18: Bacteriophages Lecture Notes

Bacteriophages

• Bacteriophages are viruses that infect bacteria.
• They are also called phages for short (from the Greek word meaning "to eat").
• Bacteriophages cannot infect eukaryotic cells.

Viruses of Bacteria

• Viruses that infect bacteria are called bacteriophages.
• Bacteriophages infect prokaryotic cells.
• Eukaryotic cells have a nucleus and are generally larger than prokaryotic cells.
• The size of a virus is around 1000 nm (1 μm).

Prokaryotic Viruses

• Binding to host cell receptor: Bacteriophages must bind to a host cell receptor.
• Crossing the cell wall: They must cross the cell wall of the host cell.
• Gram-negative hosts: Gram-negative hosts have two membranes that the bacteriophage must cross.
• Avoiding initial damage: Bacteriophages must not damage the host cell initially.
• Utilizing host resources: They utilize host nucleotides, amino acids, and ATP.
• Replication and assembly: They replicate their viral genome, build capsids, and assemble new viruses.
• Exit strategy: They exit through the cell wall, usually by lysing the host cells.

Bacteriophage Life Cycles

• Attachment: Bacteriophages attach to host cell proteins, using normal bacterial cell surface receptors (e.g., sugar uptake, signaling, conjugation).
• Genome injection: The virus injects its genome through the cell wall into the cytoplasm.
• Lytic cycle: Bacteriophage quickly replicates, killing the host cell.
• Lysogenic cycle: Bacteriophage is quiescent (dormant) and integrates into the cell chromosome as a prophage.
• Decision: The decision between lytic and lysogenic cycles is dictated by environmental cues. Events that threaten host cell survival usually trigger the lytic cycle.

The Lytic Phase (Lytic Life Cycle)

• Synthesis: Uses cell components to synthesize capsids.
• Assembly: Assembles progeny phages.
• Exit from cell:
  • Lysis: Makes protein to break peptidoglycan and bursts host cell to release progeny phage.
  • Slow release: Filamentous phages can slip individual progeny out through the cell envelope.

Coliphages: T4 and λ (Lambda)

• Coliphages: Viruses that infect E. coli.
• T4 phage:
  • Structure: Capsid head and tail.
  • Life cycle: Virulent/lytic (intemperate).
• λ phage:
  • Structure: Capsid head and tail.
  • Life cycle: Lysogenic (temperate).
• Both T4 and lambda have double-stranded linear DNA genomes.

Bacteriophage T4

• Structure: Complicated structure.
• Genes: 170 genes.
• Capsid proteins: 10 different capsid protein types.
• Tail fibers: Tail fibers bind to the host cell.
• Receptor: Receptor = OmpC porin (outer membrane protein).
• DNA injection: Long tail injects DNA.

T4 Phage Life Cycle Timeline

The entire T4 phage life cycle (from entering an E. coli cell to its destruction) takes approximately 25 minutes (at 37°C) and consists of:

• Adsorption and penetration.
• Early mRNA synthesis: Enzyme synthesis and arrest of host gene expression (starting after 2 minutes).
• DNA replication (starting after 5 minutes).
• Late mRNA synthesis and formation of new viral particles (starting after 12 minutes).
• Host cell lysis: The host cell bursts open and releases the newly formed viruses (starting after 22 minutes).

Steps of T4 Phage Infection

1. The phage particle attaches to the outer membrane of E. coli and injects its genome.
2. Early genes are transcribed and translated into proteins, including nucleases to cleave host DNA and proteins for phage DNA replication.
3. Phage DNA undergoes rolling-circle replication, generating a multigenome concatemer.
4. Late genes are expressed to make head and tail components.
5. Phage genomes are packaged into heads.
6. Heads are assembled onto tails.
7. Tail fibers are added.
8. A phage-encoded lysozyme lyses the host cell, releasing about 200 completed phage particles.

Adsorption and DNA Injection

• The phage attaches to the host cell and injects its DNA through the outer membrane, cell wall, and inner membrane into the cytoplasm.

Phage T4 Genome Replication

• Upon entry, the phage genome forms a circle.
• Early genes are transcribed.
• The phage takes control of the cell and destroys the cell chromosome.
• Uses cell nucleotides to replicate its genome.
• Uses “Rolling circle replication” for continuous replication of many copies of the genome.

Concatemer Formation

• Progeny genomes are linked in a concatemer (several genomes linked together).
• Cut so that individual linear genomes have slight overlaps.

