Plasmids - Molecular Microbial Genetics

Plasmids replicate

Independently from the chromosome

Can a bacterial cell grow normally without a plasmid?

Yes, if plasmid is lost

Functions of plasmids

• antibiotic resistance

• Virulence factors (Toxins)

• Genes to degrade organic compounds

Plasmids DO NOT encode for

• RNA polymerase

• Ribosomes

• Metabolism enzymes (glycolysis)

Cryptic Plasmids

Only encode for replication and maintenance in the host cell

Why do some bacteria have plasmids?

For gene expression

Info needed for plasmids

• ORIV

• Rep protein gene

What is the Rep protein?

Plasmid specific initiator protein

Replication of plasmids

• theta replication

• rolling circle replication

Rolling circle replication

A process where a circular DNA molecule is unwound and replicated continuously, allowing the synthesis of multiple copies of the plasmid

Primer problem

happens with linear DNA and the problem is that there is no way to copy the very ends

(ORIV site) Host Range

• what bacterial cell the plasmid will replicate (ColE1 - only replicates in E. Coli

• RK2 in E.coli has broad host range and mainly gram (-) bacteria

(ORIV site) Copy #

• ColE1 = 30-50 (relaxed plasmid)

• RK2 = 4-6 (stringent plasmid)

(OIRV site) Incompatibility

E.coli can have more than 1 type of plasmid coexist in the same host cell

“Curing” of the plasmid

• spontaneous loss of the plasmid from the host cell

• ensures that each daughter cell gets a copy of the plasmid during cell division

Prevent Curing of the plasmid 

• site-specific recombinases (separate dimers and multimers of a plasmid) (some plasmids can encode their own - P1)

• plasmid addition system (plasmid makes a toxin that kills a daughter cell that did not get a copy of the plasmid)

• partitioning (par) systems

Plasmid partitioning system

uses “Par” system in R1 to move 2 copies of the plasmid to opposite sides of the bact. cell

BACs

can splice long sequences of DNA into plasmids

Expression vector

heterologous expression of gene x

Vertical inheritance

• Gene exchange

• lateral or horizontal inheritance of DNA

Leterberg + Tatum (1947)

• horizontal

• mixed 2 parent strains of E.coli, found 2 daughter cells that were different from either parent

Conjugation Gene exchange

“one way” horizontal transfer between donor cell and replicated cell.

• “self-transmissible” 

Self-transmissible

encode all things needed to more the plasmid from donor to recipient. And is a potential donor cell

Early E.Coli Plasmid

• the F plasmid 

Stage 1 of rolling replication

• rep protein recognizes and binds to the DSO at ORIV

• rep protein splits the DNA and the DSO sequence

• rep protein recognizes the cruciform structure (top strand of plasmid) 

• rep protein “nicks” the top strand

• DNA polymerase 3 from the host cell uses the 3’ -OH as a primer

• DNA poly. copies the parent strand 

• rep protein breaks the dimer and rejoins the broken ends

Stage 2 of rolling replication

• RNA polymerase finds SSO seq. and makes a complenatery RNA primer

• DNA polymerase 3 uses primer to copy parental strand

cloning vectors in plasmid partioning

Puc 9, I DNA sequence single cut restriction site

Shuttle vectors

• allows replication in yeast cells

• 2 nm ORI

• XEP13