Lecture 15: Bacterial plasmids and genetic exchange

Question 1

What is recombination?

Answer 1

It is the rearrangement of nucleic acids to produce a new nucleotide sequence. It involves an exchange between different strands of DNA.

Question 2

What is homologous recombination?

Answer 2

It is recombination between two similar (homologous) sequences.

Question 3

What is horizontal gene transfer?

Answer 3

It is the transfer of genes or DNA pieces from one organism to another, as opposed to via cell division as is the case in vertical gene transfer.

Question 4

What are the 4 types of DNA recombination in bacteria?

Answer 4

Homologous recombination (crossing over)
Nonreciprocal recombination
Site-specific recombination
Replicative recombination

Question 5

Which forms of bacterial DNA replication require homology between DNA?

Answer 5

Homologous recombination and nonreciprocal recombination.

Question 6

Describe the major steps in crossing over (homologous recombination)

Answer 6

• Start with 2 double stranded DNA, one broken
• Break is recognized by RebBCD
• Helicase opens DNA and exonuclease pulls strands apart to get single strands
• RecA recognizes single-strnaded DNA and initiates strand transfer with other DNA
• Single-stranded DNA is paired with homologous sequence of other DNA
• DNA polymerase III uses 3’ end of single strands as a primer and fills in the gap based on the template (other DNA). Ligase fills in gaps
• Can get branch migration
• Resolvase cuts to make 2 double-stranded DNA gain.

Question 7

What are the two possible outcomes of crossing over? Explain the difference.

Answer 7

Depending on how resolvase resolves the DNA, you can either get spliced or patched DNA. In spliced, both old and new DNA are present in both DNA sets and there has been a true swap of information. In patched, only small bits of information have been swapped and the original DNA remains more or less where it started.

Question 8

Describe the major steps in nonreciprocal recombination.

Answer 8

• Start with two single-stranded DNA, a donor and a host
• If they are highly homologous, they will associate and be opened by a helicase.
• Part of the donor strand will associate with the host strand.
• An endonuclease will nick the donor strand.
• An endonuclease will nick the host strand.
• The gaps in the strand will be filled in and ligated.
  The result is heteroduplex DNA.

Question 9

The result of nonreciprocal recombination is […]

Answer 9

Heteroduplex DNA

Question 10

What are bacterial plasmids?

Answer 10

They are small, usually circular, double-stranded DNA molecules. They exist independently from the host chromosome and contain a few genes that are not essential, but are useful.

Question 11

How many copies of a bacterial plasmid can cells have? What determines this?

Answer 11

1 to hundreds depending on how easily they and their origin of replication get recognized by the replication machinery.

Question 12

What are episomes?

Answer 12

Bacterial plasmids integrated into the host chromosome.

Question 13

Give an example of an episome.

Answer 13

Fertility (F) factor

Question 14

What are the 5 families of bacterial plasmids? Briefly describe each.

Answer 14

1. Fertility factors
2. Resistance factors: carry antibiotic resistance genes
3. Col plasmids: code for colicins and bacteriocins that are used to kill other cells in the environment.
4. Virulence plasmids: produce toxins and encode genes to help resist host immune defense
5. Metabolic plasmids: carry enzymes such as the lac operon

Question 15

What is the main way through which bacterial cells evolve?

Answer 15

Via bacterial gene transfer - they pick up DNA from other sources/the surrounding environment

Question 16

What are the 3 forms of bacterial gene transfer? Briefly describe each.

Answer 16

Conjugation: cell-to-cell contact and transfer involving sex pili
DNA transformation: DNA or plasmids taken from cell environment
Transduction: passage of genetic information via bacteriophages

Question 17

Immediately after the intake of new DNA/plasmids, the cell becomes […]. Explain why.

Answer 17

Partially dipoid. This is because the cell will have two copies of the genes that the new DNA encodes for (in addition to the original genes in their genome)

Question 18

Describe the 4 possible outcomes of the intake of new DNA/plasmids into a cell.

Answer 18

1. The donor DNA is integrated into the chromosome and the cell can produce stable recombinants (integrated exogenote)
2. The donor DNA self-replicates as a plasmid and the cell can produce stable recombinants (partial diploid clone)
3. The donor DNA cannot self-replicate and the cell cannot produce stable recombinants (partial diploid clone before replication)
4. There is a host restriction such that the donor DNA is rejected and degraded right away. The cell cannot produce stable recombinants.

