Plamids and conjugation Flashcards
What is a plasmid?
- extrachromosomal DNA molecule
- circular or linear
- autonomous replication (independent of the chromosome)
- range in size from kilobases to megabases
- control their copy number (again, independent of the chromosome)
- ensure inheritance at each cell division by process called partitioning
- plasmids with same replication mechanism
can’t co-exist in the same cell – known as
incompatibility (Inc)
What forms of DNA can typically be seen on a gel?
- open circular
- linearised
- supercoiled
What partitioning occurs for small high-copy plasmids?
random plasmid partitioning
What partitioning occurs for large low-copy plasmids?
directed plasmid partitioning
What is plasmid incompatibility?
- several plasmids can co-exist in the same cell but not all can co-exist stably
- incompatibility can be due to similarities in the replication machinery
- incompatible plasmids are in the same incompatible (Inc) group
Define ‘narrow-host range’
plasmid can only replicate in related species
Define ‘broad-host range’
- plasmid can replicate in a variety of hosts
- can also be considered as promiscuous due to their ability to transfer by conjugation
How is antibiotic resistance typically spread?
by conjugation
List some characteristics that plasmids encode
- antibiotic resistance
- metal/metalloid resistance eg. Hs and As
- virulence determinants (animal and plant pathogenicity)
- bacteriocin production (bacterial toxins)
biodegradation
What is the virulence of Bacillus anthracis dependent on?
pXO1 (anthrax toxin) and pXO2 (capsule production for protection against the immune system)
What is pXO2 used as?
a live attenuated vaccine for some Bacillus strains
What does Ti stand for?
tumour-inducing
What is a bacteriocin?
an antimocrobial agent used to kill bacteria not harbouring plasmid that confers immunity/resistance to the compound
What is a transposon?
a DNA sequence with the ability to move eg. Tn3
What does IR stand for?
inverted repeat
What does bla encode?`
β-lactamase which confers resistance to ampicillin
What are the components of a plasmid vector?
- origin of replication (oriV) to replicate in the host
- selection criterion eg. antibiotic resistance or blue/white colour selection
- multiple cloining site (MCS)/polylinker
- promoter (inducible or constitutive)
What is a suicide vector?
a plasmid which is used with an oriV that is unable to replicate in the host of interest
Give an example of a suicide vector and host
pUC19 can be used as a suicide plasmid for Vibrio cholerae
What could a suicide vector be used for?
- to mutate a function if genome sequence is unknown
- transposon used to transfer gene onto suicide vector
- selection for loss of function
- clone gene into suicide vector
- transform into bacteria
- crossover and homologous recombination means that region is incorporated into chromosome
What is a shuttle vector?
a plasmid that contains two origins of replication, allowing for replication in two hosts
Give an example of a shuttle vector
Bacillus coagulans (resistance to ampicillin)
Outline the process of cloning using a plasmid vector
- DNA fragment to be cloned is enzymatically inserted into the plasmid vector
- mix E. coli cells with the recombinant plasmids in the presence of CaCl2
- allow bacteria to grow on nutrient agar plates containing ampicillin
- transformed E. coli cells will survive, while cells that do not take up the plasmid die on the ampicillin plates
- independent plasmid replication and cell multiplication lead to the development of a colony of cells, each containing copies of the same recombinant plasmid
What is the function of CaCl2 in cloning?
- it makes the bacteria competent (able to take up foreign DNA easily)
- the exposure of cells to ice-cold CaCl2 and subsequent heat shock creates pores in the bacterial cell wall
- this allows the easy uptake of plasmid DNA into the cell
- this is due to the positive Ca ion attacking both the negatively charged DNA and the lipopolysaccharide membrane
Define ‘conjugation’
the transfer of genetic material between cells by direct cell-to-cell contact
When was conjugation discovered and by whom?
discovered in 1946 by Lederberg and Tatum
In which organisms does conjugation occur?
in Bacteria and Archaea
What is required by conjugative plasmids?
two origins of replication (oriV and oriT)
What does oriV stand for?
origin of vegetative replication
What does oriT stand for?
origin of transfer
What is the role of conjugative or transfer genes?
they establish a stable mating pair and trigger DNA transport from donor to recipient via a specialised pore/channel
What is F?
Fertility factor; a narrow-host range plasmid
From which organism is F isolated?
Escherichia coli
What is RP4?
Resistance factor; confers resistance to antibiotics and is a broad-host-range plasmid
From which organism is RP4 isolated?
Pseudomonas aeruginosa (opportunistic pathogen)
In which organism is Ti plasmid found?
Agrobacterium tumefaciens
What disease is Ti plasmid involved in causing?
crown gall disease in plants (mostly fruit trees)
What is the difference between tra/trb and vir genes?
Conjugal gebes are referred to as tra/trb when conjugation occurs between prokaryotes and vir genes when it occurs between prokaryotic and eukaryotic cells
Explain the conjugation systems in Ti
two conjugation systems:
vir - involves transfer of DNA from bacterium to plant cells
tra - involved in transfer from one bacterium to another
Explain the transfer of the F plasmid?
- transfers by conjugation
- can integrate into host chromosome by recombination and thus transfer host genes
What are the main steps of conjugation?
