Plasmids

PLASMIDS

Naturally, plasmids are found in coils within a bacterial cell, the enzyme gyrase (a topoisomerase) will relax these when any modification is to take place. Plasmids are extrachromosomal genetic elements, capable of autonomous replication (replicon meaning from one point of origin) and not essential for the cell under all circumstances. Plasmids can contribute to bacterial evolution and genetic plasticity, as they can encode phenotypes.Examples of plasmid encoded phenotypes:  Conjugation - F plasmid Antibiotic resistance - R plasmid  Antibiotic and bacteriocin synthesis  Virulence - encoding toxins or enzymes capable of disrupting physiology or metabolism 

A plasmid must be able to...

Replicate  segregate - ensure each daughter cell receives at least one copy Constrain metabolic load by limiting copy number - large plasmids (1-5 copy per cell) vs small plasmid (10-50 copies per cell) Ensure host retains the plasmid - killing mechanisms in place to ensure cell does not eject plasmid  Spread to other cells - conjugation or by other mobilizable means 

Genetic map of F plasmid

F is a large, low copy plasmid  tra - mating pore and DNA mobilization functions  RepF1A - determines vegetative replication and incompatibility properties, includes: - oriV: origion of replication; regulates copy number (1-2 per cell)- par: partitioning loci- res/fcr: site specific recombination system resolves dimers- ccdA/ccdB: host killing system hok/sok - host killing system (toxin/antitoxin)  pif - protection against T7 phage (induces cell suicide)  Tn1000, IS2 and IS3 - transposable elements facilitate recombination between F and other DNA molecules 

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Rubrica: : Generalised map of F plasmid

Genetic map of ColE1 plasmid (colicin)

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ColE1 is a small, high copy plasmid oriV - origin of replication (15 copies) imm and ColE1- immunity and synthesis of Colicin  mob - nuclease required for mobilization (acts at oriT) rom - protein required for copy number control  oriT - origin of conjugative transfer  cer - sequence for site specific recombination to resolve multimers  

Partitioning of plasmids 

High copy number plasmids (ColE1) are randomly partitioned into daughter cells. Low copy plasmids are partitioned under specialised molecular machinery (3 types): ParA-like ParM-like TubZ Par M encodes actin like filaments that polymerise to form filaments. In combination with Par M, Par R encodes DNA binding adaptor protein and Par C encodes a centromere like region. Partitioning complex formed by replicated plasmids paired by Par R, bound to Par C. Par M filamentation occurs once the complex is formed, and polymerisation pushes the plasmids to opposite poles, at which point the filament depolarises. 

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Rubrica: : Partitioning schematic

Control of plasmid replication

The general principal is that plasmid replication is controlled by an inhibitor acting at oriV. As the cell size increases, inhibitor concentrations decrease and plasmid replication is initiated. Once the plasmid is replicated, more inhibitor is synthesised and replication is again stopped.In ColE1, RNA II (complementary piece of RNA to oriV) binds oriV to initiate replication. In the presence of rom gene product, RNA I binds RNA II and prevents binding to oriV. Note that both rom and RNA I concentrations are critical to regulate replication. In low copy plasmids (F plasmid), there is another system of replication control. RepA is a protein that binds to the oriV to initiate transcription, however when high concentrations of plasmids are present, RepA binds to iterons (repetitive plasmid sequences) and 'hand cuffs' plasmids together, preventing replication.

Multimer resolution 

A problem arises when a plasmid is replicated: two identical replicates of DNA can of course undergo recombination! However in addition to this, incomplete termination of replication can also produce a multimer. The issue with a multimer is that there is a substantial chance of loosing the plasmid during cell division.The F plasmid is self sufficient in its  resolution mechanism as it encodes 2 distinct systems:  ResD is a site specific recombinase that acts at fcr Tn1000 encodes its own site specific system  ColE1 however, operates under a different mechanism, taking advantage of a host encoded recombinase that acts specifically at 35bp region - cer. The host gene is names xerC.

Control of plasmid free cells 

The reason bacteria may end up rid of their plasmid is due to the metabolic stress the plasmid imposes on the cell, this may result in a slowed growth of the cell, thus plasmid free segregants outgrow plasmid bearing segregants. The cell overcomes this problem through use of 'post segregational killing systems': ColE1 containing cells secrete colicin, cells that do not contain the ColE1 plasmid do not have the imm gene and thus do not produce the immunity protein and die.  F encodes 2 host killing 'toxin/antitoxin' systems: hok/sok and ccdA/ccdB (ccdA is a stable protein that inhibits DNA gyrase, whereas ccdB is unstable inhibitor of ccdA) hok/sok killing mechanism operates much the same as ccdA/B but operates on the RNA level. Hok is a host killing peptide,  translated from a stable (20min half life) mRNA. Sok is an unstable (5 min half life) antisense that binds to Hok mRNA and prevents it being translated.