Plasmids and mobile genetic elements

What is the pan-genome?

All DNA associated with a bacterial species

Direction of gene transfer

Always one direction: donor to recipient

Why does the copy number of plasmids need to be regulated by the cell?

Many copies, especially of a large plasmid, comes with a large cost

Steps of conjugation

Cells contact via pilus, draw closer when pilus contracts

Pore forms between cells

One strand of plasmid nicked: get transfer of single-stranded DNA through rolling-circle replication

In recipient, second strand synthesis and ligation leads to dsDNA copy

Both now have the plasmid, both can act as donors

Importance of plasmids

Dynamic

Contribute to genome plasticity

Attract and distribute mobile genetic elements

Play role in bacterial evolution

Epidemiological studies of them help understand how resistance genes evolve

How are plasmids grouped?

Traditionally in Incompatibility group: ones with the same replicon that can’t stably reside in the same cell as a result

Host range is determined9 by Inc-group

Example of plasmid replication control: R1 plasmid

Normally 1-2 copies per cell

RepA initiates replication by binding oriR1; recruits DnaA that starts formation of replisome

repA expressed from P1 and P2; repressed by copB and copA

CopB represses P2

CopA RNA represses tap and repA translation

CopB repression relaxed is copy number is low: get rapid RepA production (P2 promoter stronger than P1 promoter)

Importance of CopA

Antisense RNA: why it binds and therefore blocks translation

Unstable: needs to constantly be produced

Is a measure of plasmid concentration as the amount increases depending on the number of plasmid copies

What is a multimer resolution?

When, at cell division, the second plasmid copy fails to move to the new cell, resulting in a large plasmid that can only be in 1 cell

What is the need for plasmid segregation systems? What is an example of one?

Needed for stable maintenance: makes sure that each daughter cell gets a copy

Toxin-antitoxin system an example: all cells without a plasmid are killed

How can segregation happen by elongation?

At replication, get ParM monomers forming a filament binding the plasmid copies

ParR on plasmid binds to ParM to stop the filament from falling apart

ParM filament then pushes plasmids to either end of cell, ensuring that each daughter cell will get a copy

Depolymerises

Features of IncF plasmids often found in E. coli

Narrow host range of Enterobacterales

Large plasmids with low copy number

High rate of variation; often have more than one replicon type

Definition of transposable genetic elements

DNA-segments that can move from one position to another within a genome

Random transfer

Cannot self-replicate outside of chromosome

Transfer via site-specific recombination

What enzyme is needed from site-specific recombination of transposable element?

Transposase

Overview of insertion sequences

Elements that contain only genes encoded needed for transfer of IS, flanked by inverted repeats

Smallest kind of transposon

Generate genetic variability/mutations via insertions: disrupts genes where it transposes

Overview of composite transposons

Element that contains other genes in addition to those needed for transposition

Usually to IS-elements with genes in between

Often get AMR related genes in transposons

Overview of integrons

Assembly platforms that incorporate exogenous ORFs by site-specific recombination, convert them to functional genes

Often have resistance genes

Gene cassettes in them

What three functions make up integrons?

Integrase

Primary recombination site

Outward-oriented promoter

Advantages of using nanochannels to study plasmid dynamics

Fast and easy

Small sample volume

No enzymes involved