Question 19

What determines the outcome of the intake of new DNA/plasmids into a cell?

Answer 19

Generally, the more similar to the DNA is (i.e. how similar of a cell it came from), the more likely it is to successfully integrate.

Question 20

Of the 4 possible outcomes of the intake of DNA, state whether the reslting cell will be haploid, diploid, or partially diploid.

Answer 20

Integration of DNA: haploid
Donor DNA self-replicates: partially diploid
Donor DNA cannot self-replicate: partially diploid (but future generations will be haploid)
Host restriction: haploid

Question 21

What is the fertility factor?

Answer 21

It is a conjugative plasmid that contains genes for cell attachment (sex pili) and plasmid transfer (tra region)

Question 22

A cell with an F plasmid is called a […], while a cell without one is called a […]

Answer 22

F+, F-

Question 23

Is the fertility factor an episome? Explain why or why not.

Answer 23

Yes, because it can be found associated to the chromosome or can be free-standing.

Question 24

What are the two major parts of the fertility factor?

Answer 24

Origin of transfer (oriT) and origin of replication (oriV)

Question 25

Describe the process of bacterial conjugation.

Answer 25

• The sex pilus from the F+ grabs an F- and pulls it close
• A conjugative pore forms between the cells
• The F plasmid gets nicked at its oriT and then gets replicated via rolling circle replication.
• The new strand goes into the F- cell through the pore.
• At the end up replication, replicates occurs such that the F factor becomes double stranded. Both are now F+.

Question 26

What is an HFR cell?

Answer 26

It is a high-frequency cell. It happens when, after conjugation, the F plasmid integrates into the chromosome via crossing over

Question 27

An HFR cell can lead to the creation of a […]. Explain how.

Answer 27

F’ plasmid. This happens if the F plasmid disassociates itself from the chromosome in the HFR and takes a piece of the original chromosome with it.

Question 28

How are F’ plasmids useful?

Answer 28

After the transfer of the F’ plasmid to another recipient cell, the latter will have a duplication of some genes. The duplication of DNA is useful because if the original genes are inactive or mutated, they can now operate with the new copy. Ex: lac operon.

Question 29

When does DNA transformation typically occur?

Answer 29

When bacterial cells become stressed and detect that the environment is harsh for them. They will try to pick up DNA to survive.

Question 30

Describe the process of DNA transformation.

Answer 30

• DNA from the environment by chopped up by an endonuclease.
• A piece gets picked up and brought into the cell.
• One strand is degraded
• If the DNA is highly homologous to the genome, it will likely be integrated by nonreciprocal recomination.
• If not, it will be degraded and not integrated.

Question 31

The success of DNA transformation is contingent on […]

Answer 31

High homology between the new DNA and the cell’s genome.

Question 32

How does DNA transformation differ when it’s a plasmid being picked up (vs regular DNA)?

Answer 32

They go right through the membrane and don’t get degraded, so they stay double-stranded the whole time.

Question 33

Explain how bacterial gene transfer via tranduction works.

Answer 33

• A bacteriophage infects the cell, it destroys the host chromosome and synthesizes its own DNA to put into capsids (phage particles)
• Some of the original DNA can be incorporated into the capsids, yielding transducing particles.
• If transducing particles inject themselves into another cell and the DNA is highly homogous, it will be integrated and maintained via stable gene transfer.
• If they are not highly homologous, you could either get abortive transduction or unsuccessful gene transfer.

Question 34

What is the difference between abortive transduction and unsuccessful gene transfer in transduction?

Answer 34

Both involve the DNA from the transducing particle being recognized as foreign. But successful gene transfer involves it being degraded right away, while abortive transduction involves the DNA staying free-floating and gradually getting degraded over time.

Question 35

What are the two possible outcomes of DNA transformation for linear DNAA? Explain.

Answer 35

Stable transformation or unsuccessful transformation. In stable transformation, the new DNA is incorporated into the chromosome. In unsuccessful transformation, the new DNA is simply degraded.

Question 36

What are the two possible outcomes of DNA transformation for plasmids?

Answer 36

Stable transformation or unsuccessful transformation. In stable transformation, the new plasmid is able to remain in the cytoplasm, which in unsuccessful transformation, it is degraded.