Plasmids

Plasmid replication is usually carried out by?

Host cell DNA polymerases

Plasmids encode genes involved in regulating the rate of plasmid replication and thus determine…?

Plasmid copy # within the cell

If the replication of 2 plasmids is controlled by the same genes, what does this mean?

Plasmids can’t be maintained together in same cell = incompatible

Plasmids that are incompatible with one another are ___ and belong to what group?

closely related, Inc group (“incompatible”)

What method do many plasmids use to transfer from donor cell to recipient cell? i.e. what is the process called?

Conjugation

How is conjugation (transfer process) mediated in plasmids?

By products of tra genes, some of which interact with the oriT region of the plasmid to initiate the transfer of a single strand of DNA, whereas others form structures such as pili, which aid in the transfer of DNA of recipient cell.

What can conjugative plasmids do?

1. Conjugate themselves between bacterial cells

2. Can sometimes integrate into bacterial chromosome and transfer part of the chromosome between bacterial cells

In conjugative plasmids, what are the donor and recipient cells called?

Males, females (respectively) 

Conjugative plasmids - donor cells vs recipient cells

Compare & contrast

Only donor cells have sex pilus, which is a structure encoded by some of the plasmid-encoded tra genes and helps to bring the donor and recipient cells together.

Once the donor and recipient cells have been brought together, what happens next?

One strand of plasmid is nicked at the oriT by a tra-encoded endonuclease, the resulting single strand is unwound from the helix, and is transferred into recipient cell. Strand that’s transferred is replaced in the donor cell by replication, and the complementary strand is also synthesized in the recipient cell.

T/F - under appropriate conditions, plasmids can spread rapidly within and between bacterial populations by transduction.

F - under appropriate conditions, plasmids can spread rapidly within and between bacterial populations by conjugation.

How is it that plasmids can integrate into the chromosome and mediate conjugation as part of the chromosome?

1. Some plasmids, such as F, have insertion sequencies (IS).

2. Strains that have F integrated into the chromosome are called Hfr (“high frequency of recombination”)

Some plasmids, such as F, have insertion sequencies (IS). What is this and how does it allow plasmids to integrate into the chromosome and mediate conjugation as part of the chromosome?

Specific DNA sequences that are also found on the chromosome, and homologous recombination can therefore occur between plasmid & chromosome at these sequencies.

Strains that have F integrated into the chromosome are called Hfr (“high frequency of recombination”). What is this and how does it allow plasmids to integrate into the chromosome and mediate conjugation as part of the chromosome?

A given Hfr strain will be able to transfer a particular gene at a characteristic frequency. Genes that are nearest site of insertion in the direction of transfer are transferred at the highest frequency.

In an Hfr strain, why are genes that are nearest site of insertion in the direction of transfer are transferred at the highest frequency?

Genes nearest the oriT of the integrated F will be most likely to be transferred into the recipient cell before DNA strand is broken.

Sequential nature of gene transfer can be demonstrated using?

Interrupted mating experiments, which can be used to map genes.

To detect a chromosome conjugation event between donor and recipient cells, appropriate markers and selection is needed. What does this usually entail? (discuss markers used & selection)

Combo of antibiotic resistance & auxotrophic markers. Conditions for selection must be such that desired transconjugant bacteria are able to grow, but donor and recipient aren’t able to grow.

Sometimes, an integrated F plasmid is able to excise from the chromosome, and sometimes when it does this, it brings along part of the chromosome as well. What does this result in?

Plasmid carries chromosomal genes. This is called a F’ plasmid.