Plasmids

• ❑ Natural plasmids:L

  ✓ Occur naturally in bacteria

  ✓ eg ColE1 in E. coli
• ❑ Artificial plasmids

  ✓ Engineered, usually from parts of natural plasmids

  ✓ eg pBR322, pUC19
• oriV - Origin of Replication


  A specific DNA sequence on the ColE1 plasmid where DNA replication begins. It is recognized by cellular proteins that initiate the replication process, allowing the plasmid to replicate and produce multiple copies within a bacterial cell.
• oriT - Origin of Conjugal Transfer


  A region of the ColE1 plasmid that is recognized by proteins involved in conjugation, a process by which the plasmid can be transferred from one bacterial cell to another. The oriT facilitates the transfer of the plasmid DNA during bacterial mating or conjugation.
• rom - Copy Number Control


  The rom (regulation of maintenance) region of the ColE1 plasmid is involved in controlling the copy number of the plasmid within a bacterial cell. It contains regulatory elements that influence the frequency of plasmid replication, thereby affecting the number of plasmid copies present in the cell.
• mob - Nuclease for Mobilization


  The mob (mobilization) gene encodes a nuclease enzyme that is involved in the mobilization of the ColE1 plasmid. This enzyme helps to process the plasmid DNA for transfer during conjugation, allowing the plasmid to be efficiently transferred to recipient cells.
• colE1 - Colicin E1 Toxin

  Colicin E1 is a protein toxin produced by certain strains of Escherichia coli bacteria. The colE1 gene on the plasmid encodes this toxin. Colicins are bacteriocins, which are antimicrobial peptides that can kill or inhibit the growth of closely related bacterial strains.
• imm - Immunity to Colicin E1

  The imm gene on the ColE1 plasmid encodes a protein that provides immunity to the toxic effects of colicin E1. Bacterial cells harboring the ColE1 plasmid are protected from the cytotoxic effects of colicin E1 due to the presence of this immunity protein.
• Antibiotic resistance can be spready easy and quickly between bacterial cells due to the plasmid exchanging and conjugation.
• General Characteristics of Plasmids:

  ❑ Usually circular dsDNA, occasionally linear or made of RNA

  ❑ Multiply only inside a host cell

  ❑ Single or multiple copies

  ❑ Size varies enormously

  ❑ Carry a few to several hundred genes

  ❑ Some carry genes that affect the properties of the host cell ❑ Host range varies widely

  ❑ Transferability vs mobilizability
• ❑ Small plasmids are usually high copy number:

  ✓ Replication is faster than cell division

  ✓ Random segregation into daughter cells

  ❑ Large plasmids are usually low copy number

  ✓ Replication co-ordinated with chromosome

  ✓ Replication and partitioning is controlled or plasmid is lost
• Plasmid compatibility:

  ❑ Several different plasmids may be found in the same cell at any time

  ❑ Different plasmids must be compatible

  ❑ Plasmids in the same family (i.e. incompatible group) CANNOT co-exist in the same cell

  ❑ Incompatible plasmids cannot coexist and one will be lost ❑ Competition for the same replication or partitioning machinery

  ❑ >30 incompatibility groups in E. coli
• In Escherichia coli (E. coli), a common model organism used in molecular biology research, there are more than 30 known incompatibility groups. Each incompatibility group represents a distinct set of plasmids that cannot stably coexist within the same bacterial cell.
• ParMRC System (E. coli R1 plasmid)


  A plasmid system containing three main components: ParM, ParR, and ParC
• ParM


  • An ATPase protein that polymerizes into filamentous structures, similar to actin filaments found in eukaryotic cells
• ParR


  • A DNA-binding protein that recognizes specific sequences (parC) on the plasmid DNA
• ParC


  • Centromere-like sites on the plasmid where ParR binds
• ParMRC system during cell division


  1. ParM filaments polymerize and push apart, exerting force on the plasmid DNA bound by ParR at the ParC sites

  2. This dynamic process helps to actively segregate the plasmid copies to opposite poles of the cell, ensuring their distribution to daughter cells during cell division
• TubZRC System (Bacillus)


  A plasmid system containing three main components: TubZ, TubR, and TubC
• TubZ


  • A GTPase protein that forms filaments resembling microtubules found in eukaryotic cells
• TubR


  • A DNA-binding protein that recognizes specific sequences (tubC) on the plasmid DNA
• TubC


  • Centromere-like sites on the plasmid where TubR binds
• TubZRC system during cell division


  1. TubZ filaments polymerize and depolymerize, actively segregating the plasmid copies to opposite poles of the cell in a manner similar to the ParMRC system

