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MICR3330 > 11 - Transformation, plasmids and conjugation > Flashcards

11 - Transformation, plasmids and conjugation Flashcards

1
Q

Core genome

A

Represents the genes present in all strains of a species

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2
Q

Accessory genome

A

Genes that are found in many, but not all bacterial stains within a species

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3
Q

Pan genome

A

Entire gene set of all strains of a species. It includes genes present in all strains (core genome) and genes present only in some strains of a species (accessory genome).

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4
Q

Closed genome

A

The more stains we sequence, we do not find any more genes

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5
Q

Open pangenome

A

The more we sequence strains, the more extra genes are found

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6
Q

Horizontal gene transfer (HGT)

A

Transfer of genes from one mature, independent organism to another

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7
Q

Three mechanisms of HGT

A
  • Transformation (transfer of free or naked DNA)
  • Conjugation (transfer of DNA via cell to cell contact)
  • Transduction (transfer of DNA within a virus (phage)
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8
Q

Fate of DNA

A
  • Plasmids replicate
  • Regions of homology align between the incoming DNA and the host DNA which results in homologous recombination = acquisition of new DNA into the host genome
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9
Q

Requirements of transformation

A
  • Free or naked dsDNA (linear or circular)
  • A transformable bacterial cell must be competent (bind and protect DNA from nuclease)
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10
Q

Two possible outcomes of transformation

A
  • Integration by nonreciprocal recombination (stable transformation)
  • Degradation (unsuccessful transformation)
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11
Q

Competence

A
  • Ability to bind and take up DNA into bacterial cell
  • Competence is a regulated phenotype (expression of competence proteins which make up the transformation machinery)
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12
Q

Steps in natural transformation

A
  1. DNA bound to surface of cell and endonuclease nicks DNA
  2. One strand degraded by nuclease
  3. DNA associates with other Com proteins
  4. ssDNA enters cell and recombines with the chromosome
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13
Q

Transformation machinery (gram neg)

A
  • PilQ
  • PilE
  • ComE
  • N
  • ComA
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14
Q

Transformation machinery (gram pos)

A
  • ComGC
  • ComEA
  • N
  • ComEC
  • ComFA
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15
Q

PilQ

A

Channel across outer membrane

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16
Q

PilE/ComGC

A

Moves DNA across cell wall

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17
Q

ComE/ComEA

A

DNA binding

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18
Q

N

A

Nuclease forms ss DNA

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19
Q

Coma/ComEC

A

Channel across cell membrane

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20
Q

ComFA

A

Moves DNA into cytoplasm

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21
Q
  1. DNA bound to surface of cell and endonuclease nicks DNA
A
  • Random collision of DNA and cell
  • ComG provides access to ComEA
  • DNA is DNase-sensitive at this stage as the DNA is still sitting on the bacterial surface
22
Q
  1. One strand degraded by nuclease
A
  • DNA is fragmented by a surface endonuclease (NucA)
  • Endonuclease causes double stranded breaks
23
Q
  1. DNA associates with other Com proteins
A
  • Eclipse phase
  • dsDNA is converted to ssDNA by N
  • DNA is now irreversibly bound to the cell
  • Process of transformation is now
    DNase-resistant (as DNase needs dsDNA)
24
Q
  1. ssDNA enters cell and recombines with the chromosome
A

Recombination occurs via general (nonreciprocal) recombination which is recA and homology-dependent

25
Q

Why is one strand of DNA degraded during transformation

A

Energy from degradation of one strand of DNA is used to draw the other single strand into the cell (via channel protein ComEC and ComFA)

26
Q

What is decrease in transformation efficiency with decrease in relatedness of bacteria due to

A
  • Specificity of homology dependent recombination (closely related bacteria share more homology)
  • Specificity of DNA uptake (unlikely that an unrelated bacterium will have many copes of DUS)
27
Q

Types of plasmids

A
  • Fertility (F) plasmids
  • Resistance (R) plasmids
  • Virulence plasmids
  • Metabolic plasmids
28
Q

Plasmids

A
  • Circular dsDNA
  • Replicate independently in cell cytoplasm
  • Carry genes not essential to host but may be useful in certain environments (part of accessory genome)
  • During transformation, converted to linear ssDNA, converted to circular dsDNA by host machinery
29
Q

Discovery of bacterial sex factors

A
  • Creation of nutritional mutants (auxotrophs) in E. coli (prototroph)
  • Mating these two strains revealed transfer of chromosomal markers from donor to recipient
30
Q

