Lecture 15 - Gene Transfer

What is bacterial transformation, and how did Griffith’s experiment show evidence of a transforming principle?

• Transformation is when a bacterium takes up free DNA from its environment

• Griffith mixed live R bacteria (no capsule) with heat-killed S bacteria (had a capsule)

• The mouse died, and he recovered live S-type bacteria

  • Something from the dead S cells gave the live R cells the ability to make a capsule: Griffith called it the transforming principle

How did Avery, MacLeod, and McCarty identify DNA as the transforming principle, and what does it mean for a bacterium to be competent?

Elimination Experiment: 

• Took an extract from heat-killed S bacteria and used enzymes to break down one type of molecule at a time

• If an enzyme (DNase) breaks down a molecule (DNA) and transformation stops, that molecule (DNA) is necessary for transformation

• DNA was required for the R → S change.
  
  

• A competent bacterium is in a temporary state where it can actively take up free DNA

How do bacteria become competent?

Bacteria naturally become competent to take up DNA in a response to stress

• Stress responses: Bacteria activate competence as a response to DNA damage or when under attack from antibiotics 

• Environmental cues: Nutrient starvation, particularly a lack of N, P, or C, triggers competence to obtain nutrients 

• Quorum sensing: High population densities allow bacteria to communicate and simultaneously activate competence genes to exchange genetic information

What is transduction, and how do generalized and specialized transduction differ?

Transduction is when a bacteriophage carries bacterial DNA from one bacterium to another.

• Generalized: During the lytic cycle (bursting of cell), a phage accidentally packages a piece of bacterial DNA

  • The piece can come from anywhere on the bacterial chromosome

• Specialized: Occurs during lysogenic cycle (hides in prophage and replicated), A prophage is cut out of the bacterial chromosome and accidentally takes a neighboring bacterial gene with it

  • The phage can transfer only genes near its insertion site

What is conjugation, and how do the pilus and conjugation machinery help an F⁺ cell transfer the F plasmid to an F⁻ cell?

Conjugation is DNA transfer through direct cell-to-cell contact

• The F pilus attaches to the F⁻ recipient and brings the cells togethe

• The conjugation machinery sends one strand of the F plasmid through a mating channel

• Both cells make the complementary strand, so the donor stays F⁺ and the recipient becomes F⁺

What are the three possible fates of foreign DNA taken up by a bacterium, and what must happen for daughter cells to inherit the new gene?

1. DNA may be degraded or lost

   1. Degradation and nutrient utilization - under nutrient-limiting conditions, bacteria may use DNA as a carbon and nitrogen source

      1. dsDNA- double-stranded DNA

      2. A, T, C, G - adenine, thymine, cytosine, guanine

      3. PD - phosphodiester backbone of DNA

2. DNA may integrate into the chromosome

   1. The new gene is copied along with the chromosome when the cell prepares to divide

   2. Chromosomal recombination - useful DNA from environment/other cells are integrated into bacterial chromosomes through recombination

3. DNA may replicate independently as a plasmid

   1. Maintained as a plasmid - a small, circular piece of dsDNA that is separate from chromosomal DNA, and can replicate on its own

   2. The new gene stays on a separate piece of DNA that can make copies of itself inside the cell

• For a daughter cell to inherit the new gene, the DNA carrying it must be copied and passed to that daughter cell during division (can happen through either the chromosome or a plasmid)