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BIOC0005 > Bacterial Conjugation > Flashcards

Bacterial Conjugation Flashcards

1
Q

What is bacterial conjugation?

A

The transfer of genetic material between cells by direct cell-cell contact.

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2
Q

What are three forms of horizontal gene transfer?

A

Bacterial conjugation, transduction and transformation.

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3
Q

When was bacterial conjugation discovered?

A

1946 by Lederberg and Tatum.

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4
Q

Give two examples of genetic material that can be transferred by conjugation.

A

Plasmids and transposons.

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5
Q

Give 5 examples of characteristics that conjugative plasmids can encode.

A
  • Antibiotic resistance.
  • Heavy metal resistance.
  • Pathogenicity.
  • Degradative islands.
  • Metabolic proteins.
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6
Q

What origins of replication do conjugative plasmids contain?

A
  • oriV (origin of vegetative replication).

- oriT (origin of transfer).

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7
Q

What is the difference between oriV and oriT and what is their purpose?

A

oriV:
- Origin of vegetative replication.

oriT:

  • Origin of transfer…
  • Essential for conjugal transfer… the DNA is cut here for conjugal transfer.
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8
Q

What must form before DNA transport can occur from donor to recipient?

A

A stable mating pair (i.e. mating pair formation- Mpf).

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9
Q

What is the difference between gram positive and negative bacteria?

A

Main difference is in the structure of their cell walls.

Gram negative:
- Thin peptidoglycan wall, susceptible to mechanical strain… does not retain the stain, but retains the counter stain, so appears pink.

Gram positive:
- Thick peptidoglycan cell wall… can retain the stain… appear a purple colour.

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10
Q

Name three examples of conjugative transfer mechanisms found in gram-negative bacteria.

A

F system, RP4 system and the Ti plasmid.

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11
Q

Outline the characteristics of the F system.

A
  • F (fertility factor).
  • Conjugative transfer system found in gram negative bacteria.
  • NHR plasmid, isolated from E.coli.
  • 99.2kbp.
  • 1/3 of plasmid= tra region dedicated to conjugal transfer… found downstream, of oriT where DNA nicked.
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12
Q

Outline the characteristics of the RP4 system.

A

-R (resistance factor)… confers resistance to antibiotics.
- BHR plasmid isolated from pseudomonas aeruginosa.
-Has origin of replication as ‘Rep’ not oriP.
-Has two regions for conjugal transfer:
Tra 1 (genes involved in cutting the DNA at the oriT… oriT found in tra1).
Tra 2 (Gene products involved in making the secretion system i.e. channel/ pore).
-Three antibiotic resistance genes encoded for: Tetracycline, kanamycin and ampicillin.

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13
Q

Outline the characteristics of the Ti plasmid?

A
  • Found in agrobacterium tumefaciens, involved in causing crown gall disease in plants.
    -Has two conjugative systems:
    …Vir (involved transfer of DNA (T-region) from the bacterium to the plant cells).
    …tra (involved in the transfer of DNA (the entire plasmid) from one bacterium to another.
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14
Q

How are conjugal transfer genes referred to for conjugation between prokaryotes?

A

tra/ trb genes.

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15
Q

How are conjugal transfer genes referred to for conjugation between prokaryotic and eukaryotic cells ?

A

vir genes.

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16
Q

What are the main steps in conjugation of an F-plasmid?

A
  • Formation of the type 4 secretory system.
  • Relaxosome formation.
  • Pumping of the DNA into the recipient cell.
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17
Q

What is the function of the pilus in the mating pair formation (Mpf)?

A

The pilus is a type 4 secretory system (T4SS), makes contact between the donor and recipient cell, and allows DNA to be transferred, once a stable mating pair has formed.

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18
Q

What is the relaxosome?

A

A complex of proteins that bind to oriT and cut the DNA here, for transfer.

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19
Q

What is the function of coupling proteins in conjugative transfer?

A

Synchronises Mpf with Dtr (DNA transfer) and is thought to pump the DNA into the recipient cell.

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20
Q

Outline the steps involved in conjugative transfer from an F+ cell to an F- cell.

A
  • The sex pilus establishes cell-to-cell contact between the donor and recipient cell.
  • Pilus retracts, drawing the two cells together and closing the distance.
  • Pore formation.
  • DNA strand is nicked at oriT by relaxase from the relaxosome complex.
  • Rolling circle replication replaces the DNA strand in the donor cell.
  • A complementary strand is made in the recipient from the donated strand as it is transferred.
  • DNA transfer and synthesis is complete, and the two cells separate to produce two F+ cells, i.e. where both the recipient and donor cells contain a plasmid.
  • Now recipient cell has become a donor cell… can transfer to another recipient.
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21
Q

Why is it important that the pilus retracts after the cell to cell contact has been established?

A

Because DNA being transported over longer distances can be damaged etc. as pilus breakage can occur.

