Topic A - Introduction to Bacteria and Bacteriophages

Bacteria

Small, single-celled organisms that live in communities

-Prokaryotic, no nucleus/membrane-bounded organelles

Features of Bacteria

Cytoplasmic Membrane - Contains the contents of the cell

Cell Wall - Prevents the bacteria from bursting due to changes in pressure

Capsule - Slimy, mucous layer on the outside, prevents foreign bodies from entering (antibiotics, bacteriophages)

Pili/Pilus - Extensions embedded in the cytoplasmic membrane, involved in attaching to cells, other bacteria and surfaces

Flagellum - Extension allowing bacteria to move in its environment

Chromosome - single, circular chromosome within the nucleoid region

Plasmids - Small, circular DNA molecule outside the nucleoid region

Ribosome - Complexes involved in translating proteins

Bacterial Cell Wall Structure

Bacterial cell walls contain peptidoglycan, classifying bacteria into Gram Positive and Gram Negative

Gram Positive Bacteria

Contain a thick peptidoglycan layer within the cell wall, thus does not require additional support

Gram Negative Bacteria

Thin peptidoglycan layer, requires an additional outer membrane

-Outer membrane composed of LPS that forms a lattice surrounding the peptidoglycan layer to provide structural support

-Lattice prevents chemicals from entering, thus Gram-Negative bacteria are more antibiotic resistant

Structure of Peptidoglycan

Monomer consists of 3 parts;

1. Acetylglucosamine (NAG) - sugar

2. Acetylmuramic Acid (NAM) - sugar

3. 5 amino-acid long peptide (attached to the NAM)

Peptidoglycan Formation

Two peptidoglycan monomers form a glycosidic bond, process continues to form a long repeating chain

-Two long chains attach to each other via a peptide bond between the stems (D-ala and DAP)

-5th D-ala is cleaved during peptide bond to provide energy

Crosslink by short peptides attached to NAM results in greater strength

Eukaryotic Cell Structured Compared to Prokaryotes

-Contains membrane-bound organelles, DNA contained within the nucleus

-Some contain cell walls, but not formed of peptidoglycan

-Contain a cytoskeleton (bacteria as well, but very different)

-Eukaryotes contain multiple, linear chromosomes whereas prokaryotes have a single, circular one (generally)

Bacterial Genetics

Consists of a single circular chromosome, making bacteria haploid

-Genetic information is Streamlined; most DNA contain genes, very little noncoding DNA

-Short intergenic regions, no introns and only a few repetitive elements

Reasons for Genetic Organization

Chromosome is condensed into a structed called the nucleoid

-Allows DNA to fit into the cell

-Keeps DNA organized (prevents tangling during division, regulates gene expression and facilitates DNA repair)

Bacterial Genetic Organization

Chromosome is condensed by looping and supercoiling

-Assisted by proteins (NOT HISTONES)

-Proteins constrain ends of loops which are then supercoiled to condense DNA

Gene Expression via Genetic Organization

Gene expression in bacteria is affected by cell structure

Why Use Bacteria?

-Bacteria play important role in human health/disease, environmental processes and industry

-Many molecular techniques and recombinant DNA technologies use bacteria

-Bacteria are great model organisms for studying fundamental biological processes

Examples of Bacterial Use

Luria/Delbruck - Darwin’s theory of evolution

Griffith, Hershey and Chase - DNA is the genetic material

Lederberg and Tatum - Genetic exchange and recombination

Watson and Crick - DNA structure

Benzer - Gene structure

Meselson and Stahl - DNA replication

Brenner, Jacob and Meselson - Existence of mRNA

Crick - Genetic code

Jacob and Monod - Gene regulation

Mullis - PCR

Doudna and Charpentier - CRISP

Why are Bacteria Good Model Organisms

1. Bacteria are haploid

2. Easy to grow

3. Reproduce asexually

4. Short generation time

5. Easily counted

6. Easy to isolate

7. Easy to identify

8. Easy to manipulate

9. Easy to store

Bacteria Are Haploid

One copy (allele) of each gene

-Easier to identify cells containing mutations

-But it is difficult to maintain lethal mutations

Bacteria are Easy to Grow

Grow in simple, cheap, easy to make media that may be complex (rich in biological material/organic molecules) or minimal (contains inorganic ions and glucose)

-Can grow in liquid medium (inoiculate cultures by adding cells to sterile broth), easy to grow lots of cells

-Can grow on solid medium by adding Agar to liquid medium, cells are streaked or spread onto a solid surface

Prototroph vs Auxotroph

Prototroph - Synthesize all organic molecules de novo (on its own), can grow in minimal media

Auxotroph - Cannot synthesize one (or more) essential nutrient, requires media supplemented with nutrients that can’t be synthesized

