MMG Plasmids

Plasmids

General Properties of Plasmids

  • Plasmids are small, circular, double-stranded DNA molecules

  • They are present in bacteria and some archaea, and they replicate independently of chromosomal DNA

  • Key Characteristics:

    • Physical Properties:

    • Plasmids are typically double-stranded and supercoiled

    • They are extrachromosomal, meaning they exist outside the main chromosome in the cell

    • Replication:

    • Plasmids are autonomous and self-replicating

    • Curing:

    • Plasmids can be lost spontaneously from the cell or through the action of curing agents

    • Incompatibility:

    • In the same cell, two plasmids belonging to the same incompatibility group cannot co-exist

    • Transferability:

    • Some plasmids are capable of horizontal gene transfer through the process of conjugation

    • Recombination:

    • Certain plasmids, known as episomes, can integrate into the host chromosome

Types of Plasmids

  1. F-plasmids (“fertility”):

    • Contain tra genes responsible for transfer

    • Enable conjugation and facilitate the formation of sex pilus

  2. Col plasmids:

    • Carry genes that code for bacteriocins, which are substances that kill or inhibit the growth of closely related bacterial species

  3. Degradative plasmids:

    • Confer the ability to degrade uncommon substances, such as toluene and salicylic acid

  4. Virulence plasmids:

    • Encode factors that contribute to pathogenicity, enhancing the organism's ability to cause disease

  5. R-plasmids (resistance):

    • Encode mechanisms to resist antimicrobial agents

Plasmid Replication

  • Plasmid replication occurs prior to cell division and is synchronized with chromosomal replication

  • There are two primary mechanisms for plasmid replication:

    • Bidirectional Plasmid Replication:

    • Rolling Circle Replication:

Plasmid Partition Systems

  • Type I (pull):

    • Filaments drag one plasmid copy to each pole of the cell

    • Involves the ParABC system:

    • ParA: P loop ATPase

    • ParB: DNA binding protein

    • parC: Recognition site for ParB

  • Type II (push):

    • Filaments elongating to push each plasmid copy towards the poles

    • Involves the ParMRC system:

    • ParM: Filament protein

    • ParR: DNA binding protein

    • parC: Recognition site for ParR

Initiation of Plasmid Replication

  • Example: ColE1 plasmid

    • Initiation is mediated by the formation of an RNA primer

Control of Copy Number

  • Copy number regulation is achieved through antisense RNA mechanisms

Col Plasmids

  • Example: ColE1

    • Bacteriocin Functionality:

    • Targets closely related bacteria (usually within the same genus)

    • Example: E. coli produces colicins

    • Specific Col Plasmids include Col B, Col E, Col I, and Col V

    • ColE1 is the most characterized and is a multicopy plasmid that encodes colicin E1

  • Immune Response to Colicins:

    • Bacteria are immune to their own colicins

Virulence Plasmids

  • Virulence Factors:

    • Microbial products that enhance the ability to cause disease:

    • Enable adhesion, colonization, or invasion of host cells

    • May cause damage to host cells (toxins)

    • Allow for evasion of the host immune system

Antibiotic Resistance Plasmids (R Plasmids)

  • Encode determinants that confer antimicrobial resistance (AMR)

  • Present a significant threat to global health, particularly concerning the spread of antibiotic resistance phenotypes

  • Commonly used antibiotics for marker construction include:

    • Ampicillin

    • Chloramphenicol

    • Kanamycin/neomycin

    • Tetracycline

Mechanisms of Antimicrobial Resistance

  • Acquired Resistance:

    • Resistance emerges through the acquisition of new genes via horizontal gene transfer mechanisms including transformation, transduction, and conjugation

    • R plasmids encode products that facilitate:

    1. Target alteration

    2. Drug inactivation

    3. Reduction of drug concentrations within the bacterial cell

Resistance to Specific Antibiotics

  • Beta-Lactam Antibiotics:

    • Include agents like penicillins and cephalosporins, characterized by a four-sided beta-lactam ring

    • Mechanism of action: Prevent cross-linking of peptidoglycan (targeting the cell wall)

    • Bacterial beta-lactamases inactivate beta-lactam antibiotics (e.g., the amp gene is associated with the bla gene responsible for conferring this resistance)

  • Chloramphenicol:

    • Targets protein synthesis by blocking the formation of peptide bonds

    • Inactivated by bacterial chloramphenicol acetyltransferase (CAT), which adds two acetyl groups to the antibiotic

  • Aminoglycosides (e.g., kanamycin and neomycin):

    • Operates by targeting protein synthesis

    • Neomycin phosphotransferase (encoded by npt gene) inactivates both kanamycin and neomycin

  • Tetracyclines:

    • Target protein synthesis through binding to the 16S rRNA and the 30S subunit of ribosomes

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

Learning Objectives

  • Upon completion of the assigned readings and lecture, students should be able to:

    • Define important terms such as plasmid, replicon, copy number, bacteriocin, ColE1 plasmid, and colicin

    • Describe essential properties and the five types of bacterial plasmids

    • Clarify concepts such as incompatibility, mobilizability, and transferability as they pertain to plasmids

    • Detail the two primary methods for plasmid replication

    • Explain the major mechanisms of plasmid partitioning during cell division

    • Discuss regulatory mechanisms controlling plasmid copy number

    • Outline the function of ribonuclease H

    • Describe the activities of colicin E1 and colicin immunity

    • Analyze the modes of action and resistance mechanisms for ampicillin, chloramphenicol, kanamycin/neomycin, and tetracycline.