Plasmids, transformation, and conjugation II

What is RepA required for?

Initiation of replication at oriV for the replication of R100

What is CopB?

It is a repressor of repA

What does copA encode?

An antisense RNA to 80-90bp of the repA (forms a hybrid) (copA is much smaller than the repA)

What happens 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 (C)

What happens when copA levels get high?

RepA will be degraded by forming a hybrid with the copA message

Can R100 coexist with related plasmids? What is this called?

No it cannot because the copA genes are similar. Therefore they will repress the replication of the related plasmid. This is called plasmid incompatibility

What is plasmid incompatibility?

This is where plasmids of a similar nature do not allow each other to coexist. Similar plasmids with similar functions do not exist together because control of their replication is in conflict with one another

What are iterons?

They are repeated sequences (17-22 bp) of DNA

What does RepA bind?

Interons and handcuffs 2 plasmids together (on plasmid DNA)

Can coupled plasmids replicate again? What does this allow for?

No they cannot and allows for tight control of plasmid copy number

Can RepA handcuff related plasmids? What is this another mechanism of?

Yes they can and another mechanism of plasmid incompatibility

What can replication be controlled by?

Iterons (DNA repeats)

Describe the process of R100 replication

1. Low concentration of plasmid and RepA binds to ori

2. Replication takes place

3. Increasing concentration of plasmid and RepA

4. RepA will bind to iterons

5. 2 plasmids are handcuffed together and plasmids can’t get replicated because ori site is not accessible due to complex

Can plasmids regulate their own replication?

Yes

Can the mechanisms by which plasmids control their own regulation also control other plasmids in the same cell? In these cases, how many plasmids will be inherited?

Yes and only one

Are similar plasmids in the same incompatibility group?

Yes

Are naturally occurring plasmids generally stable (have been selected for that host)?

Yes

Are artificial plasmids stable or not? Will the cell grow slower if it needs to maintain a plasmid?

They are not stable and yes

What are 3 general phenomenon associated with plasmid stability?

1. Plasmid integrity

2. Partitioning

3. Differential growth rates

What do plasmids often have? What do they allow for? Is this a part of plasmid integrity?

Insertion sequences or other recombination hotspots that allow for deletions or inversions (can make broken versions of plasmids (some don’t have all the sequences or order of genes is changed)). Yes

Can a gene be intact while others are lost or in a different orientation in plasmids?

Yes

What is OriV? Does it need to be maintained? Why?

It is the origin of replication in a plasmid and yes it does or else the plasmid cannot replicate

What is a way to ensure plasmids are inherited?

Have toxin-antitoxin systems

Can a toxin kill its host?

Yes but it doesn’t because of the presence of the antitoxin

Is the toxin stable? Anti-toxin?

Yes for the toxin but less so for the anti-toxin

Does the antitoxin need to be made all the time? What happens if it is lost?

Yes it does because if the gene encoding it is lost then the toxin would be able to kill the host (become sensitive to it)

What is the CcdB-CcdA toxin-antitoxin system?

A type of partitioning system

Where are ccdB and ccdA?

They are encoded on some low copy plasmids

What does CcdB do? What does CcdA do?

CcdB inhibits topoisomerase and kills cells. While CcdA binds CcdB and neutralizes it (are counteractive components)

What do you need topoisomerase for?

To regulate DNA replication

What type of mechanism is toxin-antitoxin systems

Plasmid fidelity mechanism

Is CcdA less or more stable than CcdB?

More stable than CcdB

What happens to the cell if it inherits a plasmid encoding ccdA-ccdB? What happens to the cell if it does not inherit plasmid encoding ccdA-ccdB?

The cell will live. The cell will die