Evil spirits 
Bad Blood 
Bad air

invented first microscope
"Animalcules"

Live from Non-life
AKA: Spontaneous Generation

Pasteurization of wine/milk/juice
 Vaccines: Cholera, rabies & anthrax(fail) 
Associated with Koch germ theory

ID cause of anthrax; B. anthracis
Koch's postulates; criteria to ID a disease from a pathogen
Developed Pure culture technique and staining techniques

The cause of the disease must be:

1. found in all cases of the disease examined
2. prepared and maintained in a pure culture
3. produce originial infection after, even after several generations
4. retrievable from an inoculated animal and cultured again

Joseph Lister surgical technique

Edward Genner

Alexander Fleming

Aseptic technique using phenol on surgical wounds

Cowpox used for protection against small pox

Pencillin discovery (1928)

Communication
Evolutionary Relationships
Illustration of diversity

Archae              Eukaryota

                                    ↑←←←  ↔  →→→ ↑

                                                  ↑  

Bacteria                                       ↑

      ↓→→→→→→→→→→→→→→↑

                  Archae and Eukaryota more closely related

Lyophilized (freeze-dried)

Frozen at -80°C

Liquid N₂ (-196°C) for long term

Taxonomic Scheme

Classical

**Requires growth of organism & observation used to classify microorgansims**

structure/morphology

biochemical/physiological

cell surface

GC composition

physical characteristics: 

shape/size of colony/cell

arrangement

external features

physiological characteristics:

growth factors

metabolic end products

antibiotic sensitivites

Serological; ex. capsule

Phage typing (detecting single strains of bacteria)

Determine percent GC content by:

melting DNA; (higher GC content=higher mp)

Phylogeny: changes in DNA gene sequence over time; evolution

*Not always necessary to culture organism

Universal. All bacteria should have the gene

Identical function

Easy to work with

16S or 23S in prokaryotes

Nucleic Acid Hybridization

Nucleic Acid Sequencing

DNA fingerprinting (not used as much)

Flourescent tagged probes of complimentary DNA that hybridize with the complimentary rRNA of the bacteria.

Viewed with a Flouresence Microscope

Extract gene for interest (rRNA)

Polymerase chain reaction (amplify)

use computer to align sequences (compares squence to known organisms)

Bacterial endosymbiont of eukaryote lost ability to live independently

DNA/Ribosomes similar to bacteria

Gram Positive

 cocci: ex. S. aureus  

  streptococcus    

  staphylococcus    

 bacilli

Gram Negative

 Bacilli

  Lactose + (E. coli )

  Lactose -

Thick peptidoglycan layer

2 membranes

polysaccharide capsule

flagella: 2 support rings

stains: dark purple

S-layer attached to petidoglycan if present

sporulate

teichoic acid/lipoids form lipoteichoic acid 

Cytoplasmic Membrane

Thin peptidoglycan layer

stains: light pink

porins (pores)

flagella (4 support rings)

s-layer attached directly to outer layer

do not sporulate

Lipopolysaccharide outer layer

Archae: single-celled, no nucleus/organelles

Magnification: how big

Resolution: distinguish fine detail (smaller wave length

View living specimens at 1000x-1500x

Types: 

Brightfield: specimen is dark, field is light (heat-fixed and stained specimens)

Phase Contrast: Best for unstained, living specimens

Flourescent: uses UV light (smaller wave length=greater resolution)

Structure comparisons

Prokaryote vs. Eukaryote

Cytoplasmic membrane: BOTH

Membrane-bound nucleus: Eukaryote

Ribosomes: prokaryote (70S)/ eukaryote (80S)

Cell Wall: petidoglycan(P)/ cellulose(E)

Internal membrane-bound organelles: Eukaryote

Flagella: flagellin(P)/ Microtubules(E)

Cytoskeleton: **Actin(P) / eukaryotes

Cell Size: .3-3 microns(P)/ >3 microns(E)

Cocci

Bacilli

Spirilla

Stalked

Sheathed

Pleomorphic

Capsule: slime

S-layer: protein surface layer; protection

pili: adhesion/conjugation

flagella: motility

chemotaxis: ability to move toward attractant (positive) or away from repellent (negative)

protection

hydrated polysaccharide or polypetide

can be thicker than bacteria

Creates immune response: antigenic reaction (ex. K-antigen)

Cell Envelope

Peptidoglycan

NAG-NAM structural component

functions: maintain cell shape, resist osmotic stress

Contain Teichoic Acid in Gram POSITIVE

Lysozyme breaks down PG

dissacharides bonded in rows/columns by polypeptides (sugars linked with proteins)

Pentaglycine bridge is Gram positive

2 layers:

1) Thin layer of Peptido Glycan

2) Surrounded by outer membrane

linked by lipoproteins

membrane fluidity: short tails and double bonds (enables growth at lower temps)

lack of fluidity (growth at higher temps)

distinguishes self from environment

site of ATP synthesis

entry/exit control

synthesis site for cell wall and surface components

maintains proton motive force

Proteins*20 amino acids connected by peptide bonds that fold into 3D structures*

synthesis of cell wall components

respiratory enzyme/ATP synthesis

Transport across membrane

Protein Transport functions

diffusion, facilitated diffusion, active transport, group translocation, secretion

Diffusion: molecules move freely energy independent (hi→low)

Facilitated Diffusion: carrier protein, requires concentration gradient (hi→low)

Active Transport: works against concentration gradient, requires energy (ATP→ADP)

Group Translocation: Phosphorolation (molecule chemically modified by phospho group), requires energy

Secretion: multiple pathways

No.

