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required steps for DNA transfer
- 1. cell-cell contact of donor and recipient (more than just touching)
- 2. preparing the DNA for transfer = mobilization3. DNA transfer
- 4. reformation of circular DNA in the recipient cell
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the most common way to transfer DNA
conjugation
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conjugation
plasmids transferring DNA molecules from one cell to another
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self-transmissible plasmids can do steps ? and ? driving their own cell to cell transfer
- steps 1 and 2
- 1: tra genes enable formation of a mating pair
- 2. oriT genes determine where on the plasmid DNA transfer is initiated
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self-transmissible plasmids must have a complete set of ? genes
tra: allow the plasmids to transfer themselves into other bacteria
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mobilized plasmids can do step ? bur NOT step ?
step 2: can be transferred by other self-transmissible elements
step 1: canNOT transfer itself into other bacteria
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examples of self-transmissible plasmid
F plasmid: can integrate itself into the bacterial chromosome by homologous recombination, mutant that always expresses the tra genes
R plasmid: a conjugation factor that promotes resistance to agents such as antibiotics and bacteriophage
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what are mobilizable plasmids?
plasmids that lack genes to initiate self transfer but DO encode the functions needed specifically for transfer of their own DNA
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example of mobilizable plasmids
ColE1: inhibits other E.coli strands
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mobilizable plasmids must have ? genes
mob: proteins nick DNA at the origin of transfer (oriT) and initiate single stranded transfer of DNA into the recipient bacterium
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non-transmissible plasmids canNOT
- 1. transfer itself into other bacteria
- 2. be transferred by other self-transmissible elements
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cell to cell contact is dependent upon what system?
- mpf system (mating pair formation):
- functions as a secretion machinery for intercellular DNA transfer during bacterial conjugation
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is bacteria reproduced sexually?
NO, it reproduces asexually
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cell to cell contact sex pilus is produced from a single protein called ? a product of the ? gene
Pilin, traA
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cell to cell contact requires a ? donor cell. produced by the F plasmid is called ?
sex pilus, "F pilus"
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cell to cell contact pilus extends from donor cell and contacts recipient cell to produce the ?
mating pair
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the pilus ? bringing the cells into close contact. ? and ? stabilize cell to cell interaction
the pilus depolymerizes bringing the cells into close contact. TraN and TraG stabilize cell to cell interaction
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the actual structure of the "tube" through which the DNA passes is made of several ?
tra proteins
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DNA trasnfer is dependent on the plasmid encoded system
Dtr (DNA transfer and replication)
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the DTR sstem includes ? gene products that cause ?
include tra, cause initiation of DNA replication, DNA trasnfer, reformation of the plasmid in the recipient cell
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the F plasmid has how many different origins of replication and what are they ?
TWO: oriV (otigin of replication) & oriT (origin of transfer)
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oriV is used only for plasmid replication during the ? and promotes what ?
vegetable cell cycle, bidirectional theta-form replication
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oriT is used during ? and promotes ?
conjugative plasmid transfer, looped rolling circle replication
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to begin "looped rolling circle replication" production of ? at oriT
a single stranded nick
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looped rolling circle replication steps
- 1. leading strand is covalently linked to parental template for the lagging strand (if double stranded)
- 2. prior to precursor synthesis, the linear branch had a 5' terminus ("A" protein acted there)
- 3. rolling circle continues unabated forming concatemers (dd or dd)
- 4. circular template for leading strand synthesis never leaves the circular part of the molecule
- 5. DNA can only be nicked to start the process IF it is supercoiled
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a complex of two plasmid-encodes proteins ? bind to ?
Tral and TraY, oriT
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? is called the relaxase, when it nicks at oriT, it becomes convalently attached to the ? end of DNA
Tral, 5'
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a plasmid that is not self-transmissible but that is mobilizable must have only the ? system
Dtr (DNA transfer replication)
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the strand the is peeled off of the rolling circle is transferred into the
recipient cell
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the ? end is transferred into the recipient cell first
5' end
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the ? protein, bound to the 5' end is transferred into the recipient cell an remains in the ?
region of cell-to-cell contact
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the 5' end is help at the ? while the strand that is peeled off into the recipient
cell fusion point
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strand DNA synthesis takes place in the donor cell
leading
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DNA-pol (I/II/III) used the 3' end as a primer and proceeds around the plasmid until it gets back to oriT
DNA pol III
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strand DNA synthesis takes place in the recipient cell
lagging
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? are produced and ? are synthesized
RNA primers, okazaki fragments
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when the leading strand repilcation has gone all te way around the circle:
Tral nicks at oriT again, releasing the entire displaced strand, ligation in the donor cell by Tral regenerates a complete double stranded circle
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synthesis of the complementary strand
the product of one other tra gene is a primase, it makes RNA
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after competed conjugation the recipient is now what?
transconjugant
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transconjugant
carries out F plasmid replication, initiated at oriV, each time it divides, has now become a potential donor, will exhibit "surface exclusion" aka entry exclusion
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a cell exhibiting surface exclusion expresses the ? and ? protein on its surface
TranS and TraT
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a cell exhibiting surface exclusion can/can't form mating pairs with other f-plasmids
CAN'T
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a cell exhibiting surface exclusion can/can't be a recipient of the same plasmid or any other plasmid transferred during conjugation
CAN'T; F cells can't serve as a recipent to other F cells, but they CAN however serve as recipient to a plasmid not similar to the F plasmid
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a cell exhibiting surface exclusion can/can't still be a recipient for conjugation that uses a pilus constructed from a different pilin
it CAN
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