-Ex; Proline auxotroph needs proline added to the media

Bacteria Reproduce Asexually

Divide by binary fission

-One cell divides to produce two genetically identical cells (clones)

Colony - Visible cluster of bacteria originating from division of a single bacterium

Bacteria have Short Generation Times

Generation Time - Time rqeuired for organism to reach maturity and produce offspring

Generation time for common model organisms;

-E.coli - 20 minutes

-D. melanogaster - 10 days

-A. thaliana - 6 weeks

-M. musculus - 10 weeks

Bacteria can be Easily Counted

Viable plate count procedure

1. Serially dilute bacterial culture

2. Spread on solid medium and incubate

3. Count colonies and calculate original concentration (Co)

Co = Number of Colonies/Volume plated x 1/Dilution

Bacteria are Easy to Isolate

Colony Purification - Isolation of individual strain/mutant from mixture of colonies on a plate

-Colony purification procedure:

1. Identity and pick colony of interest

2. Streak on new plate for single colonies

3. Repeat 1-2 times = pure culture

Mutants are Easy to Identify

Selection - Bacterial population placed under condition where only those with desired genetic traits can grow (can select one cell out of billions on a single plate)

Screening - Systematically examine individual mutants within a population to identify those with desired genetic traits

-Can look at thousands of mutants on a single plate

Bacteria are Easy to Genetically Manipulate

Done via;

Transformation - Uptake of new DNA from surroundings

Conjugation - Transfer of DNA directly from cell to cell

Transduction - Transfer of DNA to cell via bacteriophage infection

-Facilitates mutant analysis, genome mapping, and genetic modification

Bacteria are Easy to Store

Frozen in cryoprotectant stored at -80C, thawed or later use

-Some bacteria form stable spores, can be freeze-dried and stored indefinitely

Bacteriophage

Viruses that infect bacteria

-Only multiple in actively metabolizing bacterial host cells (therefore, not alive)

-Must abundant biological entity, structurally diverse

-Classified based on structural properties

-Biochemically simple: Only nucleic acids and protein

Features of Bacteriophages - Head

Found in all phages

-Functions as a protective covering for nucleic acid

-Varies in shape/size

Features of Bacteriophages - Tail

Found in some phages

-Functions to penetrate cell envelope and transfer nucleic acid during infection

Features of Bacteriophages - Nucleic Acid

May be DNA or RNA, but NOT both

-Single or double stranded

-Linear or circular

Often contain modified bases to protect from nucleases during infection

-Ex; Hydroxymethylcytosine (hydroxyl group on the cytosine base)

Lytic Life Cycle

Infecting phage converts bacterium into a phage “factory” to produce more phage

-bacterial cell bursts and dies, releasing new phage into the environment

Lysogenic Life Cycle

Phage DNA is integrated into bacterial chromosome

-No new phages are produced

Lawn and Plaque

Lawn - Continuous layer of bacteria that covers the surface of solidified growth medium (no visible colonies)

Plaque - region of clearing that develops in a lawn of sensitive bacteria due to lytic replication (zone of lysis)

Bacteriophage Stock/Lysate

Solution of identical bacteriophage particles

-Easy to produce;

1. Pick phage from single, well isolated plaque

2. Mix phage with culture of sensitive bacteria

3. Bacteria will lyse and release phage into culture supernatant

4. Will produce lots of progeny phage

Why Use Bacteriophages

1. Turn over 40% of all bacteria daily; major impact on various aspects of microbiology

2. Can carry foreign DNA to new bacterial hosts (transduction), contributes to bacterial evolution

3. Foundation of many genetic principles

Bacteriophages as Good Genetic Models

1. Haploid

2. Short generation time

3. Multiply clonally

4. Phage can be easily counted

5. Easy to cross phage strains

6. Easy to select mutants

7. Simple system with small genomes

Bacteriophage are Haploid

-Genome has one copy of each gene

-Effect of mutations seen immediately

Short Generation Time

100-fold increase in 20 minutes

Multiple Clonally

All phage in a plaque are genetically identical

-Easy to produce a pure stock

Phage can be Easily Counted

Titre; concentration of a phage stock

Number of Plaques - number of phage plated

Co = Number of Plaques/Volume plated x 1 / Dilution

Easy to Cross Phage Strains

Co-Infection - infect a susceptible bacterial cell with two different mutant phages

-Both mutant phage inject genetic material into the same cell; allows for study of mutations

Recombination - Interaction of mutant genes

Complementation - Interaction of mutant proteins

Easy to Select Mutants

Millions of phages can be mixed with bacteria and grown under selective conditions

-Only desired phage mutant will replicate (ex; host range mutants)

Simple Systems with Small Genomes

Easy to connect gene to function