Lecture on Plasmids

Chapter 4: Plasmids

What are Plasmids?

• Definition: Plasmids are small, circular DNA molecules that exist within bacteria separately from their chromosomal DNA.

• Functionality:

  • Important for bacterial adaptation and evolution.

  • Serve as useful tools in molecular biology.

• Variability:

  • Can vary in size.

  • Most often circular double-stranded DNA, but can also be linear.

  • Copy number varies; bacteria may contain multiple types of plasmids.

• Genetic Encoding:

  • Encode RNA and proteins.

  • Replicate alongside the host cell growth, distributed to daughter cells.

• Characteristic:

  • Do not encode essential functions for bacterial growth; defining characteristic is their origin of replication, which differs from that of chromosomes.

Naming Plasmids

• Traditional Naming: Based on the phenotype they impart to bacteria carrying them.

  • Examples:

  • R-factor plasmids: Confer resistance to multiple antibiotics.

  • ColE1: Encodes a gene for colicin E1, a bacteriocin that kills non-carrying bacteria.

  • Tol: Carries genes for toluene degradation.

  • Ti: Responsible for tumor induction in plants.

• Modern Naming:

  • Plasmids are given alphabetic and numeric designations; similar to bacterial strains.

  • Examples:

  • pJAT101, pJAT102 (modified), pJAT103 (different modification).

Why Have Plasmids?

• Selective Advantage:

  • Enzymes for utilizing specific carbon sources.

  • Resistance to antimicrobial substances (e.g., antibiotics).

  • Synthesis of antibiotics, toxins, and proteins aiding infection in higher organisms.

• Ecological Niche:

  • Allows bacteria to occupy larger ecological niches and contributes to the success of the plasmid itself.

• Chromosomal Gene Distribution:

  • Keeping plasmid-borne genes separate can enhance competitiveness and reduce chromosome sizes, sharing the burden of species preservation.

Plasmid Structure

• Physical Characteristics:

  • Generally covalently closed circular without free ends, promoting supercoiling.

  • Negatively supercoiled, similar to chromosomes, relieving stress during replication and transcription.

• Agarose Gel Mobility:

  • Supercoiling helps plasmids migrate faster on agarose gel during electrophoresis.

Replication of Plasmids

• Autonomous Replication:

  • Plasmids are also referred to as replicons that replicate autonomously.

• Requirements for Replication:

  • Must have at least one origin of replication (ori site) and code for proteins necessary to initiate replication.

  • Most plasmids encode one or a few initiation proteins; remaining are borrowed from the host (e.g., ligases, primases, helicases).

• Mechanisms of Replication:

  • Theta Replication: The most common mechanism where the two DNA strands open at the ori region, creating a fork-like structure.

  • Rolling Circle Replication: Involves a nick made by Rep protein at a palindromic sequence, proceeding with DNA synthesis using displaced single-stranded DNA as a template.

Theta Replication

• Mechanism:

  • Formation of a replication fork at the origin of replication.

  • RNA primer initiates replication that advances in one or both directions around the DNA.

  • This is the prevalent mechanism for plasmid replication.

Rolling Circle Replication

• Overview:

  • Plasmids employing this method are known as RC plasmids.

  • Nicking Process:

  • Rep protein activates at a double-strand origin (DSO), creates a nick and uses a 3'-OH to serve as a primer for DNA polymerase III.

  • Displaced single-stranded DNA assembled by different host mechanisms.

  • Control of Plasmid Number:

  • The production level of Rep protein influences the overall plasmid quantity within the cell.

Functions of the ori Region

• Gene Location:

  • Genes necessary for plasmid replication reside close to the ori region.

• Determinants:

  • Host range: Different types of bacteria that the plasmid can replicate within.

  • Copy number: Average count of that plasmid per cell.

  • Incompatibility: Ability of multiple plasmids to coexist within the same bacterial cell.

Host Range of Plasmids

• Narrow Host Range:

  • Can replicate only in E. coli and closely related species (e.g., ColE1 plasmid type).

  • Examples: pBR322, pET, pUC.

• Wide Host Range:

  • Can replicate across various bacteria, both gram-positive and gram-negative.

  • Examples: RK2, RSF1010.

  • Broad host range plasmids must encode all proteins essential for the initiation of replication.

Determining Host Range

• Methodology:

  • Largely by trial and error; requires plasmid introduction to other bacteria around a transformation system or electroporation.

  • Resistance genes may be used for selection.

• Self-Transmissible Plasmids:

  • Some plasmids can be introduced into other cells via conjugation.

Incompatibility of Plasmids

• Definition:

  • Not all plasmids can coexist within a given cell.

  • Plasmids are classified as part of the same incompatibility (Inc) group if they cannot coexist stably.

• Example:

  • RP4 (IncP group) and RSF1010 (IncQ group) can coexist; RP4 cannot exist with another IncP plasmid.

Determining Inc Groups

• Process:

  • Involves assessing if a plasmid can coexist with a known compatibility group by measuring curing rates upon introduction of a new plasmid.

Regulation of Copy Number

• Purpose:

  • To prevent overproduction or underproduction of plasmid copies.

• Types of Plasmids:

  • Relaxed Plasmids: Have high copy numbers (e.g., ColE1); regulated by mechanisms inhibiting replication when concentration exceeds a specific threshold.

  • Stringent Plasmids: Low copy numbers (e.g., F plasmid); replicate once or very few times per cell cycle, maintaining precise control over replication processes.

Copy Number Regulation via RNA Processing (ColE1 Plasmids)

• Role of RNA I and RNA II:

  • RNA I interferes with RNA II processing, impacting replication.

  • RNA II is required for DNA replication and forms an RNA-DNA hybrid at the origin of replication but requires cleavage by RNase H to continue.

RNA I Inhibition of RNA II Processing

• Mechanism:

  • RNA I inhibits RNA II processing through complex formations, stabilizing through interactions with exposed regions and forming double-stranded RNA (dsRNA) that interrupts necessary folding required for DNA binding.

Regulating Copy Number via Translation of Rep Protein (R1 Plasmid)

• Significance of RepA:

  • RepA is vital for initiation of replication; controlled by its own antisense RNA that pairs with it, thereby modulating its protein synthesis.

RepA Expression Control

• Promoter Dynamics:

  • RepA is transcribed from dual promoters; upon plasmid entry and initial replication, different regulatory mechanisms come into play post-uptake.

COPA Antisense RNA Role

• Complementary Action:

  • More plasmid presence translates to increased CopA RNA, leading to repression of RepA expression assistance through pairing and cleavage by ribonuclease.

Regulating Copy Number via Coupling (Iteron Plasmids - pSC101)

• Iteron Sequences:

  • Contain repeated DNA sequences that dictate regulation mechanisms and coupling behaviors enhancing control of plasmid replication through RepA interactions.

Coupling Mechanism of RepA and Iterons

• Functionality:

  • High RepA levels facilitate dimer formation, binding to two iterons across different plasmids, effectively inhibiting their replication by coupling them together.

Mechanisms to Prevent Curing

• Plasmid Addiction:

  • Some plasmids have encoded systems that result in bacterial cell death upon losing the plasmid, exemplified in the Phd-Doc toxin-antidote system in P1 plasmids.

• Partitioning:

  • Ensures that each daughter cell receives at least one plasmid copy during cell division, reducing the incidence of plasmid loss.