Phosopholipids: fatty acid composition, molecule attached to phosphate

Differences used for classification

one circular chromosome in the cytoplasm containing supercoiled DNA that is not membrane bound, additional DNA present as plasmids

Gram positive

most resistant biological structure known

Resting Stage; non reproductive

Polar: one flagella at one end of rod

Bipolar: 2 flagella, on opposite ends

Peritrichous: 4 flagella

Lophotrichous: multiple flagella at one end

*H-antigens

Phospholipids: polar head, nonpolar tail

Amphiphatic: assembles into bilayers

Archael Membranes

No peptidoglycan

Cytoplasm

rudimentary cytoskeleton

contains ribosomes

semiconservative: each strand is a template for DNA

Initiation

Elongation

Termination

DNA replication (E. coli):

Initiation

Origin: ori C (Adenine/Thymine rich)

DnaA (initiator protein) binds and unwinds DNA at ori C

uses energy (ATP→ADP), forms replication bubble

Helicase seperates unwound DNA into leading strand (3'→5') and lagging strand (5'→3')

DNA Pol I:

reads fragments, replaces RNA nucleotides with DNA nucleotides

DNA Pol III:

Needs DNA template, Nucleotides, Primer

DNA replication

Elongation

Template strand (leading) always 3'-5'

DNA pol III adds DNA on template strand in 5'-3' direction following RNA pol

Okazaki fragments synthesize short pieces in the 5'-3' direction on lagging strand

DNA pol III falls off

DNA pol I cuts of RNA nucleotides and replaces with DNA nucleotides

Finally DNA ligase seals gaps

DNA replication

Termination

2 ways:

Tus protein recognize ter sites

Bacteria have a cyclic chromosome

DNA is methylated (protection): masks DNA to not destroy good DNA

Methylation proteins recognize new strand (unmethylated) and old strand (methylated) and repairs errors

*DNA not located on the chromosome

replicates like DNA with pol, but on smaller level

Must be rapid and efficient

Semiconservative

DNA synthesis occurs in the 5'-3' direction

Methylation protects/aids mutation recognition

Multiple rounds of replication 

Conjugation: bacterial sex; plasmids copied and transferred to another bacterium by sex pilus

Selective advantage: Can spread genes 

Types: 

transmittance: F plasmids, fertility

Antibiotic resistance: R plasmids

Xenobiotic degradation: oil eaters

Virulent

Plasmids can be isolated and studied

At higher temperatures, plasmids can be 'cured' from the organism, then see if the trait is lost

Curing: growing bacteria at a higher temperature so it throws plasmid out because it's not necessary

Amplifies specific regions of DNA

in vitro (out of cell) replication of target DNA

Needs: DNA, Taq polymerase, dNTP, DNA primers

1) Heating: Melt H-bonds

2) Annealing: primers find complimentary bases

3) Elongation: DNA polymerase

Recombination: trade chromosomes

Homologous Recombination: Rec proteins help incorporate new DNA

Non-Homologous Recombination: rare in bacteria, 2 random ends of DNA glued by ligase

Mobile element or "jumping gene"

Jumps to a different place on the chromosome and copies itself

Plasmids can spread with transpons

Solid/Liquid

defined media: uses pure chemicals in known concentrations

complex media: ingredients present, but not chemically defined

General Purpose: complex, nutrient agar/broth; general growth

Differentiation: blood agar; bacteria lyse RBCs

Selective: MacConkey agar; selective for Gram negative, first step in ID of bacteria

Enrichment: contains components that promote growth; *defined medium

X-axis: Time

Y-axis: log #of visible cells

phase 1: lag phase

Phase 2: log phase (exponential phase)

Phase 3: stationary phase

Phase 4: Death phase

*organisms in nature are usually stressed

Dormacy: spore formation in bacteria

Reproduction: spore reproduction in molds

Habitat Selection: most important (food!)

Alter Metabolism: shut down non-essential proteins, induce proteins that aquire nutrients

Adhesion

Metabolic break down

**Releases energy**

Biosynthesis: Synthesis of necessary cell components for growth; building up

**Requires energy**

Specific enzymes and Regulated enzymes

Nutrients, Energy & Reducing Power: Required for survival

Metabolic energy is stored as membrane potential (ATP)

Reducing power stored as NADH or FADH₂

Polymers→Monomer→End products

(building blocks)

polymers are insoluble and broken down by secreted enzymes then the monomers are transported by active transport/group location

eg. Cellulose (insoluble) → Cellubiose (soluble)→ Glucose (soluble)

Once transported inside the cell, they are burned (catabolic) and captured as ATP, NADH, FADH₂ 

Glycolysis: catabolic reax, break down glucose and stored; glucose in→pyruvate out

Entner Douderoff: Catabolic reax; glucose→ATP, not as effective

Pentose Phosphate: Generates Ribose; used for DNA/RNA

Kreb's cycle: central hub

substrate level phosphorylation: catabolic; coupled directly to ATP production

Uses electron transport chain (ETC)

often implies aerobic catabolism; **not always (anaerobic respiration)

Polymers formed from Monomers

Amino Acids → Proteins

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