Conjugation

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Conjugation
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The Sex Pilus
-Some E. coli strains contain Fertility plasmids
(F)
- Carries the information required for its own
transfer
- DNA is NOT transferred through the pilus
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Overview of Conjugation
1. Plasmids are either self-transmissible or
mobilizable.
2. The transfer systems are encoded by the tra
genes (contained on the plasmids themselves).
3. Tra systems are linked to their
incompatibility (Inc) group. F-type ? IncF, RP4
plasmids ? IncP
4. Plasmids that have tranfer systems that allow
transfer of DNA to unrelated species are known as
promiscuous plasmids. IncW plasmids, IncP
plasmids, IncN.
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Mechanism of self-transmissible transfer
1. A site on the plasmid, known as the origin of
transfer (oriT) is nicked by a specific
endonuclease (one of the tra gene products).
2. A pore is formed between the two cells and
only ONE strand of DNA is passed through to the
other cell (5 first).
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3. The single strand in each cell undergoes
replication to form double stranded DNA.
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Mechanism of plasmid mobilization
1. The mob plasmid cannot transfer without
another plasmid
2. The other plasmid may or may not be a
self-transmissible plasmid but MUST contain tra
functions (cell contact, nicking).
3. If the other plasmid is self-transmissible it
may also transfer.
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Molecular Mechanism of Transfer by F-plasmid
1. F-plasmid is 100kb takes about 5 min to
transfer.
2. Sequences required for transfer are located
within a 33.3-kb transfer region (IncF1)
3. Pilus synthesis and assembly -Nicking
initiation of transfer -mating pair (pore)
formation -transfer of DNA
-mating-aggregate stabilization -surface
exclusion -regulation
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F-Pilus
1. Extracellular filament that extends from
surface.
-main structural component is a single subunit
(pilin) -tip protein
2. Pilus recognizes various receptors on the host
cell (lipopolysaccharide, protein
(OmpA)). -mating pair formation may occur
differently on solid or liquid media
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Major Functions During Transfer (gt40 genes)
1. TraI has nuclease helicase (ATP) activity.
Function enhanced by TraY IHF
TraI is a transferase ? covalent attachment of
the 5 end of the DNA to the protein
2. TraD active transport, binds DNA, ATP/GTP
binding sites
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Surface Exclusion
1. Reduces conjugation among cells carrying
closely related plasmids (5 exclusion groups
identified to date).
2. TraT outer membrane protein that blocks
mating-pair formation TraS block DNA transfer
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Chromosome Transfer formation of Hfr strains
(high frequency recombination)
In rare instances a plasmid (F) can integrate
into the chromosome.
There are two mechanisms of integration 1.
Homologous recombination 2. Transposition
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Chromosome Transfer Hfr ? Recipient cell
(Part 1)
1.
2.
3.
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Chromosome Transfer Hfr ? Recipient cell
(Part 2)
3.
4.
5.
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tra region of the F plasmid
regulation
Transfer genes
traJ
activator
traY/traI DNA nicking and unwinding
Channel?
Direction of transfer
oriT traM J YALEKBPVRC WU N trbCDE traF trbB
traH G ST D I/Z
finP
Surface exclusion
pilin
Senses that mating pair formed
Negative regulator transcript
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Notes on Chromosome transfer
1. Rate of transfer is constant it takes 100 min
to transfer the entire chromosome. Typically only
relatively short stretch of DNA are transferred.
2. Chromosome transfer usually does not result in
conversion of recipient cell to F.
3. In chromosome transfer donor DNA integrates
into the host genome by recombination or
transposition (in plasmid transfer this does not
occur).
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Formation of Prime Factor Plasmids
1. Plasmids that leave the genome carrying
chromosomal DNA are known as prime factors.
2. They leave the chromosome by homologous
recombination, resulting in a deletion in the
chromosome
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Transposition And Non-Homologous Recombination
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1. Transposons are DNA elements that hop or
transpose to different places on the DNA.
2. Transposition is the movement of the
transposon.
3. This process requires special protein factors
particularly to cut and ligate the DNA ?
transposase
4. NO homology is required between the transposon
and the target sequence.
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1. Bacterial transposons contain inverted
repeats at either end.
2. The transposase is encoded by sequences within
the transposon (i.e. between the inverted
repeats).
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1. The smallest transposons are called insertion
sequence (IS) elements. 750-2000 bp.
2. Most bacteria contain several IS elements and
often more than one copy on the chromosome
3. During insertion a small portion of the target
sequence is duplicated.
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2 IS elements of the same type can form a larger
transposon called a composite transposon.
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Composite transposons can move independently OR
as a unit
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Summary on Transposons
1. The smallest transposons are called insertion
sequences.
2. IS elements have inverted repeats at either
end and a transposase gene in between, they
typically do NOT have any resistance markers.
3. Two IS elements (having 4 inverted repeats)
form a composite transposon.
4. Non-composite transposons have short inverted
repeats, a transposase resistance marker.
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Molecular mechanism of transposition (I)
Cut Paste Model
1. Enzyme ? transposase
2. Both strands transpose
3. Transposon leaves donor DNA? only one copy of
transposon exits
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Molecular mechanism of transposition (II)
Replicative transposition
1. transposase
Note the cuts are on different strands
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Molecular mechanism of transposition (II)
Replicative transposition
2. Ligation to target ends
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Molecular mechanism of transposition (II)
Replicative transposition
3. 3-ends prime replication
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Molecular mechanism of transposition (II)
Replicative transposition
4. Formation of co-integrate and resolution
(resolvase TnpR)