Flashcards in Plasmids and Conjugation Deck (37):
What is incompatibility?
The inability of plasmids to exist in the same cell if they have the same replication mechanisms
What are the three molecular forms that plasmids may take?
supercoiled, linear, open-circle
What is the copy number and how does the copy number of a plasmid affect partitioning?
Copy number is the number of plasmids per cell. Small high-copy number plasmids have random plasmid partitioning whereas large low-copy plasmids are directed into each daughter cell
What are the two types of host range in plasmids?
Narrow: plasmid will only replicate in related species
Broad: plasmid will replicate in many hosts
Give an example of a broad host range plasmid
RP4, can grow in gram -ve or gram +ve bacteria
What 5 characteristic may plasmids encode?
antibiotic resistance, metal(oid) resistance, virulence determinants, bacteriocin production (antimicrobial-kills bacteria without plasmid), biodegradative properties
What is a transposon (give an example)?
A DNA sequence with the ability to move to other parts of the genome e.g. bla (beta-lactamase), encodes ampicillin resistance
What 4 things would a useful vector plasmid need?
orin which replicates in host, antibiotic resistance (or B/W selection to determine uptake), multiple cloning sites, promoter (inducible/constitutive or strong/weak)
What are the advantages and disadvantages of a strong promoter?
adv: gene expression is consistent and high
dis: constitutive expression, not inducible, so not controllable
What makes a suicide vector and what is it purpose?
A suicide vector occurs if a plasmid has an oriV which cannot replicate in host of interest. Kills host if the host doesn't take plasmid into it's genome
How does a suicide vector work?
If a host cell takes up a plasmid but does not incorporate the plasmids DNA into it's genome the plasmid will circularise and express genes which result in cell death
What is a shuttle vector and what is it used for?
A plasmid which propagates in two different species, can be used to clone a gene in one species and then transfer to another
What two origins of replication must conjugative plasmids contain?
oriV and oriT
Give three examples of conjugative systems (plasmids) with some extra detail
F plasmid: fertility factor, narrow host range
RP4: broad host range, pseudomonas
Ti plasmid: agrobacterium tumefaciens, vir and tra systems
What are the names of the sets of genes responsible for conjugal transfer between prokaryotes? What are the names of the sets of genes responsible for conjugal transfer between prokaryotes and eukaryotes?
What are the 4 steps involved in conjugation?
1. Mating pair formation (Mpf) - a pilus forms between 2 cells
2. Retraction - pilus retracts bringing cells closer together,, plasmid strand nicked at oriT (by relaxase)
3. DNA transfer (Dtr) - relaxase unwinds DNA & transfers strand through pilus, rolling circle replication replaces donor DNA strand
4. Replication - complementary DNA strand synthesised in recipient
What is the role of the coupling protein in conjugation?
The coupling protein synchronises mating pair formation with DNA transfer and pumps the DNA across
What is the role of relaxosome? And where is it coded?
The relaxosome is a protein complex made up of relaxase and accessory proteins which facilitates plasmids during conjugation. Relaxase initiates conjugation by nicking the plasmid (at the nic site)
Where and which genes encode the relaxosome?
tra gene in the oriT
What are the 4 stages of the relaxosome formation?
1. TraJ binds to inverted repeat closest to nic site
2. TraI binds to TraJ-oriT complex (at nic site)
3. TraK binds to bent region of oriT
4. TraI-TraJ-oriT complex stabilised by TraH
What is another word for the sex pili? And what is it's appearance (?
What is another name for TraI?
During conjugation, in which order do the 4 Tra proteins bind?
TraJ --> TraI --> TraK --> TraH
How does the binding of TraI initiate conjugative transfer?
by cleaving a specific phosphodiester bond in oriT
How does relaxase control the DNA through the conjugative channel?
Bound to leading end of DNA (5') and pilots strand through
What is the role of the type IV secretion system? Which plasmid contains it's genes? What are the name of the subunits?
Translocates DNA/proteins across bacterial cell envelope (in and out), Ti plasmid (tumour inducing), vir subunits (virB, virD, virE
In A. Tumefaciens' T4SS what is the general role of subunits: (VirD4, VirB11, VirB4), (VirB3, VirB8, VirB10, VirB6) and (VirB1, VirB7, VirB9, VirB2, VirB5)?
D4, B11, B4: energetic components - bind ATP (B11 also forms channel)
B3, B8, B10, B6: inner membrane channel/scaffold proteins - form and make channel work (B3 assembles pilus, and translocates substrate, B6 mediates DNA substrate transfer, B8 positions other VirB's, B10 links inner/outer membrane VirB's)
B1, B7, B9, B2, B5: outer membrane channel subunits (B1 assembles T4SS by degrading peptidoglycan layer, B7 stabilises VirB's, B9 channel and pilus assembly, B2 monomers of pilus - pilin, B5 pilus component & substrate transfer
What are the varying names for the coupling protein in Ti, RP4 and F? What is it's role?
ATPase, also interacts with relaxosome & T4SS
Give a rough overview of the two main processes involved in conjugation
A single T-strand is generated from the plasmid which is then passed through the pilus into the receiving cell
How does Agrobacterium tumefaciens infect a plant (6 steps)?
1. A wounded plant releases phenolic plant signals
2. VirA (sensor histidine kinase) detects signals and phosphorylates itself then transfer P to VirG (activating)
3. Activated VirG leads to T-DNA synthesis and Vir gene transcription
4. VirB1-11 assemble T4SS which VirD2(relaxase)-T-DNA, VirE2, VirE3 and VirF pass through
5. VirE2 coats VirD2-T-DNA (protease protection) and VirE3 aids T-DNA nuclear import
6. VirF integrates T-DNA into the host genome (along with relaxase - VirD2)
What occurs in F plasmid but not Ti plasmid and why?
Entry exclusion (inability to transfer into another cell w/ same plasmid) occurs with F/RP4 plasmid but not Ti plasmid, this is because F/RP4 have entry exclusion systems but plant cells don't
What are the two genes responsible for entry exclusion in RP4/F and how do they exclude entry?
TraT - blocks mating pair formation
TraS - blocks DNA entry into cytoplasm
What is an Hfr cell?
A bacterium with a conjugative plasmid (e.g. F) integrated into it's genome
What is an episome? Give an example of an episome
An episome is a plasmid which can integrate into a host genome and transfer chromosomal genes e.g. F plasmid
What are the steps in Hfr?
1. Episome (plasmid - F) integrates into donor genome
2. Conjugation occurs but only part of the episome is transferred to recipient (pilus cuts off before all sent over)
3. Insertion sequence of episome is homologous to recipient genome so recombination occurs
Only some of the episome genes are transferred so recipient would remain to be F- (and not Hfr)
What is a non-conjugative but mobilisable plasmid? What genes is it missing?
A plasmid which is unable to conjugate itself but may transfer its genome with other conjugative plasmids. Does not contain Mpf genes/coupling protein genes