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Microbiology lecture

Feb 17, 2017

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Page 1: Microbiology lecture

Ilana Kovach

Page 2: Microbiology lecture

Cell Structure

& Function

Chapter 3

Page 3: Microbiology lecture

Prokaryote EukaryoteDNA: In Cytoplasm

of cell

Organelles: Have

Ribosomes

Size & Organizations:

Single celled

Domains/ Kingdoms:

Archaea & Bacteria

DNA: In nucleus linear

chromosomes

Organelles: Membrane

Organelles

Size & Organizations:

Bigger more Organized

Domains & Kingdoms:

Eukarya

VirusesCannot Reproduce

Outside Cells

No Metabolism

outside Host cell

RNA or DNA never

Both

Debate living or non

living

Page 4: Microbiology lecture

Arrangement of Prokaryotic Cells

Cocci multiple Planes Bacilli single Planes

Coccus Diplococci Staphylococci

Streptococci Sarcina

Tetrad

Coccobacillus Bacilli

Diplobacilli Palisades

Streptobacilli

Page 5: Microbiology lecture

3 main Prokaryotic Cell shapes

Cocci

Spirals 1. Spirochetes Flexible

2. Spirillum Inflexible

Rods “Bacillis” Coccobacillus “Short Rod”

Vibrio “Curved Spiral”

Pleomorphic“Does not Have a shape”

Page 6: Microbiology lecture

External Structures of Bacterial Cells

Glycocalyx Made from Polysaccharides, polypeptides or Both*Function: protect cells from drying

Capsule firmly Attached

*Functions: Protection & Virulence Factor

Slime LayerLoosely attached (Water soluble)

*Functions: Protection & Aattachment Biofilms

Flagella

Structure

*Filament “Flagellin”rotate 360º

*Hook

*Basal Body

Arrangements*Amphitrichous “Axial Filament” Cork screw shapeVirulence Factor

*Peritrichous“Flagella everywhere”

*Monotrichous “Polar”

*Lophotrichous “Tuft”

FunctionRotate 360º “Boat propellers”

(H+) or (Na+) power “Counterclockwise or clockwise”

Runs (+)Tumbles (-)

Fimbriae & Pili

FimbriaeStiff vessels “Velcro” Virulence Factor helps attach

*think Fringe

Cranberry Juice E coli fimbriae can’t attach

Pili ˜Typically one-Few

*Regular ‘Pseudo Movement”*Conjugation= Process used to transfer from one DNA to another (Resistance to Antibiotic)

Page 7: Microbiology lecture

Bacterial Cell Walls

PeptidoglycanNAM (N-acetylmuramic acid)NAG (N- acetyl glucosamine)*attached by Tetrapeptide Cross Bridge

Gram Negative Thin Layer of Peptidoglycan + outer membrane

Periplasmic Space: Between inner & outer Membrane contains

peptidoglycan & periplasm

Outer Membrane (Envelope)

-Lipopolysaccharides/Endotoxin (Lipid + Sugar)

Lipid A

released from dead/damaged cells

may trigger fever, vasodilation, inflammation, shock,

(DIC)

-Porins

-other Proteins

Gram Positive Thick Layer of Peptidoglycan

Teichoic acids & lipoteichoic acids present

Acid Fast Cells Mycolic Acid “wax/hydrophobic”

Resistance

Special Staining Procedure

Page 8: Microbiology lecture

Gram Positive Gram Negative

Teichoic Acid

Lipoteichoic Acid

Peptidoglycan Layer (cell wall)

Cytoplasmic Membrane

Integral Protein

Peptidoglycan Layer of (cell wall)

Cytoplasmic Membrane

Outer Membrane of Cell

Porin Periplasmic Space

Lipopolysaccharide Layer(LPS) Containing Lipid A

Page 9: Microbiology lecture

Bacteria Without CELL

WALLs

Mycoplasma Sterols in Cell Membrane

Chlamydia Cell membrane + outer membrane (no

Peptidoglycan)

Page 10: Microbiology lecture

Bacterial Cytoplasmic Membrane

2 Main

Components:

1. Hydrocarbon Tails

“Hydrophobic”

2. Phosphate Heads

“Hydrophilic”

