Plasmids and conjugation

Mechanisms of horizontal gene transfer

• transformation

• Transduction

• Conjugation

Conjugation

• process of moving genetic material via direct cell to cell contact

• Usually plasmids

Experiment which identified conjugation

• lederburg and Tatum

• Mixed culture A and B

• B could synthesise the nutrients which A couldn’t and vice versa

• When mixed together, the colony was able to grow on minimal media

• It could synthesise all the nutrients needed

Davies experiment

• further evidence for conjugation

• Colony A and B in U shaped tube separated by semi permeable filter (impermeable to anything of cell size)

• Suction or pressure applied to one end, while the other is blocked to mix the non cell components

• The colonies didn’t grow on minimal media

• When the filter was removed, the colony could grow on minimal media

• Ruled out cross feeding, transduction and transformation

Conclusion of Davies experiment

• metabolites , DNA and phage could pass through the filter , hence weren’t responsible for the wild type reversion

• Reversion of the mutants to the wild type must require cell to cell contact

Plasmids

• Not part of main chromosome

• Mostly circular, but can be linear

• Replicate independently of chromosomal DNA

• No extracellular form, naked if not in cell (unlike phages with a protein coat)

Plasmids within the same host

• have different copy numbers, controlled by the plaid

• Can be incompatible

• Cells contain many non related plasmids

Incompatible plasmids

• often related, sharing common replication mechanisms

• Means they cannot coexist

Episomes

• special plasmids

• Can integrate into host genome

Curing

• plasmid being lost from host

• Either spontaneous

• Or in response to a chemical

Roles of plasmids

• carries useful, non housekeeping genes

• Antibiotic resistance

• Virulence factors eg toxins

• Bacteriocins

Bacteriocins

• proteins which kill or inhibit closely related species

• Not as broad spectrum as antibiotics

• Eg by forming pores in the membrane or degrading DNA

Conjugative plasmids

• plasmids able to conjugate

Nature of conjugative plasmids

• not all plasmids

• Themselves encode the gene that allows for transfer to other cells

• Some only transfer to same species

• Others transfer to other species

Tra

• genes responsible for transfer

F pilus

• sex pilus

F of f pilus

• fertility factor

Purpose of f pilus

• unidirectional transfer of DNA from donor to recipient

• Encoded by F plasmid (conjugative)m

Integrative plasmid

• eg F plasmid

• Can integrate into host a number of locations into genome or exist as free plasmid

Transfer of plasmid process

• Donor looks for recipient

• Contact is made via F pilus

• F pilus shortens to pull cells together

• The cells have a partial fusion , a mating bridge

• Plasmid is transferred , both cells

Why are they called fertility factors

• both cells have the plasmid and hence the F pilus

• Both go on and repeat the process to transfer the F pilus

How are plasmids transferred

• As single stranded DNA

Process of plasmid transfer

• one strand is nicked

• Nicked strand is unrolled from DNA and transferred

• Becomes circular once transferred

• Other strand is synthesised in both cells

Copying of plasmids

• by rolling circle replication

Leading strand - rolling circle replication

• one strand nicked at DSO of replication

• 3’ end serves as primer, DNA pol binds

• After whole circle of plasmid has been replicated , the original strand, now displaced, is released

• New strand is ligated to heal nick

Lagging strand - rolling circle replication

• The displaced single strand is circularised and ligated

• Replication initiated as SSO of replication

• RNA primer starts DNA pol off

• Once whole plasmid ds has been synthesised, its ligated to heal the nick

DSO

• double stranded origin (of replication)

SSO

• single stranded origin (of replication)

Spreading of F plasmid

• rapid

• Whatever other genes (alongside f pilus) are encoded will be spread too

Requirements of F plasmid spreading

• mating bridge must stay open

• Usually 2 mins at 37 degrees

• Often doesn’t happen at lower temps since takes longer, mating bridge must be sustained

• Sensitive to shaking/agitation

• KEY - MATING BRIDGE MUST STAY INTACT

HFr Acronym

• high frequency recombination strains

What is a HFr (not acronym)

• can partially transfer genome

• Derived from strain with F plasmid

• Occurs when F plasmid has integrated into genome , becoming an episome

• Cell produces F pilus BUT has no plasmid to transfer

HFr transferring their Genome

• plasmid within genome is still nicked

• 3’ end unrolled and resynthesised within genome

• The replaced strand is transferred to the other cell via the F pilus

• Complementary strand synthesised

Fate of HFr strain transferred DNA

• usually breaks before entire chromosome is transferred

• Because its long and the ss structure is unstable

What happens to the DNA from HFr once transferred INTO recipient

• cannot circularise since not all of the f plasmid is present , some other genes are present and some are missing

• Mostly degraded as a bacterial defence against viruses

• Occasionally, recombination takes place (DNA integrates into genome)

The new strain after HFr transfer

• NOT f+, since only part of F plasmid genome is transferred due to short mating bridge time

• Gene transfer stops when mating pair break apart

Merodiploid

• haploid strain that is diploid in some genes

Gene transfer resulting in merodiploid cells

• plasmid strand is transferred by rolling circle replication

• Mating pair break apart before whole genome is transferred

• Strain can becomes temporarily diploid is 2 alleles of a gene are then present

• Recombination occurs, genome is haploid again as survivally favoured genes dominate

• Genome carries new traits

Time of entry mapping

• allows ID of order of genes on a chromosome

• Further away genes from origin of replication, less likely transfer due to duration of mating bridge

• High % recombinants carrying trait = closer to origin of replication

What can HFr strains become

• F’ strains

How are F plasmids reformed from genome

• excised from genome

• Some chromosomal genes end up in the plasmid (F’)

F’ plasmid

• imprecise excision of plasmid from genome

• Chromosomal genes end up in plasmids

What can F’ strains mate with

• F- strains

• Recipient has own copy of gene in genome and new copy on plasmid

• Becomes merodiploid

• Can recombine to gain new traits