  2. This process ensures the faithful partitioning of plasmids to daughter cells during bacterial cell division
• sPlasmid addiction systems:

  ❑ Kills cells that lose the plasmid (addiction)

  ❑ Variety of mechanisms

  ➢ Toxin/antitoxin systems ✓ eg hok/sok system of E. coli R1 plasmid
• Types of plasmids:

  ❑ By ability to be transferred to other bacteria

  ➢ Conjugative plasmids e.g., F plasmid

  ✓ Sexual transfer via a pilus

  ➢ Mobilisable plasmids

  ✓ Cannot initiate conjugation

  ✓ Can be transferred by conjugation in the presence of a conjugative plasmid

  ➢ Non-mobilisable plasmids

  ✓ Only transferred by natural transformation or transduction
• Types of plasmids:

  ❑ By function

  ➢ Fertility F-plasmids

  ✓ tra genes confer ability to express sex pili and conjugate

  ➢ Resistance (R) plasmids

  ✓ Provide resistance to toxins or antibiotics

  ➢ Col plasmids

  ✓ Encode bacteriocins for killing other bacteria

  ➢ Degradative plasmids

  ✓ Allow metabolism of unusual substances

  ➢ Virulence plasmids

  ✓ Convert bacteria into pathogen
• Which of the following statements correct about plasmids incompatibility?

  • Plasmids of the same incompatibility group have very similar DNA sequences in their replication and partition genes, although the other genes they carry may be very different.
  • A P-type plasmid will happily share the same cell with a plasmid of the F-family
  • It is quite possible to have two or more plasmids in the same cell as long as they belong to different families.
• Fertility plasmids:• E. coli F-plasmid or F-episome • Conjugative • Can integrate into the chromosome (and transfer)
• Based on your asynchronous learning, which of the following statements IS/ARE TRUE about R-plasmids?

  • R-plasmids confer resistance to toxins and antibiotics on the host cell .
  • R-plasmids use the hok/sok addiction system to prevent the loss of the plasmid from the host cell.
  • The more copies of the R-plasmid per cell, the higher the resulting level of antibiotic resistance.
• Resistance plasmids:

  • Carry one or more antibiotic resistance genes

  • Usually in conjunction with virulence determining genes

  • Often conjugative/mobilisable

  • Multiple resistance genes arranged in cassettes

  • Multiple drug resistance (MDR)

  • Positive selection in presence of antibiotics

  • Used for artificial plasmids
• Col plasmids:

  • Bacteriocins reduce competition in the environment

  • Colicins are toxic to E. coli

  • Bind to outer membrane receptors and gain access to the inner membrane or cytoplasm

  • Pore-forming colicins disrupt the proton-motive force

  • Nucleases digest genetic material
• Colicin-producing bacteria also produce immunity proteins that protect them from the toxic effects of their own colicins. These immunity proteins bind to the colicins and prevent them from exerting their toxic effects on the producing cells.
• The Tol and Ton systems are two related systems found in Gram-negative bacteria, such as Escherichia coli, that are involved in maintaining the integrity of the outer membrane and facilitating the transport of molecules across it.
• Degradative plasmids;• Many microorganisms can digest a variety of unusual compounds• Genes responsible usually encoded on a plasmid• TOL plasmid of Pseudomonas putida pWW0• Xylene• Naphthalene• Camphor

  Also contribute to bioremediation.
• • Degrades toluene and xylenes to acetaldehyde and pyruvate → TCA cycle
• • Tumour inducing (Ti) plasmid of Agrobacterium tumefaciens:

  • causes crown gall disease in a variety of plants;

  • Fruit

  • Vegetables

  • Ornamental plants

  • Roses

  • Shrubs

  • No treatment
• Plant infection by virulence plasmid:

  • wounded plant produces acetosyringone
  • acetosyringone activates virulence genes
  • Vir (virulence) proteins synthesise T-DNA
  • t-DNA integrates with host cell DNA
  • causing auxin and cytokinin synthesis= tumour
  • Opines formed serve as a source of nutrients for Agrobacterium tumefaciens.
• Origin of Replication (ori)


  Specific DNA sequence in plasmid vectors that is recognized by the host organism's replication machinery, allowing the plasmid to replicate autonomously within the host cell, independent of the host chromosome
• Selectable Marker


  Gene in plasmid vectors that typically confers antibiotic resistance or another selectable trait, allowing researchers to easily select for cells that have taken up the plasmid
• Multiple Cloning Site (MCS)


  Region in plasmid vectors that contains multiple unique restriction enzyme recognition sites, allowing for the insertion of DNA fragments in a controlled manner