3 Main Functions:

1. Selectively Permeable

2. Energy Production (PMF)

3. Photosynthesis

(Photosynthetic Prokaryotes

Passive

Transport*Diffusion

*Facilitated Diffusion

*Osmosis

Active

Transport *Requires the cell to expend

ATP

*Active Transport

*Group Translocation

Page 11: Microbiology lecture

Cytoplasm in Bacteria

Cytosol Inclusions“Pockets of Chemicals”

Ribosomes“Protein Sythesis”

Prokaryotes 70s (Subunits made of 50s & 30s)

*S= Svedberg unit

Eukaryotes 80s

Cytoskeleton “Framework”

Page 12: Microbiology lecture

Membrane Bound Organelles in Eukaryotes

Nucleus, Endoplasmic

Reticulum

Golgi Bodies

Lysosomes

Peroxisomes

vacuoles

Vesicles

Mitochondria

Chloroplasts

Page 13: Microbiology lecture

Mitochondria &

Chloroplast in common

with Prokaryotes?

Both have double membranes and their own DNA Both

reproduce by Binary Fusion (which is also how bacteria

reproduces) It is theorized that those are the two

organelles that took part in the

endosymbiotic theory.

This is because they both have their own DNA, divide

separately from the cell, and look very similar to

bacteria's. According to the theory, they were once free

living organisms that started a mutualism with an early

eukaryotic cell, and they've been inseparable ever since

Page 14: Microbiology lecture

Arrangement of Prokaryotic Cells

Cocci multiple Planes Bacilli single Planes

Coccus Diplococci Staphylococci

Streptococci Sarcina

Tetrad

Coccobacillus Bacilli

Diplobacilli Palisades

Streptobacilli

Page 15: Microbiology lecture

Endospore FormationStep 1: DNA replicated

Step 2: DNA aligns Cells long Axis

Step 3: Cytoplasm Membrane Invaginates to form forespore

Step 4: Cytoplasmic Membrane Grows and Engulfs Forespore within a second membrane. Vegetative cell’s

DNA disintegrates

Step 5: A cortex of calcium & dipliconic acid is deposited between the membranes

Step 6: Spore Coat Forms around the endospore

Step 7: Maturation of endospore; completion of spore coat & increase in resistance to heat and chemicals by

unknown processes

Step 8: Endospore Released from original cell

Page 16: Microbiology lecture

Endospores (Bacillus & Clostridium)

Outer Spore Coat (Exosporium)

Outer Spore Coat

(Exosporium)

Spore Coat

Outer

Membrane

Cortex

Spore Core

Inner

Membrane

Page 17: Microbiology lecture

Prokaryotes

Reproduction

1) Binary Fission

Step 1: Cell Replicates its DNA

Step 2: Cytoplasmic Membrane Elongates Seperating DNA molecules

Step 3: Cross wall forms Membrane Invaginates

Step 4: Cross Wall forms completely

Step 5: Daughter Cells

2) Snapping Division Palisades (V-shapes)

Page 18: Microbiology lecture

Rules for Naming Bacteria

Genus + Species

Italicize or underline

1st name Capital Letter

Page 19: Microbiology lecture

Microbial

Metabolism

Chapter 5

Page 20: Microbiology lecture

Cellular Respiration

Pathway ATP produced

ATP Used

NADH produced

FADH2

Glycolysis 4 2 2 0Synthesis of Acetyl-CoA & Krebs Cycle

2 0 8 2

Electron Transport Chain

34 0 0 0

Total 40 2Net Total 38

Alternative Pathways

ATP produced

Electron Carriers

Products

Pentose Phosphate

1 ATP 2 NADPH 5 Carbon Precursor metabolites

Entner-Doudoroff

1 ATP 2 NADPH Other precursor metabolites

Alternate pathway’s

Page 21: Microbiology lecture

Kreb Cycle

Cellular Respiration

Glucose

C02

Pyruvic Acid

Acetyl CoA

Glycolysis

22

2

22

NADHFADH2

C02

Electron Transport

Chain

4

62

2

Ubiquinone's Cytochromes

Metal containing Proteins Flavinones

e-

Proton GradientATP

synthase 34

Final Electron Acceptor

Fumarate

Nitrate

Sulfate

Oxygen

Fermentation

NADH

NAD+

Ethanol

C02

+

Lactic Acid

Acetic Acid

Decarboxylation

Goes TO

oxidized

Main PURPOSE

Anaerobic

Aerobic

H+

Energy From e-Transfer Pumps & H+ across Membrane

Protons re-enter through channel

Page 22: Microbiology lecture

5. DHAP rearrange to form G3P

Energy Investment Stage

C C C C C CGlucose

C C C C C CGlucose 6- Phosphate

P

CP PC C C C CFructose 1, 6- Bisphosphate

ADP

ADP

Lysis Stage

Fructose 1, 6- Bisphosphate

P PC C C C C C

CCC

Dihydroxyacetone Phosphate (DHAP)

