Bacteriophages: Structure, Life Cycles, and Applications

Bacteriophages

Introduction to Bacteriophages

Bacteriophages, often called "phages," are viruses that specifically infect bacteria. The term "phage" is derived from the Latin word for "eat," reflecting their ability to destroy bacterial cells. Bacteriophages are highly specific to their bacterial hosts and do not infect eukaryotic cells, including humans.

• Bacteriophage Structure: Typically consist of a capsid (head) containing the viral genome, a tail sheath, tail fibers, collar, and endplate.

• Size: Capsid head is approximately 100 nm.

• Host Range: Infect only bacteria, not eukaryotic cells.

Prokaryotic Viruses

Mechanisms of Infection

Phages must overcome several barriers to infect prokaryotic cells. Their infection process involves specific interactions with bacterial cell surface receptors and crossing the cell wall.

• Attachment: Bind to host cell receptors (e.g., proteins involved in sugar uptake, signaling, conjugation).

• Entry: Must cross the cell wall; Gram-negative hosts require crossing two membranes.

• Replication: Utilize host nucleotides, amino acids, and ATP to replicate viral genome and assemble new viruses.

• Exit: Usually lyse host cells to release progeny phages.

Bacteriophage Life Cycles

Attachment and Genome Injection

Bacteriophages attach to specific bacterial cell surface receptors and inject their genome into the cytoplasm, often described as "shooting" the bacteria.

• Attachment: Utilizes normal bacterial proteins as receptors.

• Genome Injection: The viral genome is injected through the cell wall into the cytoplasm.

Lytic and Lysogenic Cycles

Bacteriophages can follow two main life cycles: lytic and lysogenic. The choice between these cycles is influenced by environmental cues.

• Lytic Cycle: Rapid replication and destruction of the host cell.

• Lysogenic Cycle: Viral genome integrates into host chromosome as a prophage and remains dormant until induced.

• Environmental Cues: Stress or damage to the host cell often triggers the lytic cycle.

The Lytic Phase (Lytic Life Cycle)

Steps in the Lytic Cycle

During the lytic cycle, phages use host cell machinery to synthesize viral components, assemble new phages, and exit the cell.

• Synthesis: Use cell components to synthesize capsids and assemble progeny phages.

• Lysis: Production of proteins that break peptidoglycan, leading to cell bursting and release of phages.

• Slow Release: Filamentous phages may exit the cell gradually without immediate lysis.

Coliphages: T4 and λ (Lambda)

Overview and Structure

Coliphages are bacteriophages that infect E. coli. T4 and lambda are well-studied examples with distinct life cycles and genetic structures.

• T4 Phage: Virulent/lytic, double-stranded linear DNA genome, complex structure with capsid head and tail.

• λ (Lambda) Phage: Temperate/lysogenic, double-stranded linear DNA genome, capsid head and tail.

Bacteriophage T4

Structure and Infection Process

T4 phage has a complex structure and a well-defined infection cycle in E. coli.

• Genome: 170 genes, 10 different capsid protein types.

• Attachment: Tail fibers bind to OmpC porin (outer membrane protein).

• DNA Injection: Long tail injects DNA into the host.

T4 Life Cycle Timeline

• Adsorption and Penetration: Phage attaches and injects DNA.

• Early mRNA Synthesis: Enzyme synthesis and host gene expression arrest (2 min).

• DNA Replication: Begins after 5 min.

• Late mRNA Synthesis: Formation of new viral particles (12 min).

• Cell Lysis: Host cell bursts, releasing viruses (22 min).

Genome Replication and Packaging

• Rolling Circle Replication: Continuous replication of many genome copies.

• Concatemer Formation: Progeny genomes linked together.

• Packaging: DNA cut with offset, packaged into capsids with slight overlaps (103% genome size).

Phage Particles Self-Assembly

Assembly Process

Late genes direct the assembly of phage particles and production of lysis proteins.

• Capsid Formation: Head polymerizes around DNA, tail fibers and long tail made.

• Assembly: Head, tail, and tail fibers assemble.

• Lysis Protein: Destroys cell wall, releases progeny.

