L11: Specialised transduction and transposons

Question 1

Temperate bacteriophages

Answer 1

• Can adopt two lifestyles…
• Virulent (lytic)
• Lysogenic
• Occasionally, lytic cell cycle is induced (e.g. under stress), prophage will excise the genome
• Lysogenic cycle important for specific transduction

Question 2

Linear genome of bacteriophages

Answer 2

• 48 Kbp
• Mostly double stranded, with 12 nt 5’ single strand ends
• Circularisation mediated by these complementary ends annealing

Question 3

Possible fates for circularised lambda genome

Answer 3

1. Lytic: Circularised lambda DNA replicated by rolling-circle mechanism to produce a concatemer of linear DNA.
   Concatemer cut at cos sequence by Ter enzyme to produce lambda genome sized molecule (packaged into phage particles, back to linear w/ 5’ overhangs as in phage)
2. Lysogenic: Site specific recombination. Occurs between bacterial attB site and attP in the lambda DNA
   Both contain a spacer region, O, that is flanked by different integrase binding sites (B, B’ and C, C’) (O is common to both att sites)
   -> results in attL and attR sites in prophage; composite
   - Forward = integration
   - Backward = excision

Question 4

Mixed phage lysate

Answer 4

• Results from aberrant excision of prophage form chr.; very rare
• Some prophage DNA remains in bacterial chr., excised bacteriophage lacks some prophage genome (d defective), but carries bacterial DNA from region flanking integration site
• Contains wt lambda phage and lambda d gal+ phage
  -> this phage mediates specialised transduction (can’t integrate att site as it lacks attP, and can’t replicate/enter lytic cycle
  -> integrates by recombination w/ homologous chr. DNA (transfers gal⁺ gene into reciient bacteria)
  -> limited to transducing gees close to att site

Question 5

Application of co-transformation frequency

Answer 5

• Transformation: uptake of ‘naked’ DNA; transformed DNA exchanged into (recombined) recipient genome
• Co-transformation frequency can be used to map bacterial gene order

Question 6

Transposable elements (aka transposons)

Answer 6

• Pieces of DNA that can move around genome and insert at target sites by transposition
• Found in all organisms and can comprise large parts of genome; move to different places in genome or between chr. DNA and extrachromosomal elements (e.g. plasmids w/in same cell)
• NEVER found independently

Question 7

How do transposons move?

Answer 7

• Excision and integration:
  Does not require sequence homology. Results in target-site duplications (small sequences at insertion site that become duplicated due to DNA repair mechanisms)
• Replicative transposition

Question 8

Main types of transposon (x3)

Answer 8

1. DNA-only cut and paste element
   Have terminal inverted repeats (TIR) that are recognised by encoded transposase
2. Long terminal repeat elements (LTR); retroviruses or similar. Encode several proteins including a reverse transcriptase (euk only)
3. Non-LTR; can be aut. or non-aut, encode proteins with a range of activities
   - Autonomous transposons encode the proteins needed to move the DNA (e.g. LINE); non-autonomous transposons rely on proteins made my an autonomous element (e.g. SINE)

Question 9

Where are DNA-only cut and paste transposons found? Relevance for bacteria?

Answer 9

• Widespread in bacteria and euk
• The only type of transposon found in bacteria

Question 10

Site specific recombination

Answer 10

• Circular lambda DNA integrates into bacterial genome at specific site; rec. occurs between bacterial attB site and attP in the lambda DNA
• Both contain a spacer region, O, that is flanked by different integrase binding sites (B, B’ and C, C’)
• On rare occasions, prophage is excised aberrantly
  -> some prophage DNA remains in bacterial chr., excised bacteriophage is d defective

Question 11

Defective gal+ phage (Specialised transduction; its limitation)

Answer 11

• Cannot integrate at att site (lacks attP)
• Cannot replicate and enter lytic cycle
  -> integrates by rec. w/ homologous chr. DNA
• Specialised transducing phage transfers gal+ gene into recipient bacteria
  -> gal- to gal+
• Limited to transduction of genes close to att site

Question 12

Transposable elements + mutation

Answer 12

• Major source of mutations (insertion of transposon -> KO of gene function)
• Insertion can affect genes, gene regulation, chromatin structure, genome stability and evolution

Question 13

Transposable elements between species (give examples)

Answer 13

• Vary widely in transposon presence and composition
• Maize: 85% transposon
• E.coli: ~1% transposon

Question 14

Types of bacterial DNA-only transposons

Answer 14

1. Insertion sequences (IS elements; must have short ITRs)
2. Composite/compound transposons; pair of IS elements flank another gene. May be in same (e.g. Tn9) or inverted orientation (e.g. Tn10, Tn5). Often carry genes for drug resistance. Either whole composite transposon or just the functional IS can transpose
3. Non-composite; TnA family. Large, not dependent on IS-type elements.
   Independent units; genes for transposition as well as genes for encoding drug resistance (e.g. Tn3, Tn1000).
   -> TIRs flank several genes

Question 15

Cut-and-paste mechanism

Answer 15

• Most DNA-only transposons move in this way
• Element excises completely, inserts into target (using small amount of replication to repair the join sites)

Question 16

Nick-and-paste mechanism

Answer 16

• In bacteria only, some DNA-only transposons move in this way
• Transposon remains attached to donor DNA and is joined to target (forming cointegrate, eventually resolved into 2 molecules, each containing a transposon)

Question 17

Transposition process

Answer 17

• Occurs in transposome structure
• TIR DNA recognised by transposase
• Transposases oligomerise, bringing transposase ends together, activating transposon cleavage from the background DNA
• Transposon/cleaved transposon complex binds to target DNA. adn 3’ ends attack target DNA, and join at staggered positions
• ssDNAs filled by host repair

Question 18

Transposons in episomes/plasmids

Answer 18

• Transposons present in E.coli chr. as well as F factors and R plasmids
• In F plasmid, IS in F factor is required for formation of Hfr strains
  -> generated as a result of recombination between IS in F plasmid and IS in bacterial chr.
• R plasmids: Antibiotic resistance genes typically sit within transposons