P

1. Glucose (Substrate- Level Phosphorylation)

2. Glucose Molecules Rearranged for form fructose 6 phosphate

3. Fructose 6 phosphate Phosphorylated to form Fructose 1, 6-Biphosphate

4 . Fructose 1, 6-Biphosphate splits to form (DHAP & G3P)

Glyceraldehyde 3 Phosphate (G3P)

C C CP C C CP

Page 23: Microbiology lecture

Energy Conserving Stage Glyceraldehyde 3 Phosphate (G3P)

C CCP P C CC

P2NAD+2NADH2

C CCCCCP P P P

STEP 6: 2 Inorganic phosphates are added to (G3P) & 2 NAD+ are reduced to NADH

Two 1, Phosphoglyceric Acid

ADP

22

Two 3 Phosphoglyceric Acid

CCC P C C C P

STEP 7: 2 ATP are phosphorylated by substrate level to form 2 ATP

H202

C C C C C C

PP

Two Phosphoenolpyruvic Acid (PEP)

STEP 8 & 9: Remaining Phosphates moved to middle carbon & water removed from each substrate

ADP2

2

C C C C C C

STEP 10: 2 ATP are phosphorylated by substrate level to form 2 ATP

Two Pyruvic Acid

Page 24: Microbiology lecture

Formation of Acetyl-CoAC

C

C

Pyruvic Acid

OOH

C02

C

C H

Acetate

NADHNAD+

CoA

Acetyl-CoA

Decarboxylation

C

C

CoA

Fermentation

C

C

C

C02

Pyruvic Acid

Lactic Acid

NAD+

NADH

Acetaldehyde

NADH NAD+

Ethanol

Page 25: Microbiology lecture

Krebs Cycle

Acetyl-CoA

CoA

C

C

C

C

C

CC

C

C

OOH

OOH

OOH

Citric Acid

1 2

CoA

C

C

C

C OOH

OOH

OOH

C

IsoCitric Acid

NADH

NAD+

C02+3

C

C

C

C

C

OOH

OOHα-ketoglutaric Acid

NADH C02+C

C

C

C

CoA

Succinyl-CoA

OOH

ADP

GDP

Acetyl-CoA

CoA

CoA

FAD+

4

5

C

C

C

C OOH

OOH

Succinic Acid

6

C

C

C

CHOO

OOH

Fumaric Acid

FADH2

7

H20

8C

C

C

C

Malic Acid

OOH

OOH

C

C

C

C

OOH

OOHOxaloacetic Acid

NADH

Start Here

Page 26: Microbiology lecture

H20

Electron Transport Chain

Flavoproteins Ubiquinone

Cytochromes (b) Cytochromes (c)

NAD+

NADHFADH2

FAD+

H+

++

H+H+

e- e-

e-e-

e-e-

H+

H+

H+H+

H+H+

H+H++

+

+

+ +

+

+

+

+

-

-

-

-- -

-

-

-

-

𝟏

𝟐o

e- e-

ATP synthase

ADPP+

H+

H+

H+ H+

1

2

4

3

Page 27: Microbiology lecture

Microbial

Growth &

Nutrition

Chapter 6

Page 28: Microbiology lecture

Light (Photo-) Chemical Compounds (Chemo-)

Carbon Dioxide (Auto-)

Photoautotrophs

Plants Algae & Cyanobacteria use H20 to reduce CO2, Producing O2 as a by-product

Green sulfur Bacteria and Purple sulfur bacteria do not use H20 nor O2

Chemoautotrophs

Hydrogen, Sulfur and Nitrifying bacteria, some archaea

Organic Compounds (Hetero-)

Photoheterotrophs

Green Nonsulfur Bacteria and purple nonsulfure Bacteria, Some Archaea

Chemoheterotrophs

Aerobic Respiration: Most animals, fungi and protozoa and many bacteria

Anaerobic Respiration Some animals, Protozoa, Bacteria, and archaea

Fermentation; Some bacteria, Yeast and Archaea

Energy SourceC

arb

on

Sou

rce

Page 29: Microbiology lecture

Lag Stage

Log Stage Stationary Stage

Death Stage

Microbial Growth Curve

Page 30: Microbiology lecture

Toxic forms of Oxygen Neutralizing Toxic forms of

Oxygen

Singlet Oxygen Formed during photosynthesis as electrons are boosted to higher state

