Lecture 11: Conjugation Flashcards
Conjugation
The process of a plasmid transferring to a recipient cell. Plasmid is retained in donor cell too.
F plasmid
-Fertility plasmid: Able to mediate transfer of itself into new recipient bacteria
Transfer of F plasmid
Tra genes synthesize sex pilus and type IV secretion system for DNA transfer
What structure is formed by tra genes
Genes encode for a transfer apparatus, a sex pilus that can be used to exchange and move DNA across 3-4 membranes
Steps in conjugation
-Pilus extends
-Recognizes and binds to a receptor on the surface of recipient
-Pilus retracts
Coupling proteins (TraD)
-Proteins that provide the specificity so that only certain plasmids are transferred.
-They bind the transfer apparatus and the DNA
Does the DNA go through the pilus or through fusion of cells
Not known. Some evidence suggests through the pilus. Rolling circle replication pumps DNA out of cell using the coupling protein
Genes on F plasmid
- Proteins of conjugate pilus (Type IV)
- Proteins of Tra machinery for DNA transfer
- Origin of transfer (oriT)
- Origin of replication (OriV)
- Exclusion proteins (TraS TraT) to prevent conjugation with another F+ cell
- Addiction: Post segregational killing protein
Regulation of tra genes
-An activator is produced after DNA entry
-As a second protein (FinO) accumulates, it stabilizes an antisense RNA that shuts down expression of the activator.
Why are genes like tra regulated at all
To reduce cost to the host. And to reduce exposure of the pilus to infection by certain bacteriophages
Plasmid mobility for non-self-transmissible plasmid
Using another self transmissible plasmid in a different helper cell can allow a plasmid to become mobilized towards recipient
Side effect of conjugation
-Sometimes a plasmid will insert into the chromosome and transfer part of the chromosome to the recipient cell.
-F plasmid recombines into the chromosome causing an Hfr (High frequency recombination) Strain causing high amounts of chromosome DNA transfer
R plasmid
Naturally occurring plasmid that carries genes which confer resistance to antimicrobial drugs. Some can be transferred from one bacterial cell to another by process of conjugation
Inter-kingdom conjugation
Usually, conjugative plasmids are within the same species, but they can be trans-genus
Argobacterium tumefaciens
-Plant pathogen
-Creates tumors or “galls”
-Transfers Ti plasmid to plant by conjugation
-Ti plasmid directs production of growth hormone and octopine synthesis
-Involved in genetic engineering of plants
Process of Argobacterium tumefaciens plasmid transfer
- Argobacterium transfers the Ti plasmid to the plant by conjugation
- The plasmid is recombined into the plant chromosome
- Plant expresses Ti genes
- Argobacterium feeds on the compounds produced by the plant
Genes the Ti plasmid has
-Bacterial transfer
-Plant transfer
-Self-Replication
-An oriV for self-replication
-oriTs for transfer-replication
onc & ops genes for Ti plasmid
-Onc genes encode enzymes that synthesize plant hormones (Stimulate growth of plant cells)
-Ops genes encode enzymes for opine biosynthesis (Modified amino acids)
Oncogenes in Ti pladmids
Considered non-essential so that can be replaced with any preferred DNA of interest
Making transgenic plants
-Cut out a piece of the leaf
-Float in plate containing argobacterium strain with engineered Ti plasmid
-Incubate on plate containing plant regeneration medium
-Excise germinating shoots and transplant to plates containing kanamycin
