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BIOC2001 Dina > Plamids and conjugation > Flashcards

Plamids and conjugation Flashcards

1
Q

What is a plasmid?

A
  • 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)
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2
Q

What forms of DNA can typically be seen on a gel?

A
  • open circular
  • linearised
  • supercoiled
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3
Q

What partitioning occurs for small high-copy plasmids?

A

random plasmid partitioning

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4
Q

What partitioning occurs for large low-copy plasmids?

A

directed plasmid partitioning

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5
Q

What is plasmid incompatibility?

A
  • 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
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6
Q

Define ‘narrow-host range’

A

plasmid can only replicate in related species

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7
Q

Define ‘broad-host range’

A
  • plasmid can replicate in a variety of hosts

- can also be considered as promiscuous due to their ability to transfer by conjugation

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8
Q

How is antibiotic resistance typically spread?

A

by conjugation

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9
Q

List some characteristics that plasmids encode

A
  • antibiotic resistance
  • metal/metalloid resistance eg. Hs and As
  • virulence determinants (animal and plant pathogenicity)
  • bacteriocin production (bacterial toxins)
    biodegradation
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10
Q

What is the virulence of Bacillus anthracis dependent on?

A

pXO1 (anthrax toxin) and pXO2 (capsule production for protection against the immune system)

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11
Q

What is pXO2 used as?

A

a live attenuated vaccine for some Bacillus strains

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12
Q

What does Ti stand for?

A

tumour-inducing

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13
Q

What is a bacteriocin?

A

an antimocrobial agent used to kill bacteria not harbouring plasmid that confers immunity/resistance to the compound

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14
Q

What is a transposon?

A

a DNA sequence with the ability to move eg. Tn3

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15
Q

What does IR stand for?

A

inverted repeat

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16
Q

What does bla encode?`

A

β-lactamase which confers resistance to ampicillin

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17
Q

What are the components of a plasmid vector?

A
  • 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)
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18
Q

What is a suicide vector?

A

a plasmid which is used with an oriV that is unable to replicate in the host of interest

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19
Q

Give an example of a suicide vector and host

A

pUC19 can be used as a suicide plasmid for Vibrio cholerae

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20
Q

What could a suicide vector be used for?

A
  • 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
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21
Q

What is a shuttle vector?

A

a plasmid that contains two origins of replication, allowing for replication in two hosts

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22
Q

Give an example of a shuttle vector

A

Bacillus coagulans (resistance to ampicillin)

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23
Q

Outline the process of cloning using a plasmid vector

A
  • 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
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24
Q

What is the function of CaCl2 in cloning?

A
  • 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
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25
Q

Define ‘conjugation’

A

the transfer of genetic material between cells by direct cell-to-cell contact

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26
Q

When was conjugation discovered and by whom?

A

discovered in 1946 by Lederberg and Tatum

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27
Q

In which organisms does conjugation occur?

A

in Bacteria and Archaea

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28
Q

What is required by conjugative plasmids?

A

two origins of replication (oriV and oriT)

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29
Q

What does oriV stand for?

A

origin of vegetative replication

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30
Q

What does oriT stand for?

A

origin of transfer

31
Q

What is the role of conjugative or transfer genes?

A

they establish a stable mating pair and trigger DNA transport from donor to recipient via a specialised pore/channel

32
Q

What is F?

A

Fertility factor; a narrow-host range plasmid

33
Q

From which organism is F isolated?

A

Escherichia coli

34
Q

What is RP4?

A

Resistance factor; confers resistance to antibiotics and is a broad-host-range plasmid

35
Q

From which organism is RP4 isolated?

A

Pseudomonas aeruginosa (opportunistic pathogen)

36
Q

In which organism is Ti plasmid found?

A

Agrobacterium tumefaciens

37
Q

What disease is Ti plasmid involved in causing?

A

crown gall disease in plants (mostly fruit trees)

38
Q

What is the difference between tra/trb and vir genes?