Photosynthesis organisms Contain Carotenoids

Superoxide

Anion

Produced by incomplete reduction of oxygen during respiration

Superoxide Dismutase (Enzyme)

Peroxide

Anion

Formed when superoxide anions are neutralized Peroxidase and/or Catalase (Enzymes)

Hydroxyl

Radical

Formed from ionizing radiation& incomplete reduction of hydrogen peroxide

Peroxidase and/or Catalase Antioxidants

Page 31: Microbiology lecture

Direct MethodsViable Plate count: Lot of Bacteria; Dilute low enough to count serial Dilutions ~ (30-300); Live Only

Membrane Filtration: Bacteria cannot go through filter; therefore filter retain cells ~Live Only

Microscopic Count: special slide & coulter counter; Does not distinguish Dead or alive

Indirect MethodsMetabolic Activity: nutrient consumption & Waste Production (Color change); Live only

Dry Weight: Desiccated (dried) waste weighed; does not distinguish dead or alive

Turbidity: More turbid; Use spectrophotometer to measure absorbance of more bacteria; Does

not distinguish alive or dead

Page 32: Microbiology lecture

Obligate (Strict) Aerobes

Requires O2

Undergo Aerobic Respiration Oxygen final Electron Acceptor

Microaerophil Require lower O2 levels (2-10%)

Undergo Aerobic Respiration Limited ability to detoxify hydrogen peroxide and superoxide Radicals

Facultative (Facultative Anaerobes)

Can Live either Presence OR absence of O2 (Prefer Oxygen)

Aerobic Respiration Anaerobic Respiration Fermentation

Aerotolerant Can live in either Presence or Absence of Oxygen (Doesn’t Prefer Oxygen)

Never use Aerobic metabolism Have enzymes that neutralize toxic oxygen

Obligate (Strict) Anaerobes

O2 is Deadly

Use anaerobic Metabolism Lack Enzymes to neutralize toxic oxygen

Oxygen Requirements

Page 33: Microbiology lecture

PsychrophilesCold ~ (-5- 20º); optimal: 10º

MesophilesModerate ~ (15º-45º); optimal: 37º

ThermophilesHot ~ (Low 40’s – 80’s)

Hyperthermophiles HOT!! (60’s and Above)

Page 34: Microbiology lecture

Osmotic Pressure (Isotonic, Hypertonic, Hypotonic)

Hypertonic Solution: No Cell Wall Crenate

Cell Wall Plasmolyzed

Hypotonic Solution: No Cell Wall Bursts or Lyses

Cell Wall Rigid Enough to Hold

*Salt Meat, Sugar Jelly, Pickles never Spoil

Required for Metabolism

Two physical effects of water: Hydrostatic & Osmotic Pressure

Hydrostatic Pressure Barophils (Extremophile) - Organisms that live under Extreme pressure

Membranes & Enzymes depend on Pressure to maintain their 3-D

functional Shape

Classification Factors

Page 35: Microbiology lecture

Other Classification Factors

Salt Concentration Halophiles-able to live in High salt concentrations; Dead Sea

Obligate Halophiles – Require Salt up to 30% concentrations

Halotolerant- Able to tolerate high salt concentration; Sweat on skin

pH Neutrophils (6.5-7.5) Most Microbes

Acidophils (Acidic habitats)

Alkalinophils (Alkaline Habitats up to 11.5)

Capnophils – Thrive in presence in areas of high concentration of CO2

Page 36: Microbiology lecture

Microbial

Genetics

Chapter 7

Page 37: Microbiology lecture

DNA Replication DNA RNA1. Phosphate2. Deoxyribose Sugar3. Nitrogen Base

1. Phosphate2. Ribose Sugar3. Nitrogen Base

4 Bases: (A) adenine (T) thymine (G) guanine (C) Cytosine 4 Bases: (A) adenine (U) Uracil (G) guanine (C) Cytosine

Hydrogen Bond Adenine & Thymine (2 bonds) Guanine & Cytosine (3 bonds)

Single Stranded!!!

Transcription Translation

Page 38: Microbiology lecture

Plasmids

Fertility Plasmids:

Pili Conjugation

Plasmids are small molecules of DNA that replicate

independently of the chromosome.

