Lecture 11 Flashcards
What are the 2 life cycles bacteriophage lambda
Lytic cycle and lysogenic cycle
What is the first step of temperate bacteriophage transduction
Linear chromosome circularized in bacteria by annealing complementary ends and ligation
Lytic pathway
- Phage injects DNA
- Phage DNA circularises
- New phage DNA and proteins assembled into new phages
- Cell lyses, releasing the phages
Lytic cycle fate
lambda DNA replicated by rolling stone mechanism to form concatamer of linear DNA
Concatamer cut at cos sequence by ter enzyme
Produces lambda genome sized molecules - packaged into phage particles
Lysogenic pathway
Daughter cell with prophage has phage DNA circularised
Phage DNA integrates into bacterial chromosome becoming a prophage
Bacterium reproduces to copy prophage - transmits it to daughter cells - cell division produces populations of bacteria with prophage
Lysogenic pathway
- Site specific recombination with lambda DNA integrating at specific site in bacterial genome
- Recombination between attB and attP site
- attP and attB have spacer regions (O) - flanked by different integrase binding sites (B, B’, C, C’)
- Recombination at O
How is integrated prophage usually excised from chromosome?
Recombination between core O sequences of attL (B O C’) and attR (C O B’)
Prophage excision from chromosome can occur abnormally.
Result: Some prophage DNA stays in bacterial chromosome.
Excised bacteriophage is defective, lacking some prophage genome.
Excised bacteriophage carries bacterial DNA from the region around integration site
- Results in mixed phage lysate - contains wt lambda phage and lambda d gal+ phage
Lambda d gal+ phage mediate what?
Specialised transduction
Defective lambda d gal+ phage
- Defective phage:
can’t integrate at att sites
can’t replicate and enter lytic cycle
integrates by recombination with homologous chromosomal DNA
Specialised transducing phage
Transfer gal+ into recipient bacteria
Recombined into recipient chromosome - gal- to gal+
Specialised transduction limited to transducing genes close to att site
Transformation - uptake of ‘naked’ DNA
bacterial host with Donor DNA (a+, b+, c+)
DNA isolated
Recipient bacterium (a-, b-, c-) transformed by recombination
Transformants can be:
a-, b-, c- - No transformation from donor DNA
a+, b-, c- - a+ transformation
a+, b+, c-
a-, b+, c-
a-, b+, c+
a-, b-, c+
Why never a+, b+, c+, or a+, b-, c+?
Distance between a and c too great
What are transposons?
Pieces of DNA that move around the genome (or between chromosomal DNA and extrachromosomal elements) and insert at target sites by transposition
What organisms are transposons found?
All and form large part of genome
What can transposon insertion affect?
Genes, gene regulation, chromatin structure, genome stability, evolution
How can transposons move?
Excision and integration
Replication
3 main types of transposon
Insertion sequences - Bacterial DNA-only transposon - code for enzyme needed for transposition, flanked by short inverted terminal repeats
Composite transposons
Non-composite transposons
Composite transposons
- Pair of IS elements flank another gene
- Same (Tn9) or inverted orientation (Tn5 or Tn10)
- Often carry genes for drug resistance
- IS10 transpose alone
- Can be flanked by pair of IS elements but still transpose
Non-composite transposons
TnA family
Large, ~5kb transposon, not dependent on IS-type elements
Independent units, genes for transposition and drug resistance
Tn3, Tn1000
Terminal inverted repeats, generate 5bp direct repeat at target site
Transposition mechanism
DNA-only transposons move by cut-and-paste mechanism - element excises and inserts into target, using small amount of replication to join sites
- Some in bacteria move by nick-and-paste - attached to donor DNA and joined to target, forms cointegrate which resolves to 2 molecules each containing a transposon
- Few transposons can move by both
Where does DNA cut-and-paste transposition occur?
Transpososome structures
Describe transposition at molecular level
- terminal inverted repeat DNA recognised by transposase
- transposases oligomerize - transposon ends together
- Transposon cleavage activated
- Transposase/cleaved transposon complex bind target DNA - 3’ end attacks target DNA, join at staggered positions
- ssDNA filled by host repair
How are Hfr strains generated
recombination between transposons on F-plasmid and bacterial chromosome
