Plasmids, transformation, conjugation

What are plasmids?

They are extrachromosomal elements and are usually circular

Can plasmids be linear? Are plasmids part of the chromosome?

Yes in rare cases. No

What do plasmids encode? What are two examples?

Various factors that allow growth in new environments. Antibiotic resistance (encode antibiotic resistance cassettes) and virulence (virulence factors).

What is virulence?

It is the power of disease

Why are plasmids important? How can they be moved around?

This is because they can be shared among species (can be moved between cells). Horizontal and vertical plasmid transfer

Can a cell survive without a plasmid?

Yes because it encodes non essential genes

What can some strains of E.Coli use a plasmid for?

Adhesion

What are colicins?

They are antimicrobial proteins secreted by bacteria and kill other bacteria by forming pores or by entering the cell and acting as nucleases.

What were colicins originally found in?

Coli bacteria (E.Coli)

Are colicins toxins? What group are the pore forming and nuclease colicins?

Yes and group A colicins

Does the colicin/toxin hurt the bacteria that is secreting it?

No it does not hurt the host because the plasmid that encodes the toxin also makes the immunity product that will neutralize the toxin (encoded in pairs)

What are colicin systems encoded by?

Plasmids

If the bacteria does not have the plasmid will the colicin kill them?

Yes it will

Why might we have colicins? What is there pressure to do?

This is to kill competitors in the niche. Pressure to retain the plasmid so the toxin doesn’t hurt you

What is copy number?

It is the physical number of copies within a given cell

What are the five characteristics of high copy number of plasmids?

1. Relaxed

2. Generally small size (less than 10 Kb)

3. ColE1 plasmid is best studied of this type

4. Replication is unlinked to cell division (doesn’t divide but can be replicated)

5. Random partitioning

What does ColE1 do?

It makes colicins that kill species related to E.coli

What are the five characteristics of low copy number of plasmids?

1. Stringent

2. Usually larger (can be up to 300 Kb)

3. R100 plasmid is model for this type

4. Directed partitioning systems (there is a divide)

5. Often conjugative

Why do we have a conjugative event in low copy number?

This is for a back up mechanism for ensuring it is transferred to the progeny

In replication and partitioning in high copy plasmids, what are progeny assured of? Does it matter where we partition the cell during cell division?

Inheriting the plasmid because there are many copies. No it doesn’t matter because there are a ton of plasmids

How can copy number be amplified in high copy plasmids? Why? What is this known as?

Adding a drug that inhibits protein synthesis because replication is unlinked to cell division. Known as plasmid amplification

What is an example of a drug that can be added that prevents protein synthesis? How does it inhibit protein synthesis?

Chloramphenicol and it is able to bind to ribosomes and prevent growth of the polypeptide chain. Existing proteins can still work but new ones are just not made

Can plasmids still replicate when chloramphenicol is added? Why?

Yes they can because of existing proteins (synthesis of proteins is different than activity of proteins)

Does it matter where we partition the cell during cell division when we have low copy plasmids? Do they have special partioning mechanisms?

Yes to both

What would happen if we didn’t have specialized partitioning mechanisms in low copy plasmids?

One daughter cell may not get a plasmid

What is the replication of a low copy plasmid linked to?

A chromosome to ensure at least 2 copies are present at time of division

What is regulation of copy number often controlled by?

Antisense RNA (small RNA)

What does high copy number result in?

The expression of an antisense RNA that interferes with replication

At low concentration/low copy number what is the concentration of RNAI and Rop?

They are also at low concentration

What happens as the plasmid replicates?

The concentration of RNAI and Rop protein increase as the plasmid is replicating

What is RNAII needed for?

Building at the origin (for origin of replication) (required for plasmid replication)

When there is high concentration of RNAI and Rop protein what happens?

Rop protein will help RNAI bind RNAII forming a hybrid which will prevent plasmid replication from occurring

What is cell actively doing?

Measuring the copies of plasmids it has and has Rop and RNAI to stop the process of replication