Plasmids, transformation, conjugation

Question 1

Plasmids

Answer 1

• extrachromosomal elements-usually circular, can be linear (rare)
• can encode factors that allow growth in new environments-antibiotic resistance, virulence
• can be shared among species

Question 2

Virulence

Answer 2

• shigella
• causes dysentary
• virulence genes involving invasion/adhesion encoded on a plasmid

Question 3

Colicins

Answer 3

• toxins that kill other bacteria that don’t have the plasmid you have
• secreted by bacteria, kill other bacteria by forming pores or by entering and acting as nucleases
• dont hurt the host because the plasmid that encodes the toxin also makes the immunity product that neutralizes the toxin

Question 4

High Copy number (5)

Answer 4

• relaxed
• generally small size (<10 kb)
• ColE1 plasmid, makes colicins that kill species related to E.coli
• replication is unlinked to cell division
• random partitioning

Question 5

Low Copy number (5)

Answer 5

• stringent
• larger (up to 300 kb)
• R100 plasmid is model for this type
• directed partitioning systems
• often conjugative-allows back up mechanism for ensuring it is transferred to progeny

Question 6

Replication and Partitioning in high copy plasmids

Answer 6

• progeny is assured inheriting the plasmid because there are many copies
• since replication is unlinked to cell division, copy number can be amplified by adding drug that inhibits protein synthesis (chloramphenicol)
• ->plasmid amplification

Question 7

Replication and Partitioning in low copy plasmids

Answer 7

• replication of plasmid linked to chromosome, ensures at least 2 copies present at time of division
• specialized partitioning mechanisms help ensure plasmid is distributed to daughter cells

Question 8

Regulation of Copy Number

Answer 8

• controlled by antisense RNA
• high copy number results in the expression of an antisense RNA that interferes with replication
• when plasmid reaches high copy number, rop is being made (b/c encoded on plasmid)
• rop helps RNA1 (antisense) binds RNAII (coding strand)

Question 9

Replication of R100

Answer 9

• RepA is required for initiation of replication at OriV
• CopB is repressor of repA
• CopA encodes antisense to 80-90 bp of the repA message
• when a plasmid enters a cell there is no CopB and little CopA so you make lots of repA until you achieve the copy number
• R100 cannot coexist with related plasmids because the copA genes are similar
• they repress the replication of the related plasmid-plasmid incompatibility

Question 10

Control of replication by DNA repeats (iterons)

Answer 10

• repeated sequences of DNA
• RepA binds iterons and handcuffs 2 plasmids together
• these coupled plasmids cannot replicate again-tight control of plasmid copy number
• RepA can also handcuff related plasmids-another mechanism of plasmid incompatibility

Question 11

Mechanism of RepA iterons

Answer 11

• RepA builds up and binds plasmids together
• handcuffed plasmids do not get replicated
• the origin becomes inaccessible due to steric hindrance

Question 12

Plasmid Incompatibility

Answer 12

• plasmids can regulate their own replication
• the same mechanisms can control other plasmids in the same cell
• in these cases only 1 plasmid will be inherited
• the plasmids are in the same “incompatibility group”

Question 13

Plasmid Stability

Answer 13

-naturally occurring plasmids are generally stable (they have been selected for that host)
-artificial plasmids are often unstable
3 general phenomenon associated:
-plasmid integrity
-partitioning
-differential growth rates

Question 14

Plasmid Integrity

Answer 14

• plasmids often have insertion sequences or other recombination hotspots that allow for deletions or inversions
• one gene may be intact while the others are lost or in a different orientations

Question 15

Toxin-antitoxin systems

Answer 15

• ways to ensure plasmids are inherited
• toxin can kill host but doesn’t because of the presence of antitoxin
• the toxin is usually stable and the antitoxin is less so
• the result is that the antitoxin needs to be made all the time to keep up, so if the gene encoding it is lost the toxin can kill the host

Question 16

Partitioning Systems-CcdB-CcdA toxin-antitoxin system

Answer 16

• CcdA (antitoxin) is less stable than CcdB (inhibits topo)

- cells that don’t get plasmid don’t express CcdA and they die

Question 17

Partitioning Systems-Sok and hok

Answer 17

• hok encodes protein that is toxic to host
• sok makes antisense RNA that prevents hok TL
• if plasmid is lost, hok is made and cell dies

Question 18

Differential Growth Rate

Answer 18

• expression of many genes from a large plasmid is a burden if these genes are not required
• selective pressure is to lose plasmid
• facultative pathogens are good examples: shingles virulence plasmid is 200 Kb
• high expression of virulence plasmid genes at 37 degrees
• plasmid is lost at high rate if virulence is not required

Question 19

Transformation

Answer 19

• uptake of naked DNA
• Griffiths experiment used transformation to show DNA was genetic material
• some strains are naturally competent to take up DNA
• cells can be made chemically competent

More desperate ways to transform DNA:

• biolistic transformation-shoot in DNA on gold particles
• electroporation-shock in the DNA using a capacitor

Question 20

Competence (5)

Answer 20

• naturally competent bacteria become competent in response to growth phase (usually late log phase)
• in some cases, competence is linked to diffusible factors (quorum sensing)
• cells have proteins that bind preferred DNA sequences (usually from related or same species)
• naturally competent bacteria have dedicated structures and systems to allow for uptake of DNA
• DNA enters as ssDNA (s poor target for REs so not degraded)

Question 21

Joshua Lederberg

Answer 21

• worked on conjugation

- discovered transduction

Question 22

Self transmissible plasmids

Answer 22

encode machinery to initiate conjugation and transfer plasmid

Question 23

mobilizable plasmids

Answer 23

can be transferred but need Tra functions supplied in trans

Question 24

Donor and Recipient

Answer 24

Donor-has plasmid

Recipient-gets plasmid

Question 25

Transconjugant

Answer 25

-if transfer is successful progeny is transconjugant

Question 26

Conjugation-Bacterial Sex

Answer 26

• donor contains plasmid that encodes transfer functions (tra genes)
• donor forms pilus and brings cells in close contact
• pilus forms bridge to transfer DNA
• non-conjugating plasmids can be transferred too if they have mob genes and boom site (oriT)
• in Hfr transfer the fertility factor is integrated into the chromosome, the entire chromosome can be transferred to the recipient

Question 27

What do you need for conjugation?

Answer 27

Tra genes:

• Dtr component (DNA transfer and replication)-genes needed to process the DNA
• Mpf (mating pair formation): genes that make sex pilus

-oriT

Question 28

Dtr Relaxase

Answer 28

• an endonuclease that cuts at nic site in oriT and makes covalent bond with 5’ end of DNA
• gets transferred and DNA goes along for ride
• relaxasome binds oriT
• simplest mobilizable plasmids have just an oriT

Question 29

Mating Pair formation

Answer 29

• relaxasome binds oriT
• coupling of donor and recipient sends signal to relaxasome
• single strand cut and 5’ end bound by relaxase
• rolling circle replication and transfer to recipient

Question 30

Transfer of a resistance plasmid

Answer 30

• have plasmid encoding amp resistance and a streptomycin resistant recipient
• mix culture and plate w/ strep and amp
• plasmid containing recipient resistant to strep and amp

Question 31

Argobacterium and T-DNA

Answer 31

• trans-kingdom mechanism of DNA transfer
• agrobacterium tumefaciens- plant pathogen, causes crown gall tumour
• Type IV secretion system to transfer DNA from bacteria to plant host
• exploited to transfer DNA into fungi, malaria, etc