Lysogenic Bacteriophage

Temperate Phages and Lysogeny

Temperate phages can integrate their genome into the host DNA, forming a stable relationship as a prophage.

• Lysogeny: Viral genome integrates into host DNA; host cell is called a lysogen.

• Dormancy: Prophage DNA is mostly dormant until induced (e.g., DNA damage).

• Single Prophage: Only one lysogenic virus of a particular type per host cell.

Temperate Coliphage Lambda (λ)

Structure and Replication

Lambda phage has a double-stranded DNA genome with cohesive ends (cos sites) and can undergo both lytic and lysogenic cycles.

• Genome: 48.5 kb, 50 genes, linear with 12nt complementary single-stranded regions (cos sites).

• Receptor: Porin protein in E. coli.

• DNA Circularization: Linear DNA circularizes at cos sites inside host.

Replication of λ Genome

• θ Replication: Initiates at ori site, bidirectional.

• Rolling Circle Replication: Produces long chains of concatenated genomes.

• Packaging: Cutting at cos sites generates linear form for packaging.

Lysis vs. Lysogeny Decision

Regulation of Life Cycle

Lambda phage can choose between lysogenic and lytic cycles based on environmental conditions. Integration and excision of the prophage are tightly regulated.

• Lysogeny: Integration into host genome, dormant state.

• Lysis: Excision and entry into lytic cycle upon induction.

Comparison of λ and T4 Phage

Key Differences

The following table summarizes the main differences between λ and T4 phages:

	λ	T4
Life cycle	Usually lysogenic, can be lytic when induced	Always lytic
DNA Structure	Linear when injected; circularizes via cos sites	Linear when injected; circularizes with terminal repeats but then replicates as concatemers
DNA Replication	θ replication; Rolling circle replication forms concatemers	Rolling circle replication; forms concatemers
Cutting of concatemers for head packaging	Cuts at cos sites	Cuts according to DNA lengths (103% of the genome size)

Transduction

Generalized Transduction

Generalized transduction occurs when phages accidentally package host bacterial DNA during the lytic cycle, transferring it to another bacterium.

• Process: Host DNA is incorporated into phage particles and transferred to new cells via homologous recombination.

Specialized Transduction

Specialized transduction involves the transfer of specific bacterial genes adjacent to the viral attachment (att) sites during lysogeny and subsequent induction of the lytic cycle.

• Process: Only genes near att sites are transferred when prophage excises incorrectly.

Infection Outcomes

Variability in Infection

Not all phage infections result in host cell death. Some phages use slow-release replication, allowing host cells to survive and grow slowly.

• Slow-release Replication: Progeny phages exit gradually through the cell envelope.

• Lytic Burst: Host cell is lysed, releasing many phages at once.

Culturing Bacteriophage

Methods and Plaque Formation

Bacteriophages are cultured using agar plates and bacterial lawns. Plaques indicate areas where phages have lysed bacterial cells.

• Procedure:

  1. Mix dilute phage with agar and E. coli (top agar).

  2. Make serial dilutions.

  3. Pour mixture onto petri dishes with bottom agar.

  4. Incubate overnight; E. coli grows, phages infect and lyse cells.

• Plaques: Cleared areas where E. coli cells have been lysed by phage infection.

• Plaque Forming Units (pfu): Each plaque originates from a single virion.

Key Terms and Concepts

• Bacteriophage: Virus that infects bacteria.

• Lytic Cycle: Viral replication resulting in host cell lysis.

• Lysogenic Cycle: Viral genome integration and dormancy in host DNA.

• Prophage: Integrated viral genome in host DNA.

• Concatemer: Long DNA molecule composed of repeated genome units.

• Transduction: Transfer of bacterial DNA via phage infection.

• Plaque: Clear area on bacterial lawn indicating cell lysis by phage.

Important Equations

• Rolling Circle Replication:

$\text{Synthesis of complementary DNA strand}$

• Plaque Forming Units (pfu):

$\text{pfu/mL} = \frac{\text{Number of plaques}}{\text{Dilution factor} \times \text{Volume plated (mL)}}$

Additional info: The notes expand on the mechanisms of phage infection, genome replication, and laboratory techniques for culturing and quantifying bacteriophages, providing context for their role in microbial genetics and biotechnology.