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22
Q

Give the general dimensions of the F-pilus (sex pili).

A
  • 1-20um long (but conjugation doesn’t happen over long lengths so the pilus contracts.
  • 8nm in diameter.
  • Composed of 7.2 kDa pilin subunits… just one protein type makes up the pilus.
  • Pore size through which the DNA is transferred through is 2nm… enough space to accomodate a ssDNA and the protein (relaxase), which attaches to end of DNA once it nicks the DNA at the oriT.
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23
Q

Compare the morphology of the F pilus and the RP4 pilus.

A

F-pilus:

  • Long and sturdy… can do conjugation in a liquid broth/ medium further apart.
  • Part of the pilus has lipids associated with the protein structure, with each subunit… thought to play a role in DNA transfer, as changes the charge of the pore, and facilitates transfer.

RP4:
-Short pili… need to do conjugations on hard filter.

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24
Q

What model can be used to describe relaxosome formation?

A

The RP4 model.

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25
Q

What are the components of the relaxosome?

A
  • Relaxase (TraI… essential).

- Accessory proteins (TraJ… essential… and TraH… helper, stabilising the complex).

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26
Q

What is the function of the relaxase component of the relaxosome?

A

Relaxase is a key enzyme, which nicks the DNA strand and unwinds the DNA (i.e. like a helicase), to produce a single stranded DNA molecule ready for transfer.
If the gene for relaxase is mutated, no conjugation occurs, indicating it is a vital part of conjugation.

27
Q

What is the function of the accessory proteins found in the relaxosome complex?

A
  • TraH stabilises the complex.
  • Accessory proteins vary between different transfer systems.
  • If the TraH is mutated, then will still get conjugative transfer, but will just be slower… indicating it is not essential part of the process, but does help it along.
28
Q

Outline the stages of a relaxosome formation.

A
  • TraJ binds to the inverted repeat closest to the site of DNA cleavage (the nic site).
  • TraI binds to the TraJ-oriT complex.
  • TraI (relaxase) cleaves a specific phosphodiester bond in the oriT, initiating conjugative transfer. The TraI stays bound to the Transfer strnad, and pilots the DNA through the conjugative channel.
  • TraH stabilises this TraI-TraJ-oriT complex.
  • The oriT is the entire region of the TraJ, TraI and nic site.
29
Q

How does the intrinsically bent region in oriT facilitate relaxosome formation?

A

It allows it to wrap around a core of several subunits of TraK, which enhances the fraction of plasmids that can be captured and cleaved at the nic.

30
Q

What two vir operons are involved in conjugation in the Ti-plasmid?

A

The virB and virD operons.

31
Q

What proteins are encoded by the virB operon and what is their functions?

A

VirB1-> VirB11… encodes proteins involved in the Mpf.

32
Q

What proteins are encoded by the virD operon and what is their function?

A

VirD1->VirD5.

  • VirD1 and D2 are essential for Dtr.
  • VirD4 is a coupling protein (component of the T4SS).
33
Q

What are VirD1 and VirD2 equivalent to in the RP4 system?

A

traI and traH.

34
Q

Outline the main components of the T4SS.

A

VirD4, the pilus, the relaxosome cutting the oriT, and coupling proteins.

35
Q

What proteins are exported independently of DNA in this conjugation system, and what is their function?

A
  • VirE2, VirE3 and VirF.

- They are important for the processing of DNA when it gets into the plant cell .

36
Q

What are T6SS?

A

These are like inverted bacteriophage systems, that aim to ‘kill’ other bacteria that it is not related to, by shooting out toxins that kill the bacteria.

37
Q

What is the purpose of the T4SS?

A

To mediate the translocation of macromolecules (DNA and proteins) across cell envelopes of bacteria.

38
Q

What genes in the Ti plasmid refer to the T4SS?

A

The virB1->virB11 genes.
These are located in a single operon, and are transcribed as a single transcriptional unit.
There are equivalent genes in other virulent systems, but are called different things.

39
Q

Where are the VirB/ VirD4 subunits in A.tumefaciens localised?

A

On and across the membrane… come together to form the T4SS.

40
Q

Outline the main components of the T4SS.

A
  • Energetic components (VirD4, VirB4 and VirB11).
  • Core channel components (VirB3, VirB6, VirB8 and VirB10).
  • Pilius components (VirB2, VirB5) and outer membrane channel subunits (VirB1, VirB7 and VirB9).
41
Q

What subunits are involved in the energetic components of the T4SS? What is the characteristic of these proteins?

A

VirD4, VirB4 and VirB11.

These are large proteins (>70kDa), with conserved motifs, and are involved in ATP binding.

42
Q

What subunits are involved in the core channel components (i.e. inner membrane channel/ scaffold proteins) of the T4SS? What is the characteristic of these proteins?

A

VirB3, VirB6, VirB8 and VirB10.

