Study Guide on Bacteriophages and Viral Lifecycle

Bacteriophages

What is a Virus?

  • Definition: A virus is defined as a small infectious agent that replicates solely inside the living cells of other organisms.

  • Size: Most viruses exhibit a diameter ranging from 10 to 300 nm.

  • Host Types: Viruses can infect various types of organisms, including animals, plants, and microorganisms (including bacteria).

Components of a Virus

  • Virion: The complete, infectious form of a virus outside the host cell, which can also be in the process of infecting a new cell.

    • Genetic Material: May consist of either DNA or RNA.

    • Capsid: A protein coat that encases the viral genetic material.

    • Envelope: A lipid layer that surrounds the capsid, found in some viruses.

What are Bacteriophages?

  • Definition: Bacteriophages, often referred to as phages, are viruses that specifically infect bacteria.

  • Genetic Composition: Similar to other viruses, bacteriophages possess either DNA or RNA as their genetic material.

  • Capsid Structure: The capsid serves as the protective protein coat that surrounds the viral DNA.

  • Procapsid: This is the precursor form of the capsid, which is necessary for the assembly of the infectious virus particle.

Formation of Viable Virus Particles

  • DNA Fitting into Procapsid: For successful assembly, the viral DNA must be the correct size to fit into the procapsid. If the DNA is oversized, it cannot fit properly.

  • Rolling Circle Replication: This process describes a mechanism of unidirectional nucleic acid replication, allowing the rapid synthesis of numerous copies of circular DNA molecules. The DNA is typically synthesized into a concatomer: a long and continuous DNA molecule composed of repeated sequences linked in series.

  • Enzyme Functionality: To ensure the viral DNA fits into the procapsid, an enzyme is produced that cleaves the DNA into appropriately sized fragments. Notably, this enzyme is also capable of cleaving host DNA into smaller pieces, resulting in fragmented host genetic material.

The Lytic Cycle of Bacteriophages

  • Example: P1 is a bacteriophage with double-stranded DNA (dsDNA) that infects E. coli and various other bacteria.

  • Overview of Phage Infection: The lytic cycle results in the eventual lysis (destruction) of the host cell, releasing newly formed virions.

Phage Lifecycle Stages

  1. Attachment: The phage attaches to the host cell via tail fibers.

  2. Penetration: The virus penetrates the host cell; phage lysozyme opens the cell wall, and the tail sheath contracts to inject the phage DNA into the host.

  3. Biosynthesis: The phage DNA directs the host cell's machinery to synthesize viral components.

  4. Maturation: Newly synthesized viral components are assembled into complete virions.

  5. Release: The host cell membrane is disrupted by phage lysozyme, leading to cell lysis and release of new virions.

Key Structures of T-Even Bacteriophages

  • Capsid (Head): 65 nm

  • Tail Fiber

  • Sheath

  • Baseplate

  • Pin

  • Total observable dimensions of the phage: approximately 40 nm (tail fiber) and other associated components.

The Lysogenic Cycle of Bacteriophages

  • Overview: During the lysogenic cycle, the phage genome integrates into the bacterial chromosome, existing as a dormant state for generations.

  • Prophage: A viral genome that has been inserted into the bacterial chromosome and can either exist integrated or as a plasmid.

  • Induction of Lytic Cycle: If the lysogen (the bacteria harboring the prophage) experiences stress, the integrated phage genome can be excised from the bacterial chromosome, thus triggering the lytic cycle and resulting in the eventual lysis of the host cell.

Life Cycle of Temperate Phages

  1. Attachment: Phage attaches to host and injects DNA.

  2. Replication: While in the lysogenic state, the host cell and viral DNA replicate and divide without causing immediate harm.

  3. Induction: The viral genome can trigger a transition to the lytic cycle when activated, leading to cell lysis and release of new phages.

Laboratory Cultivation of Viruses

  • Growth Medium: Viruses like bacteriophages must be cultivated within living cells.

  • Plaques: Bacteriophages can form plaques on a lawn of bacteria; these plaques represent clear zones in a bacterial growth medium resulting from bacterial cell lysis.

Calculation of Viral Titer

  • Definition: Viral titer is the lowest concentration of a virus that still effectively infects cells.

  • Titer Calculation Formula:

    • extPFU(PlaqueFormingUnit)/mL=racextNumberofplaquesperplateextVolumetaken(inmL)imesextDilutionofsampleplatedext{PFU (Plaque Forming Unit)/mL} = rac{ ext{Number of plaques per plate}}{ ext{Volume taken (in mL)} imes ext{Dilution of sample plated}}

  • Example Calculation:

    • If 100 µl (0.1 mL) of a sample with a dilution factor of 10^-6 is plated and produces 54 plaques:

    • extTiter=rac54(0.1extmLimes10−6)=rac541imes10−7=54imes107=5.4imes108extPFUext{Titer} = rac{54}{(0.1 ext{ mL} imes 10^{-6})} = rac{54}{1 imes 10^{-7}} = 54 imes 10^{7} = 5.4 imes 10^{8} ext{ PFU}