Plasmids II Flashcards
Transformation
Uptake of short fragment of naked DNA by transformable bacteria
Transduction
Bacteriophages -< transfer of DNA from 1 bacteria to another
What is an auxotroph and prototroph?
Auxotroph has nutrients missing, prototroph can synthesis own material
F episome
A plasmid that can also traffic in and out of chromosomes
Characteristics of F episome
5% of E.coli genomic size
2 oriR’s:
- oriV : ‘free’ plasmid one copy/bacterium
- oriT: cis-acting element at beginning of transfer region where transfer f plasmid initiated
tra region: 33kb with 40 genes -> allow integration via homologous recombination
Tra operon
mostly transfer + replication
- 12 genes for modification + assembly for piin in sex pilus
- stabilisation of T4SS
- arranged in loci as tra +trb
What do the different tra’s do?
traA - pilin traI - relaxase traD - coupling protein ATPase traY - DNA binding TF traM - protein interactions tra N,G, ompK -> formation and stabilisation of mating pair
Mechanism of conjugation
- Pilus from F+
- Pilus retracts -> proximity
- Cell pairs stabilise
- Relaxosome formation + concomitant rolling circle synthesis
- DNA transfers through T4SS at beginning of rolling circle rep.
- Transferred DNA becomes double stranded
What does the relaxosome consist of?
Relaxase (traI) + accessory proteins (traY, traM, IHF)
How is the relaxosome formed?
It forms at the oriT of -ve supercoiled DNA -> monomer relaxase covalently links 5’ end generated during phosphodiester bond hdrolysis
What happens after the relaxosome is formed?
- Relaxase, dimer, nicks oriT at nic
- relaxase also unwinds DNA
- relaxase-bounds DNA is bound by coupling protein traD + traM
- TraM mediates relaxosome-transferase contact transport through T4SS
A Hfr strain forms when
F episome integrates host chromosome through insertion sequences
How does f episome replicate in hfr strain?
Uses tra operon but can’t replicate fully. Conjugation starts at oriT but paired cells are not as stable so rolling circle only partially transfers chromosome.
When you receive from an hfr strain, you’re still…
F - , + partial chromosome can’t replicate so it’s either lost/recombines with host
In antibiotic resistance, instead of f factor it’s
r factor e.g. shigella
Bacteriocins are
bacterial secretions that inhibit the growth of closely related bacterial strains
What is an example of a bacteriocin?
Co1E1 plasmid inhbiits shigella -> it’s a channel forming TM protein with copy # 10-15 (high) since plasmid rep. not necessarily coupled to chr. rep.
CoIEI is non-transferrable by bacterial mating since…
mating pair formation genes absent
mob+ can be transferred if
combined with conjugate plasmid (F factor. R factor)
Why is it the normal frequency of recombination higher in hfr strains?
F factor integrated into chromosomal DNA so contains oriT which can be nicked to allow recombination
Give the name of the vectors in increasing size limit:
Plamid, phage, cosmid, BAC, YAC, MAC