These are proteins found in the inner membrane, and contribute to the formation and activity of the channel.

43
Q

What subunits are involved in the outer membrane channel and pilus of the T4SS?

A

VirB1, VirB2, VirB5, VirB7 and VirB9.

44
Q

What is the function of VirD4?

A
  • It is a coupling protein that interacts directly with the relaxosome and hydrolyses ATP.
  • It sits in the membrane and points to the cytoplasmic side of the bacteria, with most of the protein in the cytoplasm.
45
Q

What is the function of VirB11?

A

Hydrolyses ATP for conjugation and form as homohexamer rings that contain a central cavity (channel).

46
Q

What is the function of VirB4?

A

Hydrolyses ATP for conjugation and is involved in energising the assembly or activity of the channel.

47
Q

What is the function of VirB3?

A
  • It interacts with VirB4 and VirB2 (e.g. pilin)m and is involved in the pilus assembly pathway and substrate translocation.
  • It is primarily found in the inner membrane but has a portion in the periplasm.
48
Q

What is the function of VirB6?

A

-Interacts with the DNA substrate, and mediates its transfer.
-Interacts with components of the
channel (i.e. other proteins).
-It mainly just spans the inner membrane.

49
Q

What is the function of VirB8 and VirB10?

A
  • They interact with channel proteins.
  • VirB8… involved in position of other VirB proteins, and spans the inner membrane and periplasm.
  • VirB10… spans the periplasm and interacts with several other VirB components, linking the proteins to the inner and outer membranes.
50
Q

What is the function of VirB1?

A

-Acts as a hydrolase within the periplasmic space, facilitating T4SS assembly by degrading the peptidoglycan layer found in gram negative bacterial cells… allowing the T4SS to form from the inner to the outer membrane.

51
Q

What is the function of VirB7 and VirB9?

A

VirB7- It is a lipoprotein and stbalises several VirB subunits.
VirB9- Is required for channel assembly and pilus biogenesis.

They are both localised to the outer membrane.

52
Q

What is the function of VirB2 and VirB5?

A

VirB2 pilin is processed to form a cyclic peptide= building blocks for pilus polymerisation.
VirB5- exported and forms a component of the pilus and also contributes to substrate transfer.

53
Q

What is the function of F-propilin and its features?

A
  • It directs export across the membrane.

- 121 amino acids, with a 51 amino acid leader sequence.

54
Q

What system is used by the Ti plasmid to transfer DNA between bacterium?

A

The tra system.

55
Q

What system is used by the Ti plasmid to transfer DNA between plants and bacteria?

A

The vir system.

56
Q

Outline the interaction between a wounded plant and agrobacterium tumefaciens.

A

-Wounded plant releases signalling molecules which are detected by A. tumefaciens.
-Signalling molecules binds to histidine kinase in the inner membrane of the bacterium, activating it, causing it to auto-phosphorylate, and switch itself on.
-This phosphorylates virG (transcriptional regulator) which is a DNA binding protein.
-VirG then binds upstream ot the vir genes to promoter… switches on transcription of these vir genes (conjugation genes… allowing transfer of T-DNA).
-Proteins encoded by conjugation genes now aid the transfer of the T-DNA into the plant cell:
-VirB1-B11 proteins form the channel for the T4SS, through which the DNA is transported to the plant cell.
-VirD1 and D2 are important for relaxosome formation, and bind to the oriT at the end of the T-DNA border, in order to cut both ends (as only the T-DNA is transferred not the whole plasmid).
-VirE2, E3 and F are exported separately to the DNA into the plant cell:
… VirE2… protects the ssT-DNA once in the cell… coats it and protects it from degradation by plant nucleases.
… VirE3… Important for the nuclear import of T-DNA.
… VirF… Important for T-DNA integration into the genome.

57
Q

What else in encoded by the T-DNA to aid bacterial survival?

A

Opine and proteins for opine catabolism… this is a food source and allows the bacteria to utilise it.

58
Q

How are the genes on T-DNA transcribed in the plant cell?

A

They don’t have any prokaryotic promoters… use the plant’s promoters.

59
Q

Whatis entry exclusion?

A

This is a mechanism that reduces the conjugal transfer of plasmids of the same incompatibility group into cells already harbouring those plasmids.
i.e. so F+ plasmid can’t transfer intp a recipient cell already with an F plasmid.

60
Q

Does entry exclusion occur in the Ti system?

A

No.

61
Q

What proteins are involved in the entry exclusion system of F?

A

TraS and TraT.

62
Q

What is TraS and how does it result in entry exclusion?

A

It is an inner membrane protein, blocking entry after the Mpf has occurred… so the mating pair can still form, just blocks entry of the DNA.

63
Q

What is TraT and how does it result in entry exclusion?

A

It is an outer membrane lipoprotein which reduces mating pair formation.

BIOC0005 (9 decks)

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