Lecture 11 - Bacterial Genetics II

Question 1

What is specialised transduction?

Answer 1

Specialised transduction is mediated by temperate bacteriophages (e.g. bacteriophage λ)
1. The phage injects phage DNA into the bacteria
2. The phage then circularises
The lytic cycle or lysogenic cycle can then proceed

The lysogenic cycle
The phage DNA integrates into the bacterial chromosome, becoming a prophage.
The bacterium reproduces copying the prophage and transmitting it to daughter cells.
The cell divides produce a population of bacteria infected with the prophage
Occasionally, a prophage exists the bacterial chromosome initiating a lytic cycle

The lytic cycle
New phage DNA and proteins are synthesised and are assembled into phages
The cell lyses releasing the phages

Question 2

Discuss the lytic pathway for circulised λ DNA.

Answer 2

The linear chromosome is first circularised in bacteria by annealing of complementary ends and ligation. There are two possible fates for circularised λ.

1. Lytic pathway - circularised λ DNA is replicated by rolling circle mechanism to produce a concatemer of linear DNA. The concatemer cuts at cos sequence by Ter enzyme to produce λ genome sized molecules - packaged into phage particles.

The second is the lysogenic pathway.

Question 3

Describe the lysogenic pathway for bacteriophage lambda.

Answer 3

2. Lysogenic pathway - Circular λ DNA integrates into bacterial genome at a specific site - site specific recombination
   Recombination occurs between bacterial attB site and attP in the lambda DNA. Both attP and attB contain a spacer region, O, that is flanked by different integrase binding sites (B. B’ and C, C’). Site-specific recombination occurs within sequence ‘O’ - the core sequence common to both att sites.
   The integrated prophage is normally excised from chromosome by recombination between core O sequences of attL (Left) (B O C’) and attR (C O B’) (Right). On rare occasions the prophage is excised from the chromosome aberrantly- some prophage DNA remains in the bacterial chromosome and the excised bacteriophage lacks some prophage genome (d defective) but carries bacterial DNA from region flanking integration site. This results in mixed phage lysate.
   The λd gal+ Phage mediates specialised transduction and can inject its DNA into new bacteria but:
   ○ Defective phage cannot integrate at att site (lacks attP)
   ○ Defective phage cannot replicate and enter lytic cycle
   ○ Defective phage integrates by recombination with homologous chromosomal DNA
   ○ Specialised transducing phage transfers gal+ gene into recipient bacteria.
   ○ Recombined into recipient chromosome -converts gal- to gal+
   Specialised transduction limited to transducing genes close to att site.

Question 4

What is transformation?

Answer 4

Uptake of naked DNA as a method for gene transfer

Question 5

What are transposable elements and transposons.

Answer 5

• Transposable elements (aka transposons) are pieces of DNA that can move around the genome and insert at target sites by transposition.
  
  • Transposons are found in all organisms and can comprise large parts of genomes
  • Transposons move to different places in the genome, or move between chromosomal DNA and extrachromosomal elements e.g. plasmids within the same cell.
  • Transposable elements p- a major source of mutations - insertion of transposon in a gene will knock-out its function.
  • Transposon insertion can affect genes, gene regulation, chromatin structure, genome stability and evolution
    No independent form of the element

Question 6

How do transposons move?

Answer 6

Transposons can move by excision and integration or by replication
* Transposition does not requires sequence homology for insertions.
* Insertions result in target-site duplications - small sequences at the insertion site that become duplicated due to DNA repair mechanisms
* The amount of duplication is characteristic of the particular transposon

Question 7

What are the three main types of transposon.

Answer 7

1. DNA-only cut and paste element
   ○ DNA only-cut and paste elements have terminal repeats that are recognised by the encoded transposase
   ○ Autonomous transposons encode the proteins needed to move the DNA.
   ○ Non-autonomous transposons rely on proteins made by an autonomous element
   ○ DNA-only cut and paste transposons are widespread in bacteria and eukaryotes and are the only type of transposon found in bacteria.
   DNA transposons always contain an element bordered by short inverted repeats that are recognised by element-encoded transposase.
   Bacterial elements carry other genes such as pathogenicity factors and genes for antibiotic resistance (this has contributed to spread of antibiotic resistance in bacteria
   Eukaryotic DNA-only elements usually only contain the transposase.
2. Long terminal repeat elements - are retroviruses, or similar to retroviruses. They encode several proteins including a reverse transcriptase
3. Non-LTR elements can be autonomous or non-autonomous, and encode proteins with a range of activities.
   ○ Long interspersed Elements (LINE) encode proteins that mediate their own transposition (these are common in the human genome)
   ○ Short interspersed Elements (SINE) do not encode proteins for their movement, and so rely on those from LINEs

Question 8

Describe some Bacterial DNA-only transposons.

Answer 8

1. Insertion sequences
   Bacterial DNA cut and paste elements that encode their own transposition functions are called insertion sequences. Insertion sequence - codes for enzyme needed for transposition, flanked by short inverted terminal repeats.
2. Composite transposons
   In composite transposons a pari of IS elements flank another gene. IS elements may be in same (Tn9) or inverted orientation (Tn10, Tn5). Composite transposons often carry genes for drug resistance. Function IS element (IS10) can transpose alone. Composite transposon flanked by a pair of IS elements can transpose.
3. Non-composite transposons -TnA family - Large, ~5kb transposons, not dependent on IS-type elements. Independent units, genes for transposition as well as genes encoding drug resistance. Terminal inverted repeats, generate 5bp direct repeat at target site.

Question 9

Describe the mechanisms of transponsons.

Answer 9

• Most DNA-only transposons move by cut and paste mechanisms
  
  • In cut and paste, the element excises completely and inserts into the target, using a small amount of replication to repair the join sites
  • In bacteria only, some DNA-only transposons move by nick and paste
  • In nick and paste, the transposon remains attached to the donor DNA and is joined to the target from in g a cointegrate which is eventually resolved into two molecules each containing a transposon.
    In bacteria a few transposons can move by both mechanisms

Question 10

How do transposons work on the molecular level.

Answer 10

• DNA cut-and-paste transposition occurs in transposome structures
  
  • Terminal inverted repeat DNA recognised by transposase
  • Transposases oligomerize, bringing transposon ends together activating transposon cleavage from the background DNA
  • Transposase/cleaved transposon complex binds to target DNA, and the 3’ ends attack target DNA and join at staggered positions
    Single stranded DNAs are filled by host repair.