Prokaryotic genetics-50 Flashcards
Plasmids and Conjugation
What are the mechanisms of horizontal gene transfer?
-Bacterial transformation
-Bacterial transduction
-Bacterial conjugation
What is conjugation?
The process of moving genetic material (often, but not always, plasmids) via direct cell-to-cell contact.
What experiments provided evidence for horizontal gene transfer?
-Lederberg and Tatum, 1946
-Davies, 1950
What was the Lederberg and Tatum experiment?
A: met- bio- thr+ leu+ thi+
B: met+ bio+ thr- leu- thi-
Mix A and B
Wash cells.
Plate has ~10^8 cells.
Minimal medium plates.
A- and B- mixed- some progeny are wild type.
Prototrophic colonies: met+ bio+ thr+ leu+ thi+
Wild-type phenotype, therefore can grow on minimal media.
What was the Davies experiment?
Tube with A- and B- solutions.
Semipermeable filter between two solutions-allows small entities to pass but no cells.
Block one end with cotton wool- to seal it.
Apply suction or pressure to other end- to mix media via filter.
Plate bacteria from both sides on min media.
No growth i.e. remain A- and B-
Remove filter and colonies grow.
Metabolites, DNA and even phages can pass through filter. Reversion to wild type must require cell-to-cell contact.
What are plasmids?
Almost always double stranded DNA.
Most are circular, but they can be linear.
Size between 1kb to >1Mbp
Replicate independently of chromosomal DNA.
Do not have extra cellular form like phages.
What do the two experiments rule out? Why?
Cross feeding- ability of different strains to support each other.
Transformation- ability to take up naked from environment.
Transduction- use phages to move DNA from one strain to another.
Due to semi-permeable filter, phages can go though the filter but cells cannot.
What are episomes?
Special plasmids that can integrate into host genome.
What are plasmids in the same host like?
Plasmids have different copy number:
High copy number (>100)
Low copy number (1 or a few)
Plasmids can be incompatible:
-related plasmids sharing common mechanisms of replication often cannot coexist.
-light blue incompatible with one of the others so will be lost.
Cells can contain many non-related plasmids.
Curing:
Plasmid is lost from host.
Happens spontaneously OR in response to certain chemicals.
What is the role of plasmids?
Carries non-essential but often highly useful genes.
Antibiotic resistance- problematic from medical point of view, useful from biotech point of view.
Virulence factors- e.g. toxins that increase pathogenicity. E. coli hemolysin (lyses red blood cells) and enterotoxin (causes diarrhea).
Bacteriocins- proteins killing or inhibiting closely related species (not as broad spectrum as antibiotics). e.g. colicins (forms pores in membrane, degrades DNA ect.)
What are conjugative plasmids?
They encode the genes that will allow transfer to other cells, therefore can conjugate.
Some transfer only to same species.
Others are more promiscuous- transfers to other types of bacteria.
E.g. F plasmid in E. coli
Plasmid encode tra genes responsible for transfer.
All genes required for transfer ~33kbp, about 1/3 of the F plasmid.
What is F pilus?
A mating pair connected by an F pilus.
Sometimes called a sex pilus.
Unidirectional transfer (not exchange of DNA) of DNA from donor to recipient.
F stands for Fertility factor.
F plasmid can spread through F- strain quickly similar to an infectious agent.
F is integrative plasmid- can integrate in a number of locations or exist as a free plasmid.
How does pilus creates contact?
-Donor and recipient
-Donor looking for mate
-Contact is made
-Cells pull closer
-Transfer of plasmid via mating bridge
-Both cells now have plasmid
How is plasmid transferred?
Plasmid is transferred as single stranded DNA.
-F+ carries plasmid
-one strand of plasmid is nicked
-nicked strand is ‘unrolled’ and transferred
-once transferred plasmid is made circular, other strand synthesised in both cells
How is plasmid copied?
It is copied by Rolling Circle Replication (RCR) leading strand.
-One strand is nicked at double stranded origin of replication.
-3’ serves as primer for replication.
-Once a full round has been completed, ‘old’ strand is released as ssDNA.
-New strand ligated to ‘heal’ nick.
-In conjugation this ssDNA has been transferred to acceptor cell.
RCR is used by many viruses for replication.
What happens once both cells now have F plasmid? What are the conditions for this process?
F plasmid can spread through an F- culture rapidly, ensuring the cells are converted to F+. Whatever other genes are encoded by the plasmid will now be spread.
Process takes ~ 2 min at 37 degrees.
Usually doesn’t happen at 30 degrees or lower.
Sensitive to agitation.
mating bridge is only present for a certain time period
What are HFr strains?
High Frequency Recombination (HFr) strains
-Derived from F+ strain.
-F plasmid has integrated into genome (carries same genes as before, but has no plasmid to transfer) through recombination.
-This is a rare event
-F plasmid is an episome
Still produces F pili, but it has no plasmid to transfer
Can HFr strains transfer their genome?
Yes, they can transfer their genome.
-Integrated F plasmid
-Plasmid is still nicked
-Replaced strand transfer to other cell
-Usually DNA brakes before entire chromosome is transferred
-Complementary strand synthesised in other cell
What happens to the new DNA? (HFr strain and F- strain)
-DNA cannot circularise, as not all of the chromosme is transferred.
-Most is degraded.
-Occassionally recombination takes place.
Does the new strain have all of the new genes? Why?
No, not all of the F plasmid and genome is transferred.
It would take a long time to transfer the entire genome. Unlikely that the mating pair will stay together for the required time.
Hence, new strain is not F+.
When does gene transfer stop?
Gene transfer stops when mating pair breaks apart.
-Mating pair breaks apart before the whole part of the plasmid can be transferred.
-Temporarily the strain is partially diploid (merodiploid).
-After recombination it is haploid again and it carries new traits.
What is merodiploid?
Haploid stain that is diploid only in some genes.
What is the time of entry mapping?
The further gene is away from origin, the longer and less likely transfer is.
How can HFr strains become F’ strains?
F plasmid can excise from genome to become F plasmid again.
Occasionally excision is imprecise and some chromosomal genes end up in plasmid.
Can a F’ strain mate with F- strain?
Yes
-Donor has some chromosomal genes on F plasmid
-Recipient has its own copy of gene in genome and new copy on plasmid (merodiploid)
-Can recombine to gain new traits