- mating-pair formation (Mpf) - pilus formation which is a type IV secretion system (T4SS)
- signalling event that triggers DNA transfer
- DNA transfer (Dtr) which involves relaxosome formation
- coupling protein - synchronises Mpf with Dtr and facilitates DNA transfer
What is the mechanism of conjugation between two prokaryotic cells?
- cell-to-cell contact made by pilus
- pilus retracts, bringing cells closer together
- DNA strand to be transferred is nicked at the origin of transfer (oriT) by relaxase (also known as nickase)
- relaxase also acts as a helicase, unwinding DNA to be transferredd
- rolling circle replication replaces DNA strand in the donor cell
- complementary DNA strand is made in the recipient, which is now a donor
Describe the structure of the F pilus
- 1-20um long (usually 2um)
- 8nm diameter
- composed of identical 7.2 kDa pilin subunits
- pore 2nm - enough space to accommodate single-stranded DNA and protein (ie. the relaxase)
What is the relaxosome comprised of?
the relaxase and accessory proteins
What is the function of the accessory proteins involved in the relaxosome?
they stabilise the relaxosome
What is the relaxase in RP4?
TraI (essential)
What are the accessory proteins in RP4?
TraJ (essential) and TraH (acts as a ‘helper’ to stabilise the complex
What is the key enzyme in RP4?
TraI, which nicks one DNA strand and unwinds DNA (ie. acting as a DNA helicase) to form a sinfle-stranded DNA molecule ready for transfer
Outline the stages of relaxosome formation
- TraJ binds to the inverted repeat closest to the site of DNA cleavage (ie. nic)
- TraI binds to TraJ-oriT complex
- relaxosome formation is facilitated by the intrinsically bent region in oriT that allows it to wrap arunf a core of several subunits of TraK to enhance the fraction of several subunits that can be captured and cleaved by nic
- TraI-TraJ-oriT complex is stabilised by TraH
How is conjugation initiated by TraI?
- TraI is a relaxase that cleaves a specific phosphodiester bond in the oriT, initiating conjugative transfer
- relaxase is covalently bound to the 5’ end of the T-strand, piloting the DNA through the conjugative channel from 5’ to 3’
What is a phosphodiester bond?
a group of strong covaent bonds between a phosphae group and two 5-carbon ring pentose over two ester bonds
What is the Ti plamid system comprised of?
- virB operon - virB1-11
- virD operon - virD1-5
What do virB1-11 encode?
proteins involved in Mpf
What are the functions of virD1-5?
virD1 and virD2 are essential for Dtr and virD4 is a coupling protein
What is T4SS?
type IV secretion system
- mediates translocation of macromolexules across cell envelopes of bacteria
- conjugation Mpf system is a large subfamily of T4SS
- T4SS can also functino in the uptake of nucleic acids or proteins from the extracellular environment
What is the role of VirB4?
involved in energising the assembly or activity of the channel
What is the role of VirB11?
form as homohexamer rings and contain a central cavity = channel
What are the inner membrane channel/scaffold proteins?
VirB3, VirB6, VirB8 and VirB10
What is the function of the inner membrane channel/scaffold proteins?
proteins found in the inner membrane contributing to the formation and activity of the channel
What are the functions of VirB3?
- interacts with VirB4 and VirB2 (ie. pilin)
- involved in pilus assembly pathway and substrate translocation
What are the functions of VirB6?
- interacts with the DNA substrate, mediating its transfer
- interacts with the components of the channel
What are the functions of VirB8 and VirB10?
- interact with channel proteins
- VirB8 is involve in positioning the other VirB proteins
- VirB10 interacts with several other VirB components, linking proteins in the inner and outer membranes
What are the periplasmic/outer membrane subunits?
VirB1, VirB2, VirB5, VirB7 and VirB9
What is the function of VirB1?
facilitates T4SS assembly by degrading peptidoglycan
What are the functions of VirB7 and VirB9?
- localised in outer membrane
- VirB7 is a lipoprotein and stabilises several VirB subunits
- VirB9 is required for channel assembly and pilus biogenesis
What are the functions of VirB2 and VirB5?
- VirB2 pilin is processed to form cyclic polypeptides, which form building blocks for pilus polymerisation
- F propilin (121 amino acids) has a 51 amino acid leader sequence (directs export across the membrane) that is cleaved
- VirB5 is exported to the periplasm and forms a component of the pilus and also contributes to substrate transfer
What is entry exclusion?
- mechanism that reduces conjugal transfer of plasmids of the same incompatibility group into cells already harbouring those plasmids
- doesn’t occur in Ti plasmid systems
- Eex system of F and RP4 involve two proteins
- F system: TraS and TraT
What is Tras?
an inner membrane protein that blocks entry after Mpf has formed
What is TraT?
an outer membrane lipoprotein that reduces mating pairs forming
What is an episome?
a plasmid that can integrate into the host chromosome and transfer chromosomal genes from donor to recipient
Give an example of an episome
F plasmid
What is mobilisation?
Some plasmids are unable to transfer themselves but canbe transferred by other conjugative plasmids = mobilisable
Which genes do mobilisable plasmids have?
they have oriT and Dtr genes, but are missing those involved in Mpf and coupling protein
