Module 2: Inheritance and Transfer of Genetic Material

Question 1

How was the ‘transforming factor’ discovered?

Answer 1

Type IIR cells (S. pneumoniae) and IIIS DNA was combined with different enzymes to experiment what the transforming factor was.
1. protease –> IIIS colonies formed –> not protein
2. RNase –> IIIS colonies formed –> not RNA
3. DNase –> no colonies –> DNA was transforming factor

Question 2

Describe the process of transformation

Answer 2

1. a competent bacterium can bind exogenous DNA and transport it into the cell
2. exogenous DNA is bound to receptor by competence proteins ComEA and ComG
3. DNA is pulled through the channel made of ComEC by ComFA (DNA translocase)
4. one strand of DNA is degraded, surviving strand is stabilised by RecA
5. surviving strand is integrated into the chromosome of the recipient cell

Question 3

Describe conjugation in E.coli

Answer 3

1. the donor bacterium synthesises an F pilus which contacts the recipient cell
2. the pilus is retracted, bringing the membranes of the two cells into contact
3. DNA is transferred across the membrane bridge using rolling circle replication

Question 4

How was conjugation discovered?

Answer 4

Auxotrophic strains Y-10 and Y-24 (both auxotrophic for different genes) were cultured together and individually on rich medium, resulting in colonies of all growing. When grown on minimal medium, only the mixture of strains produced colonies, as they produced prototr`ophic recombinants.

Question 5

Why are F+ cells prevented from conjugation with F+ cells?

Answer 5

TraS on the F plasmid blocks TraG (pore) activity
Inhibit uptake of other F plasmids

Question 6

F’ factor

Answer 6

F factors that are excised with an adjacent portion of the chromosome being included in the F factor

Question 7

Life cycle of the virulent (lytic) T4 phage

Answer 7

1. T4 phage attaches to bacterium and injects its DNA
2. Synthesis of phage-specific mRNAs begin
3. Replication of phage DNA begins, host DNA has been degraded
4. DNA filled heads are assembled
5. phage particles are fully assembled
6. Host bacterium is lysed, releasing progeny phage

Question 8

Lifecycle of a temperate bacteriophage

Answer 8

1. injection of DNA and cyclisation of phage DNA
2. phage enters lysogenic pathway
3. involves phage DNA integrating into bacterial DNA (called a prophage)

Question 9

Compare and contrast generalised vs specialised transduction

Answer 9

1. generalised transduction:
   - mediated by lytic and lysogenic phage
   - host DNA is hydrolysed into pieces and phage DNA is packaged
   - occasionally, bacterial DNA fragment is packaged into a phage capsid
2. specialised transduction:
   - only mediated by lysogenic pahge
   - prophage DNA excises incorrectly, taking adjacent bacterial DNA with it

Question 10

Describe site-specific recombination of bacteriophage DNA

Answer 10

1. integrase catalyses recombination
2. integrase binds to att P on phage DNA
3. the DNA-protein complex formed binds to att B bringing the two chromosomes together
4. integrase catalyses the DNA cutting, using a short region of sequence homology to form a heteroduplex joint

Question 11

Describe normal prophage excision

Answer 11

1. prophage loops out, and attBP pairs with attBP
2. site specific recombination
3. chromosome is excised

Question 12

Describe anomalous excision of prophage

Answer 12

1. appears to occur via Holliday junction migration
2. prophage loops out anomalously, with attBP and attBP not paired
3. recombination excises a chromosome carrying adjacent bacterial genes

Question 13

What are the types of bacterial transposons?

Answer 13

1. insertion sequences (cut and paste transposons)
2. composite (cut and paste transposons)
3. Tn3 element (replicative transposon)

Question 14

Describe IS elements and how they are inserted

Answer 14

• compact: only contain one or two genes
• gene products are only involved in transposition
• terminal inverted repeats are found at the ends
• usually encode tranposase that is needed for tranposition

1. two strands of target DNA are cleaved at different sites
2. IS element is inserted into the gap created by staggered cleavage
3. DNA synthesis fills in the gaps on each side of the IS element

Question 15

Describe Composite Transposons

Answer 15

• created when to IS elements insert near each other
• intervening DNA usually has a drug resistant cassette

Question 16

Tn3 elements

Answer 16

• larger than IS
• contain genes not required for transposition
• have simple inverted repeats at each end
• produce target site duplication when they transpose

Question 17

Transposition of Tn3 elements
draw a diagram

Answer 17

1. transposase encoded by Tn3 catalyses the formation of a cointegrate btwn the donor and recipient plasmids
2. Tn3 is replicates so there is a copy of the element at each junction in the cointegrate
3. resolvase produced by tnpR resolves the cointegrate by mediating recombination between the two Tn3 elements
4. donor and recipient plasmids separate, each with a copy of Tn3

Question 18

What two factors do conjugative R plasmids have?

Answer 18

1. resistance transfer factor (RTF) contains genes that are required for conjugative transfer between cells
2. R-determinant contains genes for antibiotic resistance

Question 19

Describe the evolution of conjugative R plasmids
draw a diagram

Answer 19

1. Recombination occurred between IS1 elements located in a non-conjugative R plasmid and a conjugative RTF plasmid
2. produces a conjugative R plasmid with antibiotic resistance