A

Conjugal gebes are referred to as tra/trb when conjugation occurs between prokaryotes and vir genes when it occurs between prokaryotic and eukaryotic cells

39
Q

Explain the conjugation systems in Ti

A

two conjugation systems:
vir - involves transfer of DNA from bacterium to plant cells
tra - involved in transfer from one bacterium to another

40
Q

Explain the transfer of the F plasmid?

A
  • transfers by conjugation

- can integrate into host chromosome by recombination and thus transfer host genes

41
Q

What are the main steps of conjugation?

A
  • 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
42
Q

What is the mechanism of conjugation between two prokaryotic cells?

A
  • 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
43
Q

Describe the structure of the F pilus

A
  • 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)
44
Q

What is the relaxosome comprised of?

A

the relaxase and accessory proteins

45
Q

What is the function of the accessory proteins involved in the relaxosome?

A

they stabilise the relaxosome

46
Q

What is the relaxase in RP4?

A

TraI (essential)

47
Q

What are the accessory proteins in RP4?

A

TraJ (essential) and TraH (acts as a ‘helper’ to stabilise the complex

48
Q

What is the key enzyme in RP4?

A

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

49
Q

Outline the stages of relaxosome formation

A
  • 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
50
Q

How is conjugation initiated by TraI?

A
  • 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’
51
Q

What is a phosphodiester bond?

A

a group of strong covaent bonds between a phosphae group and two 5-carbon ring pentose over two ester bonds

52
Q

What is the Ti plamid system comprised of?

A
  • virB operon - virB1-11

- virD operon - virD1-5

53
Q

What do virB1-11 encode?

A

proteins involved in Mpf

54
Q

What are the functions of virD1-5?

A

virD1 and virD2 are essential for Dtr and virD4 is a coupling protein

55
Q

What is T4SS?

A

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
56
Q

What is the role of VirB4?

A

involved in energising the assembly or activity of the channel

57
Q

What is the role of VirB11?

A

form as homohexamer rings and contain a central cavity = channel

58
Q

What are the inner membrane channel/scaffold proteins?

A

VirB3, VirB6, VirB8 and VirB10

59
Q

What is the function of the inner membrane channel/scaffold proteins?

A

proteins found in the inner membrane contributing to the formation and activity of the channel

60
Q

What are the functions of VirB3?

A
  • interacts with VirB4 and VirB2 (ie. pilin)

- involved in pilus assembly pathway and substrate translocation

61
Q

What are the functions of VirB6?

A
  • interacts with the DNA substrate, mediating its transfer

- interacts with the components of the channel

62
Q

What are the functions of VirB8 and VirB10?

A
  • 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
63
Q

What are the periplasmic/outer membrane subunits?

A

VirB1, VirB2, VirB5, VirB7 and VirB9

64
Q

What is the function of VirB1?

A

facilitates T4SS assembly by degrading peptidoglycan

65
Q

What are the functions of VirB7 and VirB9?

A
  • localised in outer membrane
  • VirB7 is a lipoprotein and stabilises several VirB subunits
  • VirB9 is required for channel assembly and pilus biogenesis
66
Q

What are the functions of VirB2 and VirB5?

A
  • 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
67
Q

What is entry exclusion?

A
  • 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
68
Q

What is Tras?

A

an inner membrane protein that blocks entry after Mpf has formed

69
Q

What is TraT?

A

an outer membrane lipoprotein that reduces mating pairs forming

70
Q

What is an episome?

A

a plasmid that can integrate into the host chromosome and transfer chromosomal genes from donor to recipient

71
Q

Give an example of an episome

A

F plasmid

72
Q

What is mobilisation?

A

Some plasmids are unable to transfer themselves but canbe transferred by other conjugative plasmids = mobilisable

73
Q

Which genes do mobilisable plasmids have?

A

they have oriT and Dtr genes, but are missing those involved in Mpf and coupling protein

BIOC2001 Dina (6 decks)

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