Plasmid Biology and Genetic Transfer Methods

Plasmid Biology

• Overview of Plasmids

  • Definition: A plasmid is a genetic structure in a cell that can replicate independently of the chromosomal DNA.

  • Characteristics:

  • Typically exists as a small circular DNA strand, mainly found in the cytoplasm of bacteria or protozoans.

  • Plasmids never encode essential functions necessary for the organism's survival.

  • Concept of plasmids as 'selfish evil genetic parasites'.

Plasmid Strategies for Inheritance

• Mechanisms Ensuring Daughter Cell Plasmid Inheritance

  1. Active Partitioning:

     • Example: Plasmid pR1 of Salmonella.

     • Molecular Model:

     • Nucleation of new ParM molecules forms filaments on the plasmid.

     • Process involves conversion between ParM-ATP and ParM-ADP.

     • Stable filaments polymerize, effectively driving plasmid distribution toward opposite cell poles during cell division.

     • When plasmids hit cell poles, this triggers filament detachment and depolymerization, allowing plasmid segregation.

  2. Post Segregational Killing (Addiction Modules):

     • Address low copy number plasmid risks (stochastic loss).

     • Function of Addiction Modules:

       • Composed of two gene cassettes; one encodes a toxin (e.g., a nuclease or pore-forming toxin) and the other encodes an antidote.

       • The antidote's half-life is shorter than that of the toxin; daughter cells that lack the plasmid are killed due to the breakdown of the antidote.

Examples of Toxin-Antitoxin Systems

• CcdB:

  • Poisons DNA gyrase, creating negative supercoils in DNA.

  • Has a long half-life making it a stable protein.

• CcdA:

  • Functions as an antidote for CcdB; has a short half-life and is degraded by Lon protease.

Types of Plasmids

• Major Types of Plasmids: Table 13.1 summarizes different types of plasmids, their size, copy number, and associated hosts.

  • Type Examples:

  • Fertility Factors:

    • F factor, size: 95-100 kbp.

  • R Plasmids:

    • Examples: RP4 (1-3 kbp), R1 (80 kbp), R6 (98 kbp).

    • Resistance features include:

    • Resistance to antibiotics such as ampicillin (Ap), kanamycin (Km), Streptomycin (Sm), etc.

  • Col Plasmids and Virulence Plasmids:

    • Virulence traits linked with pathogenicity (e.g., Agrobacterium tumefaciens).

Transferable Drug Resistance

• Historical Context:

  • Case study from 1968: Guatemala epidemic due to Shigella dysenteriae.

  • Infection resistant to multiple antibiotics (chloramphenicol, tetracycline, streptomycin, and sulfonamides); resulted in significant morbidity and mortality (100,000 infected, 12,000 died).

  • Origins:

  • Began with a Japanese woman in 1955 noted for similar resistance patterns.

  • E. coli could mix with drug-sensitive Shigella, transferring resistance through R plasmids (specifically R100) via conjugation.

Mechanisms of Bacterial Gene Transfer

• Conjugation and F Factor:

  • Conjugative Plasmids:

  • Existence and functionality of plasmids like the F factor which can direct their own transfer by forming sex pili.

  • Replication Mechanism:

  • Theta Replication and Rolling Circle Replication (RCR) with distinct origin sequences (oriV for vegetative replication and oriT for transfer).

  • Process of Transfer:

  1. Attachment of sex pilus from F+ donor to recipient F- cell.

  2. Contraction of pilus forms a relaxosome bridge.

  3. Nicking at oriT allows DNA transfer and replication in both donor and recipient cells, effectively converting F- to F+.

Homologous Recombination and Hfr Strains

• Hfr Conjugation:

  • Formation of Hfr strains occurs when a conjugative plasmid integrates into the bacterial chromosome through homologous recombination (HR).

  • Processes:

  • Involves the transfer of chromosomal genes along with F plasmid material resulting in recombined cells.

Nitrogen Fixation and Symbiosis

• Plant Pathogenesis and Nitrogen Fixation:

  • Discussion of the symbiotic relationship between Rhizobium species and leguminous plants to facilitate nitrogen fixation.

  • Significance of the Ti plasmid from Agrobacterium and T-DNA in enabling plant transformation and nodulation.

Environmental and Medical Relevance

• Methanogens and Climate Change:

  • Role of methanogenic archaea in methane production and their symbiotic relations within animal digestive systems influencing methane emissions.

• Bacterial Biofilms:

  • Understanding biofilm formation in the context of medical devices, chronic infections, and the implications of biofilms in resistance to antibiotics.

  • Process of colonization, exopolysaccharide matrix formation, and resistance development through gene transfer in biofilm communities.

Conclusion

• Overall implications of plasmids in genetics, medical microbiology, and environmental science.

• Perspectives on future research areas involving plasmid dynamics and their roles in gene transfer, resistance, and environmental interactions.