Conjugation and Transduction Flashcards
What are the three major types of genetic transfer found in bacteria?
- Transformation
- Conjugation
- Transduction
Define transformation.
Genetic transfer in bacteria where DNA molecules are taken up from the environment and incorporated into the genome.
Define conjugation.
Genetic transfer in bacteria where donor DNA is transferred from one bacterial cell to another by direct contact.
Define transduction.
Genetic transfer in bacteria where DNA is transferred from one bacterial cell to another by bacterial virus.
Outline F plasmids.
F plasmids are conjugative plasmids that contain oriT, the origin of transfer, and tra genes, which code for pilus formation and transfer promoters.
It is about 100 kb in length and 1 or 2 copies are found per cell, and replicate when the cell does. It contains F factors which are transferred during conjugation, which is achieved through rolling circle replication.
F plasmids can be found in two forms: F+ or Hfr. F+ is a extrachromosomal state, whereas Hfr is integrated into the chromosome.
Describe the transferring of an F+ factor and an Hfr factor.
F+
- Donor and recipient cells make contact.
- Nuclease makes a nick in the DNA at oriT, primases assemble at the 5’ end, and replication is initiates at the 3’ end.
- The 5’ end is transferred from donor to recipient by rolling circle replication.
- The linear F plasmid then becomes circular in the recipient.
Hfr
- Donor and recipient cells make contact.
- Nuclease makes a nick in the DNA at oriT, primases assemble at the 5’ end, and replication is initiates at the 3’ end.
- The 5’ end is transferred from donor to recipient by rolling circle replication.
- The F factor and other genes on the chromosme are transferred, and are integrated into the recipient genome through homologous recombination.
- The free DNA fragments are then digest by nucleases.
Outline the differences in F transfer and Hfr transfer.
- Time: transferring the entire bacterial chromosome takes 100 minutes, the F plasmid takes 2 minutes.
- Hfr transfer is usually interrupted as the mating pair breaks apart.
- Transferring Hfr to F- cells usually results in F- cells, as the transfer is interrupted before the final part of the F factor is transferred.
- The Hfr recipient cell becomes a recombinant, as the DNA is replaced through homolgous recombination.
Define vector.
A DNA sequence that can carry another section of DNA into a host.
Examples include plasmids, bacteriophages, BACs, and YACs.
What types of mutants are useful as select and non-select markers? Give an example of each. [3]
Prototrophs, antibiotic mutants, and nutritional mutants.
A common carbon-source mutant is the lac- mutant (which cannot ferment lactose) which needs a medium supplemented with another carbon-source in order to grow. Lactose supplements selects for lac+ cells and against lac- cells. The phenotype is denoted as LacZ+, the genotype lac+.
A nutritional mutant is one that cannot synthesis all the nutrients it needs in order to function, e.g. leucine, threonine. WT: prototroph, mutant: auxotroph.
An example of an antibiotic mutant is the str-r mutant, which is resistant to streptomycin (str) and allows selection for cells with resistance and against mutants without the streptomycin gene.
Outline rolling circle replication.
Rolling circle replication is the form of replication used when the F plasmid, F+ or Hfr, is transferred from the donor bacterium to the donor.
- The tra gene codes for a specific nuclease that nicks the DNA at the oriT site, and binds primases at the free 5’ end (primes DNA for replication). Replication is initiated at the 3’ end of the nicked strand and replaces the transferred 5’ single strand.
- Lagging strand DNA synthesis in the recipient converts the transferred single strand into double stranded DNA.
- When transfer is complete the linear F strand becomes circular again. The F- cell has been transformed into an F+ cell.
- In laboratory conditions the transfer only takes a few minutes, but in nature the transfer is not so efficient.
What properties do vectors need in order to make them useful cloning vectors?
- Need to be able to have DNA inserted
- Need to be able to replicate in the host cell
- Need to be able to replicate with the inserted DNA
- Need restriction sites
How can F plasmids be used in gene mapping? What are the main issues?
Genes can be mapped through mating Hfr x F- cells, mapping gene transfer order. This is done through deliberate interruption of mating cells (BLEND!!!!). The time of gene transfer is the earliest time when the breakage no longer prevents the recombinant from appearing. This is called interrupted-mating technique.
After mating Hfr bacteria they need to be able to be tested to see if the desired recombination. This is done by using select markers, e.g. str-r, leu+, thr+, and counterselect markers which inhibit growth of the donor.
- Select markers must not be present in F- cells.
- Counterselect markers should be located in a place that most mating cells will have broken apart before its transfer.
- Hfr transfer is done starting in the middle of the F factor, therefore the whole chromosome is needed to be transferred in order for the whole F factor to be transferred.
Issues:
- Limited to only a few bacterial types, such as E. coli.
- Mapping of the whole chromosome is rare.
- They can only transfer genes next to each other, so distant genes may be missed.
- F plasmids are too large to use in genetic manipulation and gene cloning.
Outline the bacteriophage lytic lifecycle.
- Phage particle attaches to the host bacterium and inject nucleic acid into the cell.
- Phage nucleic acid is transcribed (host polymerase) and translated (host tRNA and ribosomes) into phage proteins (late genes), and enzymes that replicates the phage DNA (early genes).
- The Phage DNA turns off the host DNA.
- Another late gene produces molecules that form a lysozyme, which attacks the peptidoglycan wall.
- The newly synthesised nucleic acid is packaged into protein shells, and the particles are released from the cell, rupturing the cell.
- Some bacterial genes are incorporated into the DNA of the new host, giving and altered phenotype.
Outline the difference between a lytic and lysogenic lifecycle of bacteriophages.
Phages can have one of two different forms of lifecycle: lytic or lysogenic:
Lytic: release of mature phages causes the lysis of the cell, 102 -103 phages are released.
Lysogenic: can choose to either be lytic, or take up a semi-stable residency in the host (usually phage DNA integration into the host chromosome, forming a prophage).
The integration event that forms the prophage is reversible and is driven by phage genes. If it transfers to a lytic lifecycle, it excises itself from the genome and undergoes the lytic pathway.