Packaging of T4 DNA into Virus Heads

• The genome (between the arrows) is packaged.
• Each packaged DNA is slightly longer than the complete phage chromosome (103%).
• The sequence begins and ends at different points in different virions.

Phage Particles Self-Assemble

• Late genes transcribed.
• Capsid particles are made.
• The head polymerizes around progeny DNA.
• Tail fibers and the long tail are made.
• The head, tail, and tail fibers assemble.
• Lysis protein is made, which destroys the cell wall and releases progeny.

Lytic Cycle of T4 Phage - Timeline

• 0 min: DNA injection.
• 2 min: Early mRNA made.
• 3 min: Host DNA degraded.
• 5 min: Phage DNA replicated.
• 9 min: Late RNA made.
• 12 min: Head and tails made.
• 13 min: Heads filled.
• 15 min: Virions formed.
• 22 min: Host cell lysis.

Lysogenic Bacteriophage

• Some phages maintain a stable relationship with the host cell and stay with the host; these are called temperate phages.
• Temperate phages are capable of lysogeny.
• Integrate viral genome (prophage) into host DNA (a bacterium harboring a prophage is called a lysogen).
• Prophage DNA is mostly dormant and does not enter the lytic cycle until induced (e.g., by DNA damage/very bad environment).
• Only a single lysogenic virus of a particular type can be present in a host cell.

Temperate Coliphage Lambda (λ)

• λ phage has a capsid head and tail.
• The genome is double-stranded DNA with 48.5kb and 50 genes.
• Linear DNA with cohesive ends (cos sites).
• 12nt complementary single-stranded regions.
• DNA circularizes once inside the host.
• The lambda phage receptor in E. coli is a porin

λ Phage DNA

• λ phage DNA in the phage head is double-stranded and linear but circular in the host cell.
• Tandem repeating ends combine in the host to give a circular configuration at the ligated cos site.
• Cos recognition sites determine where the genome is cut and packaged.

Replication of λ Genome

• θ (theta; circle-to-circle) replication at the beginning.
• Initiates at the ori site.
• Bidirectional replication.
• Rolling circle replication for continuous replication of many copies of the genome.
• Long chains of concatenated genomes.
• Cutting occurs at cos sites – generate linear form for packaging 5’.

Lysogeny vs. Lysis Decision for λ Infection as a Temperate Phage

• Phage DNA circularizes at the cos sites.
• Phage DNA integrates into the bacterial genome forming a prophage.
• Phage DNA replicates with the host genome.
• Upon induction, the prophage excises from the bacteria genome.

Lambda Phage Reproductive Cycle

1. Phage attaches to host cell and inserts DNA.

2. Linear dsDNA cyclizes to circular DNA.

   Lysogeny: Phage DNA integrates into the host genome to form prophage. Integrated phage DNA replicates with the host genome. Stress induces excision of phage DNA.

   Lytic cycle: Viral DNase cleaves host cell DNA. The cell synthesizes capsid proteins. Phage recombines by re-joining the ends of its phosphodiester chain and enters the lytic cycle. The cell replicates phage DNA. DNA is packaged into capsids. Phage lyses the cell, and progeny phages are released.

Comparison of λ and T4 phage

Generalized Transduction

• Phage infects a bacterial cell and enters the lytic cycle, resulting in the creation of normal phages and transducing particles (containing host DNA).
• The transducing particle injects the host DNA into a new bacterial cell.
• Homologous recombination occurs, resulting in a transduced cell.

Specialized Transduction

• Some viruses can integrate their viral genome into the bacterial chromosome (lysogeny) at att sites.
• When entering the lytic cycle, bacterial genes adjacent to the viral attachment (att) sites are sometimes picked up and packaged into new virions.

Infection Outcomes

• Not all infections are fatal!
• Slow-release replication vs lytic burst.
• Some phages can push individual progeny through the cell envelope.
• Host cells grow slowly but don’t die.

Culturing Bacteriophage

1. Dilute phage mixed with agar and E. coli (top agar).

2. Make serial dilutions.

3. Pour mixture onto petri dishes containing bottom agar.

4. Plates incubated overnight.

   • E. coli grows.
   • Phage infect E. coli cells and multiply.
   • Plaques are formed (containing virions).

• Cloudy area: growth of E. Coli as a lawn.
• Cleared areas: plaques (E. coli killed by the phage).
• Virions infect E. coli cells.
• Plaques are formed upon the lysis of E. coli cells.
• One plaque is formed from the infection of one virion: plaque forming units (pfu).