Module 2

Central Dogma 1: Replication

DNA polymerase form 2 identical daughter DNA molecules

Central Dogma 2: Transcription

RNA polymerase produce RNA (mRNA, tRNA, rRNA)

Central Dogma 3: Translation

Ribosomes produce polypeptide proteins from mRNA

Prokaryotic Cells

Bacteria

Archaea

No membrane-bound organelles

Eukaryotic Cells

Fungi

Plants

Animals

Membrane-bound organelles

Bacteria Capsule

Polysaccharide layer outside cell membrane

Immune cell protection

Virulence factor (aid in host infection)

Bacteria Flagella

Protein appendages for motility

Identify bacteria

Pili and fimbriae for adhesion and exchange genetic material

Bacteria Chromosomal DNA

Large DNA molecule

Plasmids in nucleoid (cytoplasmic region)

Bacteria: Ribosomes

In cytoplasm

Translate mRNA into amino acid sequence

Transcription and translation in cytoplasm

Bacteria: Cell Membrane

Surround cytoplasm

Lipid bilayer

Bacteria: Peptidoglycan

Outside cell membrane

Sugar polymers and peptide chains

Protect from osmotic stress

Can have second lipid bilayer outside

Gram-Negative Structure

Outer membrane

Peptidoglycan layer

Inner cytoplasmic membrane

Gram-Negative: Outer Face of Outer Membrane

Lipopolysaccharides/endotoxins (lipid anchor and polysaccharide chain)

Release and produce strong immune response

Gram-Negative: Surface Proteins of Outer Membrane

Porins allow molecule entry

Efflux pumps expel toxic molecules

Gram-Negative: Cell Wall

Second lipid membrane

Outer membrane and peptidoglycan

Gram-Negative: Periplasmic Space

Between inner and outer membranes

Gram-Positive Structure

Peptidoglycan layer

Cytoplasmic membrane

Gram-Positive: Cell Membrane

Teichoic acids (carbohydrate polymers) anchor to peptidoglycan or cell membrane

Rigid

Gram-Positive: Cell Wall

Thick peptidoglycan layer

No outer membrane

Synthetic Antibiotics

Lab made

Ex: Sulfa drugs (chemical)

Natural Antibiotics

Produced by bacteria and fungi

Ex: Penicillin G (mold)

Semisynthetic Antibiotics

Chemically modified natural products

Bactericidal Antibiotic

Directly kill bacterial cells

Bacteriostatic Antibiotic

Prevent bacterial cell growth and replication

Host immune defences kill bacteria

Antibiotic Selectivity

Prokaryotes and eukaryotes have different cellular pathways and structures

Antibiotic Target: Cell Wall

Important for structure, replication, and osmotic stress resistance

Good selectivity (none in animal cells)

Inhibit cell wall synthesis enzymes

Bind chemical intermediates for cell wall synthesis

Cell Wall Antibiotic: β-Lactam Antibiotics

Penicillins, cephalosporins, carbapenems

Contain 4-membered β-lactam ring

Target transpeptidase forming peptide links in peptidoglycan

Antibiotic Target: Cell Membrane

Control molecule transport

Form pores in membrane

Essential metabolites leak out

Poor selectivity (in animal cells) → Recognize molecule specific to bacteria

Cell Membrane Antibiotic: Polymyxin Colistin

Kill Gram-negative

Last resort

Bind lipopolysaccharide

Disrupt membrane

Antibiotic Target: Ribosomes

For protein translation

Good selectivity (Different structure in animal cells)

Bacteria: 70S

Eukaryote: 80S

Ribosome Antibiotic: Aminoglycoside

Sugar molecule with amino side chains

Prevent ribosome reading mRNA

Produce incorrect amino acid sequences

Truncated proteins (shortened)

Ex: Streptomycin

Antibiotic Target: Nucleic Acid Synthesis

Inhibit replication and transcription enzymes

Nucleic Acid Antibiotic: Quinolones and Fluoroquinolones

Target DNA gyrase (replication)