Resistance Plasmids: Penicillin, Antibiotics

Cryptic Plasmid:

I don’t know Virulence Plasmids:

Toxin, Attachment Protein

(Cause disease)

Bacteriocin Plasmids:

Antagonistic kill other

Bacteria

Page 39: Microbiology lecture

Horizontal Gene Transfer

Conjugation in genetics: method of horizontal gene transfer in which a bacterium containing a fertility plasmid forms a conjugation pilus that attaches and transfers plasmid genes to a recipient; in reproduction of ciliates: coupling of mating cells

Transformation Method of horizontal gene transfer in which a recipient cell takes up DNA from the environment. (Dead Organisms) Griffiths with Streptococcus Pneumoniae

Transduction Method of horizontal gene transfer in which DNA is transferred from one cell to another via a replicating virus.

Horizontal Gene Transfer: Process in which a donor cell contributes part of its genome to a

recipient cell, which may be a different species or genus from the donor.

Page 40: Microbiology lecture

F+1. Donor cell attaches to a recipient cell with its Pilus 2. Pilus may draw cells together 3. One strand of F plasmid DNA transfers to the recipient 4. The recipient synthesizes a complementary strand to become F+ Cell

with a pilus; the donor synthesizes the complementary strand, restoring its complete plasmid.

Hfr1. F plasmid integrates into chromosome by recombination 2. Cell joins via a pilus 3. Portion of F plasmid partially moves into recipient cell trailing a strand

of donor’s DNA 4. Conjugation ends with pieces of F plasmid and donor DNA in recipient

cells; Cells synthesize complementary DNA strands 5. Donor DNA and Recipient DNA recombine, making a recombinant

F - cells

Page 41: Microbiology lecture

Griffiths Experiment

Discovered transformation

Strain S (Capsulated) Killed Mice

Strain S (Heat treated) did not harm mice

Strain R (No capsule) did not harm mice

Strain R + Heat treated strain S Killed Mice

Page 42: Microbiology lecture

After a virus called bacteriophage (phage)

attaches to a host cell, it injects its genome

into the cell and directs the cell to

synthesize new phages. During assembly

of new phages some host DNA may be

incorporated, forming transducing

phages, which subsequently carry donor

DNA to a recipient cell.

1. Phage injects its DNA2. Phage enzymes degrade host DNA 3. Cell synthesizes new phages that incorporate phage DNA

and mistakenly some host DNA 4. Transducing Phage injects Donor DNA 5. Donor DNA is incorporated into recipients chromosome

by recombination

Page 43: Microbiology lecture

Operons

Inducible: are not usually transcribed and must be activated by inducers such as quorum sensing polypeptides

Operon: A series of genes, a promoter, and often a

sequence controlled by one regulatory gene. The operon

model explains gene regulation in prokaryotes.

Lac operon Repressed

3’

Promotor & Regulatory GenePromotor

1 2 3

Lactose Catabolism Genes

5’ Template DNA strand

Repressor mRNA

Translation

Transcription

Repressor

RNA polymerase Can’t bind

Operator (Blocked)

Lac Operon

Lac operon Induced

3’

Repressor

3’5’

321Template DNA strand5’

Repressor cannot bind

Inducer (Allolactose from lactose)

TranscriptionProceeds

RNA for lactose Catabolism

Type of Metabolic Pathway

regulated: Catabolic pathways

production of virulence proteins

Regulating Condition: Presence

of substrate of pathway, quorum

sensing polypeptides

Page 44: Microbiology lecture

Trp

Trp

Repressible: operates in reverse fashion they are transcribed continually until deactivated by repressors

which bind to the operator and inhibit transcription trp Operon Active

OperatorPromotor

Trp Operon with five genes

Regulatory Genes

Inactive Repressor

Transcription

Template DNA strand

mRNA coding multiple polypeptides

Trp

Enzymes of tryptophan biosynthetic pathway

5’

5’

5’

3’

3’

3’

trp Operon Repressed

3’

Trp

Trp

Trp

Tryptophan(Corepressor)

Activated Repressor Trp

Operator blocked

Inactivate Repressor

RNA polymerase Ceases

5’

21 3 4 5

5 432 1

Type of Metabolic

Pathway regulated: Anabolic

pathways

Regulating Condition: Presence

of product of pathway, quorum

sensing polypeptides