Chapter 9 Bacteria and Bacteriophages

Bacteria and Bacteriophages

Genetic Transfer in Bacteria

  • Recombination in Bacteria:
      - Vertical gene transfer: Occurs within species.
      - Horizontal gene transfer: Occurs between species.

  • Three Processes for Transferring Genetic Material:
      - Conjugation
      - Transformation
      - Transduction

Escherichia coli (E. coli)

  • Growth Media:
      - Simple defined solid or liquid medium: Provides minimal essential nutrients.
      - Minimal Medium: The simplest set of chemicals needed for E. coli to grow.
      - Wild-type Strains (Prototrophs): Can synthesize all components needed for growth.
      - Complete Medium: Supplies all vitamins, amino acids, etc., needed for growth in case of mutations in biosynthetic pathways.
      - Auxotrophs: Strains unable to synthesize essential nutrients; require supplementation.

  • Manipulation: E. coli is easily manipulated using standard microbiology techniques.

Conjugation

  • Definition: A process of unidirectional transfer of genetic material from one bacterial cell to another through direct contact (from donor to recipient - transconjugant).
      - Discovery: Joshua Lederberg and Edward Tatum (1946) explored conjugation by crossing auxotrophic E. coli.
      - Importance of Cell Contact: Bernard Davis demonstrated that cell-to-cell contact is necessary for conjugation.

Sex Factor F

  • F Factor:
      - Definition: A plasmid that mediates conjugation implemented by the donor cell (F+), while the recipient is F-. Mating does not occur between individuals of the same type.
      - Structure: Contains an origin of DNA transfer (o) and genes coding for the F-pili (sex-pili), which are hair-like components that assist in mating.

Transfer of Genetic Material in Conjugation

  • Outcome of Conjugation: Following conjugation, both cells become F+.

  • Process Summary:
      1. Initial Conjugation: F+ cells conjugate with F- cells.
      2. Nicked Strand: The F factor undergoes nicking, where a strand is nicked and transfers to recipient.
      3. Strand Transfer: The nicked strand transfers to the recipient cell.
      4. Copier Strands: The remaining strands in both cells are copied.
      5. Completion of Transfer and DNA Synthesis: Finalizes the transfer of F factor, resulting in both cells being F+.

High-Frequency Recombination (Hfr)

  • Definition: Special derivatives of F+ strains that allow for recombination between bacterial chromosomes.

  • Crossover Mechanism: A crossover between bacterial chromosome and F factor leads to the transfer of bacterial genes into recipient cells.

  • Conjugation Capability: Hfr strains can conjugate with F- cells.

F′ Factor

  • Definition: Occurs when excision of the F from the chromosome is imprecise, resulting in the inclusion of a small section of the host chromosome within the plasmid.

  • Function of F′ Cells: Can conjugate with F- cells and introduce bacterial genes from another cell, turning the recipient into a partially diploid (merodiploid) organism. This process is known as F-duction or sexduction.

Gene Mapping with Conjugation

  • Early Mapping Studies: François Jacob and Elie Wollman in the 1950s used Hfr strains in interrupted-mating experiments to map bacterial genes.

Mapping Genes on Circular DNA

  • Transfer Map Overlaps: The overlaps in transfer maps from different Hfr strains aid in reconstructing the overall structure of the circular bacterial chromosome.

Transformation

  • Definition: Unidirectional transfer of extracellular DNA (e.g., plasmids) into bacterial cells, often resulting in phenotypic changes.

  • Procedure: Donor DNA is extracted, purified, fragmented, and then added to recipient cells.
      - Competence in E. coli: Unlike other strains, E. coli requires treatment to become competent for transformation.

  • Methods to Make Cells Competent:
      - Electroporation
      - Heat Shock
      - Chemical Treatment

Gene Mapping using Transformation

  • Cotransformation: This occurs when linked genes transform together at frequencies higher than expected by random events. The relationship among genes can be inferred by comparing transformation frequencies with a third gene.
      - Example: If genes p and o rarely cotransform, their order is p-q-o.

Transduction

  • Definition: The process by which bacterial viruses (bacteriophages) transfer genes from one bacterium to another. The viruses involved are referred to as phage vectors.

  • Mechanism: Once introduced into the recipient bacterium, the recombinant viral DNA undergoes homologous recombination with the bacterium's chromosome (the transductant).

Bacteriophages

  • Structure: Composed of DNA or RNA encased in a protein coat.

  • Variability in Viral Types: Variations in viral protein determine type characteristics.

  • Life Cycles of Different Phages: Phages undergo different life cycles which include:
      - Lytic Cycle (e.g., T2 phage)
      - Lysogenic Cycle (e.g., λ phage)

Lytic and Lysogenic Cycles of λ Phage

  1. Attachment: λ phage binds to bacterial cell wall.

  2. Entry: An enzyme from the phage digests the cell wall, introducing its linear chromosome into the bacterial cell.

  3. Integration (Lysogenic Cycle): The phage chromosome circularizes and can integrate into the bacterial chromosome, becoming a prophage.

  4. Cell Division: As the bacterium grows and divides, the chromosome containing the prophage replicates.

  5. Progeny Formation: Phage particles assemble within the cell and each particle contains one copy of the linear phage chromosome.

  6. Lytic Pathway: If certain conditions arise, the phage chromosome may excise from the bacterial chromosome to initiate the lytic cycle, leading to breaking down the bacterial chromosome.

  7. Production of New Phages: Viral components and new phage chromosomes are synthesized.

Generalized Transduction

  • Gene Transfer: Capable of transferring any gene from the donor bacterium to a recipient.

  • Historical Experimentation: Conducted by Lederberger and Norton Zinder (1952) using Salmonella typhimurium, demonstrating viral-mediated recombination despite physical barriers.

  • Cotransduction: Closely linked genes are typically cotransduced.

Generalized Transduction Mapping of Bacterial Chromosomes

  • Cotransduction occurs primarily when genes are situated near each other on the chromosome and are transferred together within the phage head.

  • Alternative Scenario: Genes can be carried in separate phages that infect the same bacterial cell, although this is exceedingly rare.

  • Cotransduction Rate Use: Used to calculate genetic map distances and order.

Specialized Transduction

  • Specific Gene Transfer: Specialized transduction occurs when only specific genes are transferred.
      - Mechanism: This can arise from crossover events between the circular bacterial chromosome and the circularized phage chromosome.

  • Initial Low-Frequency Transducing Lysate (LFT): Results from the induction of a lysogenic bacterium, which leads to a process known as outlooping.
      - Normal Outlooping: Produces standard phages.
      - Rare Outlooping: Can lead to the production of transducing phages carrying new genetic material.

Mapping Bacteriophage Genes

  • Phage Forms: Multiple phage forms can infect bacterial cells and undergo crossover between two distinct phage genomes.

  • Recombination Frequency: This frequency can be utilized for mapping genetic locations within the phage.

Complementation Test

  • Purpose: This test is employed to ascertain how many genes account for a specific phenotype set among mutations.

  • Genetic Interpretation: If no complementation occurs, the mutations are on the same gene; conversely, mutations that complement reside on different genes.

  • Gordon Benzer's Contribution: He referred to this functional unit as the “cistron,” identified as the minimal DNA segment that encodes either RNA or a functional “gene.”

Chapter 15 Animation

  • Additional Resource: The Complementation Test is further elucidated in an accompanying animation.