Plasmids and Plasmid Biology

Plasmid structure
Plasmid replication and copy number control
Plasmid transfer
Plasmids as tools
F plasmids
Plasmids and Plasmid Biology

Plasmids
Extrachromosomal DNA, usually circular-parasite?
Usually encode ancillary functions for in vitro growth
Can be essential for specific environments: virulence, antibiotics resistance, use of unusual nutrients, production of bacteriocins (colicins)
Must be a replicon - self-replicating genetic unit

Plasmids
Plasmid DNA must replicate every time host cell divides or it will be lost
a. DNA replication
b. partitioning (making sure each progeny cells receives a plasmid)
High copy plasmids are usually small; low copy plasmids can be large
Partitioning is strictly controlled for low copy, but loose for high cop

Plasmids
Plasmid replication requires host cell functions
Copy number is regulated by initiation of plasmid replication
Plasmids are incompatible when they cannot be stably maintained in the same cell because they interfere with each other’s replication.

CsCl gradient with ethidium bromide and UV light.
Three forms of plasmid DNA
“Old School method of purifying plasmid”

Virulence plasmids from Salmonella, Shigella, Yersinia, B. anthracis, E.coli, and others.

Plasmid replication
Plasmid replication requires host DNA replication machinery.
Most wild plasmids carry genes needed for transfer and copy number control.
All self replication plasmids have a oriV: origin of replication
Some plasmids carry and oriT: origin of transfer. These plasmids will also carry functions needed to be mobilized or mob genes.

Plasmid replication
Plasmid segregation is maintained by a par locus-a partition locus that ensures each daughter cells gets on plasmid. Not all plasmids have such sequences.
There are 5 main “incompatibility” groups of plasmid replication. Not all plasmids can live with each other.
Agents that disrupt DNA replication destabilize or cure plasmids from cells

Antisense RNA gene control.
-the RNA-RNA hybrid is very stable
-blocks most translation and tanscription
-requires RNAases to degrade
-common theme in bacterial gene regulation as we are learning

Antisense RNA: RNA-RNA hybrid blocks replication
GGCUAAUUCC Antisense RNA is also used in euks called
CCGAUUAAGG siRNA
Blocking RNA priming for DNA PolI prevents replication
RNA I-small inhibitory RNA that binds to RNAII. RNAII will act as a primer for DNA replication
Rop: plasmid encoded proteins which stabilizes the RNAI-RNAII complex
Anti-sense RNA replication control

ColE1 Replication Control-an example of primer control of replication
RNAII will serve as a primer for the replication fork.
The 3’ end is processed by host RnaseH to allow efficient RNA-DNA hybrid to form
The hybrid acts as a primer for host Pol1
As the concentration of plasmid increases, Rop does also
Rop stabilizes the RNA1-II complex
No RNA for replication priming.
ColE1 replication does not need plasmid encoded rep proteins

The events upon entry into a cell
RepA mRNA is made from Prep until copy number becomes high
CopB expression increase an Cop represses RepA expression at PrepA
CopA now is made-a 90base antisense RNA
CopA binds to 5-end of the RepA mRNA, forming dsRNA
This is recognized by host RNAaseIII and degraded.
Thus concentration of RepA protein is maintained by rate of RNA-RNA hybrid formation.

Rep-protein control -R1 family of plamsids.
Rep-protein expression controlled by antisense CopA
PcopB-encodes Rep and CopB
PcopA-encodes antisense RNA
plasmid replicates to high level
CopB levels rise, shutting off RepA production
antisense RNA from PcopA made
complexes with repA mRNA
Host RNaseIII will cleave the complex
Plasmid copy control balanced by host RNaseIII activity and transcription from the plasmid

Iteron Plasmids: Handcuffing RK2 and other broad host range plasmids
RepA is able to bind the repeat sequences upstream of the promoter region for repA.
binding causes two plasmid molecules to couple “handcuff”
prevents replication.
“copy up” mutants: mutations in RepA that are less able to bind to each other.

Incompatibility Groups
Not all plasmids can live together.
Plasmids that are able to coexist in the same cell do not interfere with each other’s replication
A single cell can have as many Inc group plasmids as it can tolerate and replicate!
Partion Locus: a region on broad host range plasmids that binds to a structure on the inner membrane of the cell to ensure proper segregation.
Plasmids labeled with fluorescent protein
-move to each daughter cell during division.
Pogliano, Joe et al. (2001) Proc. Natl. Acad. Sci. USA 98, 4486-4491

Figure 4.18
Par locus
think of this as a primitive centromere
the growing filaments push the plasmids to the opposite poles of the cells

Site-directed mutation: Suicide plasmds
Plasmid must be unable to replicate without essential replication proteins provide in trans.
It helps if the plasmid can be mobilized-oriT required
Need a selectable marker
Large or small region of homologous DNA cloned that will integrate into the chromosomal target.
Need a counter selection method to kill the donor cells
Screen for what you think is correct.
Also, merodiploid reporter strains can be constructed in this manner
Make a lacZ fusion to your promoter of interest
Clone into a suicide plasmid
Mate into recipient.
Resulting strain will harbor a duplication of the promoter region:lacZ and still have a functional copy of the gene.
Why would this be important?
Plasmids as genetic tools: Construction of Mutants

R6K: broad host plasmid.
-Pir is the essential replication protein
-pir mutants cannot replicate unless supplied in trans.
-integration into the chromosome is selected for by growth on ampicillin
How could you make targeted mutant using this method?

large (100 kb)
low copy (1-2 copies/cell)
self transmissible
requires protein synthesis (chloramphenicol-sensitive)
repE gene encodes RepE protein
RepE protein binds to origin of replication (oriS) and initiates DNA replication
RepE binds to the repE promoter and activates transcription
RepE binds to the copA/incC locus binding copies of F together via RepE – inhibiting replication (coupling)
F-plasmid

F Pilus assembly

replication
IS elements
Primitive transposon
30+ genes needed
For transfer
Genetic organization of F

F-transfer at fine detail