Rapid antibiotic resistance

Ex: Ciprofloxacin

Antibiotic Resistance: Decrease Permeability

Decrease porin proteins

Prevent antibiotic entry and reaching target

Antibiotic Resistance: Modify Target

Prevent antibiotic interaction with target

Ex: Change transpeptidase sequence (no β-lactam interaction)

Antibiotic Resistance: Inactivation

Chemically modify antibiotics

Prevent interaction with target

Ex: Produce β-lactamase enzyme inactivate β-lactam

Antibiotic Resistance: Ejection

Efflux pumps

Prevent antibiotic accessing target

Light Microscropy

Lens and light series magnify bacteria

Visualization

Resolution: Ability to distinguish fine detail

Light Microscopy: Ocular Lens

10x

Light Microscopy: Objective Lens

10x low power

40x high power

100x oil immersion

Light Microscopy: Magnification

Ocular x objective

Staining Process

1. Colour cells
2. Heat fixture on slides
3. Visible under microscope

Simple Staining

Observe entire cell

View shape and morphology

Ex: Crystal violet

Differential Staining

Different colour for different cell types

Distinguish under microscope

Ex: Gram stain

Gram Stain: Colours

Gram-positive: purple

Gram-negative: pink

Gram Staining Process

1. Crystal violet
2. Iodine (trap crystal violet in peptidoglycan of gram-positive)
3. Alcohol wash to decolourize gram-negative
4. Safranin (stain red/pink)

Special Staining

Visualize specific cell parts

Flagella, endospores, capsules

Special Stain: Flagella

Number and arrangement

Special Stain: Endospores

Protect from environmental stress

Visualize and identify

Special Stain: Capsules

Negative staining (clear areas)

Cocci

Sphere shape

Bacilli

Rod shape

Spiral

Twists

Vibrios, spirilla

Cluster: Dip-

2 cells

Cluster: Strepto-

Long linear chain

Cluster: Staphylo-

Grape-like clusters

Fluorescence Microscopy

Fluorescent molecules bind cell surface

Excitement produces fluorescence

Non-selective

Immunofluorescence

Attach fluorescent molecule to antibody to identify bacteria

Biochemical Tests

Identify bacteria through enzyme interactions with metabolites

Biochemical: Catalase Test

H2O2 + catalase-positive bacteria = O2 production (bubbles)

Biochemical: Oxidase Test

TMPD + oxidase-positive bacteria = blue

Biochemical: Indole Test

Kovac’s reagent + indole-positive bacteria = red

Genetic: Phenotypic Tests

Quick

Limited identification of unknown strain

Genetic: Genotypic Tests

More info on bacterial identity

Genetic: Sanger Sequencing

Determine 16S rRNA DNA sequences in 70S ribosome

Compare to 16S rRNA database

Specific

Genetic: Next Generation Sequencing

Determine all DNA sequences

Identify chromosome and plasmid sequences

Growth/Culture Media

Grow bacterial cultures

Different nutritional components and function

Growth Media: Chemically-Defined

Nutritional differentiation

Contain pure chemicals of known composition

Growth Media: Complex

Nutritional differentiation

Chemically complex extracts

Non-fully defined composition

Growth Media: Differential

Functional differentiation

Integrate biochemical tests

Distinguish bacterial types

Growth Media: Selective

Functional differentiation

Grow specific bacteria only

Diagnostic

Differential: Blood Agar

Complex solid media with blood

β-hemolytic: Surrounded by clear zone

⍺-hemolytic: Green by-product

ɣ-hemolytic: Brown by-product

Selective: Bismuth Sulfite Agar

Inhibit gram-positive and most gram-negative

Isolate Salmonella gram-negative bacteria

Complex: MacConkey Agar

Selective and differential

Selective: Bile salts and crystal violet inhibit gram-positive

Differential: Lactose and pH indicator differentiate Lac+ and Lac-