of

NH3~ammonia. Nitrate can be used, but** it is in a really

oxidized form. It must be reduced all the way down to Ammonia (super reduced) by Nitrate Reductase. Then it can be used by the prokaryote.

This is Assimilatory Nitrate Reduction

How does N2 give us useful nitrogen. This is Nitrogen Fixation! N2 + 8H+ + 8e- + 16 ATP Nitrogenase 2 NH3 + H2 + 16 ADP + 16 Pi Energy requirement: Very high Widespread among microorganisms

Rhizobium - lives as symbiont with plants (legumes)

Cyanobacteria Nitrogenase reaction is anaerobic

Is inhibited by O2

N2-fixing System of Streptomyces thermoautotrophicus

What else do prokaryotes do with Nitrogen Denitrification

NO3- & NO2

- converted to N2

NitrificationConvert NH3 to NO3

- & NO2-