Bacteriophages and Infection Cycles

Bacteriophages: Types

• Bacteriophages come in two main types: head-and-tail and filamentous.
• Head-and-tail phages have a head containing DNA and a tail.
• Filamentous phages have a protein capsid surrounding a DNA molecule that forms a long, filamentous structure.

Phage Infection Cycle: Virulent Phages

• Virulent phages infect bacteria.
• Specific phage enzymes coded by the phage chromosome cause phage DNA replication.
• Capsid proteins are synthesized and phage particles are assembled and released from the cell in a lytic cycle.

Temperate Phages and Prophage

• Temperate phages have a DNA (λ phage) that can integrate into the bacterial genome.
• Prophages remain dormant and indistinguishable from an uninfected cell.
• Prophages can be released from the host genome by reverting to a lytic mode.

Lysogenic Infection Cycle

• In the lysogenic cycle, a phage particle attaches to a bacterial cell and injects its DNA, which circularizes.
• The phage DNA integrates into the host chromosome.
• The phage DNA is maintained in a stable state, and cell division occurs, with the phage DNA replicated with each division.
• The phage DNA can excise from the host chromosome to begin the lytic cycle again.

Gene Transcription

• PR and P₁ get activated after circularization, enabling the synthesis of immediate early transcripts (such as N and cro), which terminate at t₁ and tr.
• N protein overrides the t₁ and tᵣ terminators, leading to the transcription of delayed transcripts cII and cIII.
• cIII protects cII from degradation by host proteases and cII activates the PRE promoter which produces repressor protein, inactivating PR and P₁, hence switching off the lambda genome .
• cII also activates the promoter PINT to mediate site-specific recombination with host proteins across att and a similar sequence in the bacterial chromosome integrating the phage.
• Other genes such as O and P cause DNA replication, Q causes activation of PR, expression of genes encoding head and tail proteins , while Int and Xis cause phage excision.

Isolation of Phage DNA

• Extracellular phage titre is low.
• To improve yields, cultures need induction, where lytic processes dominate.
• Most phage strains carry a temperature-sensitive mutation (ts) in the cl gene.
• cl+ cells in lysogeny at 30°C, enter lytic cycle at 42°C.

Plaque Assay

• Phage infection is visualized as plaques on an agar medium.
• Each plaque is derived from a single infected cell.
• Virus titers are measured in terms of "plaque forming units/mL."

Genetic Map of Bacteriophage λ

• The 49 kb λ genome includes essential features for cloning.
• Clustering of functionally-related genes, turned "on" or "off" as a group.
• Linear double-stranded DNA with two free ends and stretches of ssDNA at each end.

Cos sites and Circularization

• Cos sites are essential for circularization of the linear DNA necessary for insertion into the host genome.
• Gene A endonuclease cleaves the catenane at the cos sites.
• Additional genes can be added to the internal regions of the phage genome without altering the phage life cycle if genome size isn't significantly altered.

Cloning Strategies

• Strategies using λ phage potentially allow the insertion of up to 3 kb of new DNA without drastically altering the phage's infectivity.
• The entire genome has multiple recognition sequences, so adding DNA may not affect overall phage function.
• A 15 kb region can be replaced with new genes without affecting infectivity.

Restriction Enzyme Selection

• Using natural selection can remove almost all recognition sequences for a specific restriction enzyme from a phage genome.
• Repeated infections of E. coli with mutant phage that produce progressively fewer plaques will isolate phages with reduced numbers of specific recognition sites.

Insertion Vectors

• Vectors are used to insert foreign DNA into the phage genome, including vectors Agt10 and λZAPII which can carry up to 8kb and 10kb fragments of DNA, respectively.
• The nonessential region is removed and replaced with the foreign DNA.
• Insertional activation of specific genes can be used in cloning.

Replacement Vectors

• Contains two recognition sites for restriction enzymes to allow the insertion of new DNA.
• Digest with other restriction enzymes to prevent re-insertion of the original phage sequence into the new DNA.
• Vectors can carry larger fragments of DNA.

M13 Phage

• M13 DNA does not integrate into the bacterial genome.
• M13 DNA is circular and entirely single-stranded.
• New phage particles are assembled and released without lysing the host cell.
• M13 phage particles may be released into the medium per cell per generation can reach up to 1000.

M13 Genome

• M13 genome is small, about 6407 base pairs, circular and single-stranded DNA
• Useful for creating cloning vectors.
• Has ten genes essential to phage replication.
• A unique intergenic sequence capable of accepting 507 base pair insertions.

M13 Cloning Vectors

• Various M13 cloning vectors (e.g., M13mp1, M13mp2, M13mp7) contain polylinkers with unique restriction sites enabling insertion into other vectors.
• Using in vitro mutagenesis, unique restriction sites such as EcoRI are introduced to the lacZ' gene in the M13 vectors.

Explore the fascinating world of bacteriophages with this quiz. Learn about the different types of bacteriophages, their infection cycles, and the transition between virulent and temperate phages. Test your understanding of how these viruses interact with bacterial cells.