Possible explanations of discovery of bacterial sex factors experiment

A
  • Free DNA did not reproduce the result therefore not transformation
  • U tube experiment showed cell to cell contact betwen strain A and B was essential for DNA transfer (not transduction)
  • Conclusion: conjugation
31
Q

Functions of F plasmid

A
  • Synthesis of F pili
  • Surface exclusion
  • Stabilisation of mating pairs
  • DNA transfer
  • Regulation
32
Q

Steps of conjugation

A
  1. A bacterium with the F piluscontacts recipient cell (F-)
  2. Rolling circle replication of the F plasmid is initiated
  3. The ssDNA copy of the F plasmid transferred to the recipient cell through the conjugal bridge.
  4. The transferred ssDNA circularizes and second strand synthesis takes place to produce a dsDNA F plasmid.
  5. The two cells separate and progeny are both F+
33
Q

Step 1 molecular events: A bacterium with the F pilus contacts recipient cell (F-)

A
  • F pilus biosynthesis in donor cell
  • F pilus contacts recipient cell (specific due to recognition domain)
34
Q

Step 2 molecular events: Rolling circle replication of the F plasmid is initiated

A
  • Pilus retraction by disaggregation of the pilus fibre in the periplasm of the donor cell
  • Mating aggregate formation and stabilization mediated by TraG and TraN (conjugal bridge or mating pore)
  • Rolling circle replication is
    initiated
35
Q

Initiation of rolling circle replication

A
  • The covalently closed circular plasmid DNA is nicked at a strand specific site (nic) within the origin of transfer (oriT).
  • Nicking is catalyzed by TraI (helicase) which remains covalently attached to the 5’ phosphate group, leaving a free 3’OH group.
  • Relaxosome formed
  • DNA replication initiates at the free 3’OH by DNA polymerase III, and proceeds in a 5’ to 3’ direction
36
Q

Relaxosome

A
  • Accessory proteins (TraM, TraD and TraY) facilitate binding of the TraI to oriT
  • This complex is called relaxosome
37
Q

Step 3 molecular events: The ssDNA copy of the F plasmid
transferred to the recipient cell through the conjugal bridge.

A
  • TraD mediates association of the relaxosome with the Tra pore and transfer of the ssDNA through the Tra pore
  • 3’→ 5’ replacement strand DNA synthesis of donor F plasmid commences
38
Q

Step 5 molecular events: The two cells separate and
progeny are both F+

A
  • Tral terminates F plasmid transfer by creating a nick between the two oriT sites (one for each copy of the F
    plasmid)
  • Ds plasmids in both cells are ligated to form complete
  • Mating complex collapses and cells seperate yielding two F+ donor cells
39
Q

Hfr

A

High frequency recombination

40
Q

What is integration of F plasmid into the E. coli chromosome dependent on

A
  • Regions of shared homology (e.g. insertion elements IS, IS2, IS3)
  • RecA mediated homologous recombination
41
Q

Features of Hfr

A
  • Over 20 different Hfrs have been isolated
  • Integration is directional resulting in polarity of DNA transfer
42
Q

Uses of Hfr

A

Chromosome mapping using interrupted transfer

43
Q

Why does the frequency of
transfer of late markers decrease
over time?

A
  • Mating couples separate
  • Double cross-over events over
    long distances is less frequent.
44
Q

Excision of the F plasmid from the Hfr chromosome

A
  • Homologous recombination between IS element flanking F plasmid insertion site and IS elements in the chromosome
  • Illegitimate recombination where no homologous regions exist
45
Q

Integrated F

A

Can leave the host chromosome and carry host genes with it

46
Q

Donor and recipient cells

A
  • Donor cells contain the primary
    F prime (F’) and are haploid for
    the chromosomal region on the
    plasmid
  • Recipients carry secondary F’
    plasmids and are diploid for the
    chromosomal region carried on
    the plasmid
47
Q

Complementation studies

A
  • Secondary F’ carries chromosomal genes from donor which restore wild-type phenotype to mutant recipient
  • If the recipient has a chromosomal lac- and the F’
    carried lac+, then a recipient
    carrying this plasmid would be
    lac+ as the gene carried on the F’ would complement the defect in this locus
48
Q

Does conjugation occur in organisms other than E. coli

A
  • F factor transfer to Salmonella and Shigella, different conjugative plasmids in many other species
  • Cannot occur where the plasmid is not maintained in the recipient
49
Q

Other conjugative elements

A

Integrative and conjugative elements (ICEs)

50
Q

ICEs

A

Contain genes encoding conjugation machinery and genes
for integration into the chromosome

51
Q

Does conjugation occur in gram-positive bacteria

A

Yes, e.g. Staphylococcus aureus

MICR3330 (29 decks)

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