Chapter 23: Plasmids

Define:

• Plasmid: Self-replicating genetic elements sometimes found in both prokaryotic and eukaryotic cells. They are neither chromosomes nor part of the host cell’s permanent genome. Most plasmids are circular molecules of double-stranded DNA although rare linear plasmids and RNA plasmids are known

• Replicon: Molecule of DNA or RNA that contains an origin of replication and can self-replicate

• Copy number: The number of copies of a plasmid found within a single host cel

• Bacteriocin: Toxic protein made by bacteria to kill closely related bacteria

• ColE1 plasmid: Small multicopy plasmid that carries genes for colicins of the E group. It is used as the basis of many widely used cloning vectors.

• Colicin: Toxic protein or bacteriocin made by E. coli to kill closely related bacteria

Describe the essential properties of plasmid and the 5 types of bacterial plasmids

• They have dsDNA, are supercoiled, extrachromosomal, can self-replicate, can be lost spontaneously or by curing agents, In the same cell two plasmids belonging to the same group cannot co-exist, self-transferrable, and some (episomes) can integrate with host chromosome (recombination)

• 1. F-plasmids (fertility)

• 2. Col plasmids

• 3. Degrative plasmids

• 4. Virulence plasmids

• 5. R-plasmids (resistance)

Define the following relative to plasmids: 

• Incompatibility: The inability of two plasmids of the same family to co-exist in the same host cell.

• Mobilization: Ability of a nontransferable plasmid to be moved from one host cell to another by a transferable plasmid

• Transferability: Ability of a plasmid to move itself from one host cell to another

Describe the two methods for plasmid replication

• Bidirectional Plasmid Replication -  a process where DNA is copied in two directions from a specific origin site on a plasmid

• Rolling Circle Replication - a process that replicates circular DNA or RNA molecules in a single direction

Describe the two major ways for the partition of plasmids upon cell division

• Type I (pull): Filaments drag one plasmid copy to each pole

  • Type I ParABC

    • ParA – P loop ATPase, ParB – DNA binding protein, parC – recognition site for ParB

• Type II (push): Filaments elongate pushing each plasmid copy to a pole

  • Type II ParMRC

    • ParM – filament protein, ParR – DNA binding protein, parC – recognition site for ParR

Describe regulatory mechanisms for controlling plasmid copy number:

• negative feedback mechanism mediated by transcription of an antisense RNA from an alternative promoter

Describe the function of ribonuclease H:

• cleaves the RNA strand within a RNA-DNA hybrid molecule to generate a primer with a free 30-OH group

Describe the activity of colicin E1 and colicin immunity:

• Colicin E1 is a bacteriocin produced by Escherichia coli that kills other nearby E. coli cells by forming a pore in their outer membrane, disrupting the cell's membrane potential and leading to cell death

• Colicin Immunity is the mechanism where a bacteria producing colicin E1 also produces a specific immunity protein that prevents the colicin from harming itself by blocking its activity at the cell membrane level, essentially protecting the producing cell from its own toxin

Describe the mode of action for:

• Ampicillin: Target cell wall

  • Prevent cross-linking of peptidoglycan

• Chloramphenicol: Targets protein synthesis

  • Blocks formation of the peptide bond (C)

• Kanamycin / Neomycin (aminoglycosides): Target protein synthesis

  • Bind multiple sites on both the 30S and 50S ribosomes to prevent tRNA from forming initiation complexes (A)

• Tetracyclines: Target protein synthesis

  • Bind the 16S rRNA and the 30S subunit

Describe the resistance mechanisms to the antibiotics listed above

• Ampicillin: Bacterial β-lactamases inactivate β-lactam drugs

• Chloramphenicol: Bacterial chloramphenicol acetyl transferase (CAT) inactivates the drug by adding 2 acetyl groups

• Kanamycin / Neomycin: Neomycin phosphotransferase (encoded by npt

• gene) inactivates both kanamycin and neomycin

• Tetracyclines: Tetracycline-specific efflux pumps (encoded by tetH) export the drug from the bacterial cell