Bacteriophages Lecture Notes

Bacteriophages

• Bacteriophages are viruses that infect bacteria.

• They cannot infect eukaryotic cells.

Viruses of Bacteria

• Bacteriophages infect prokaryotic cells.

• Prokaryotic viruses must:

  • Bind to a host cell receptor.

  • Cross a cell wall; Gram-negative hosts must cross two membranes.

  • Not damage the host cell initially.

  • Use host nucleotides, amino acids, and ATP.

  • Replicate the viral genome, build the capsid, and build new viruses.

  • Exit through the cell wall, usually by lysing host cells.

Bacteriophage Life Cycles

• Attachment to host cell proteins using normal bacterial cell surface receptors, such as those for sugar uptake, signaling, or conjugation.

• The virus uses host proteins.

• The virus injects its genome through the cell wall into the cytoplasm.

• Bacteriophages can have two types of life cycles:

  • Lytic Cycle: The bacteriophage quickly replicates, killing the host cell.

  • Lysogenic Cycle: The bacteriophage is quiescent (dormant) and integrates into the cell chromosome as a prophage; it can reactivate to become lytic.

• The decision between the two cycles is dictated by environmental cues.

  • Events that threaten host cell survival usually trigger the lytic cycle and cell bursting.

The Lytic Phase (Lytic Life Cycle)

• Use cell components to synthesize capsids.

• Assemble progeny phages.

• Exit from the cell via:

  • Lysis: Makes a protein to break down peptidoglycan and bursts the 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 are viruses that infect E. coli.

• T4 phage:

  • Has a capsid head and tail.

  • Virulent/lytic (intemperate).

• λ phage:

  • Has a capsid head and tail.

  • Lysogenic (temperate).

• Both T4 and lambda have double-stranded linear DNA genomes.

Bacteriophage T4

• Has a complicated structure.

• Consists of 170 genes.

• 10 different capsid protein types

• Tail fibers bind the host cell.

  • Receptor = OmpC porin (outer membrane protein).

• A long tail injects DNA.

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

  • 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).

  • The host cell then bursts open and releases the newly formed viruses (starting after 22 minutes).

T4 Phage Infection Steps

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.

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 the genome by “rolling circle replication.”

• Continuous replication of many copies of the genome.

• Progeny genomes are linked in a concatemer (several genomes linked together).

• Cut with an offset 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 are transcribed.

• Capsid particles are created.

• 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, 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: temperate phages.

• Temperate phages are capable of lysogeny.

• Integrate the viral genome (prophage) into host DNA (a bacterium harboring a prophage is called a lysogen).

• Prophage DNA is mostly dormant.

• They don’t enter the lytic cycle until induced (e.g., by DNA damage or a 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: capsid head, tail

• Double stranded DNA genome – 48.5kb with 50 genes

• Double-stranded DNA genome (48.5 kb with 50 genes).

• Linear DNA with cohesive ends (cos sites).

• 12-nt 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 is 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 replication (theta; circle-to-circle) at the beginning.

  • Initiates at the ori site.

  • Bidirectional replication.

• Rolling circle replication.

  • Continuous replication of many copies of the genome.

  • Long chains of concatenated genomes.

• Cutting occurs at cos sites to generate the linear form for packaging.

λ Infection as a Temperate Phage

1. Phage DNA circularizes at the cos sites.

2. Phage DNA integrates into the bacterial genome.

3. Phage DNA replicates with the host genome.

4. Upon induction, phage DNA excises.

Phage Lambda Reproductive Cycle

1. Phage attaches to host cell and inserts DNA.

2. Linear dsDNA cyclizes to circular DNA.

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

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

Comparison of λ and T4 Phage

Generalized Transduction

• Involves the lytic cycle during which phage particles can accidentally package host DNA.

• When the phage infects a new bacterial cell, it transfers this bacterial DNA.

• The transduced DNA can then be integrated into the recipient's chromosome via homologous recombination.

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.

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 are 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 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).