Plasmids, Transposons and Gene Transfer

• Study notes for Plasmids and Transposons, Genetic Exchange and Recombination, and Mechanisms of Gene Transfer are provided.

Plasmids

• Closed circular or linear DNA molecules capable of independent replication within a bacterial cell.
• They range in size from 2 kb to 100 kb.
• Plasmids encode functions for their own life cycles and influence host cell physiology.
• Can increase host cell pathogenicity in plants and animals.
• Can give host cells resistance to antibiotics, heavy metals (R plasmids), mutagenic agents (ethidium bromide) and disinfectants (formaldehyde).
• Linear Streptomyces plasmids have a racket frame structure with terminal proteins attached to the 5’ ends, protecting the ends and completing replication.
• Juxtaposition proteins bring plasmid termini together by binding to palindromic symmetry regions.
• The ori near the plasmid center initiates bidirectional DNA replication.
• Linear plasmids from Borrelia contain terminal hairpin telomeric structures of inverted repeats with nick sites.
• Copy number can range from 1–2 per chromosome to over a hundred (e.g. pUC series).
• Maintenance is the ability of a plasmid to survive in the host bacterium; can be narrow (e.g. ColE1, pBR322, pUC18) or wide (e.g. P-type plasmids).
• Plasmids transfer from host to host via the conjugative plasmid with the oriT and tra genes.
• Mobilizability means plasmids cannot transfer themselves (e.g. ColEI) but can be mobilized by transmissible plasmids (e.g. F or ColV)

Plasmid Incompatibility (Inc) Groups

• Incompatibility is the failure of two plasmids to be stably inherited and maintained in the same cell line without selective pressure.
• Incompatibility is connected to regulation of copy number and segregation.
• Plasmids of one incompatibility group share replication control functions (trans-acting).
• Each plasmid can control the replication of the other, but one plasmid is lost during random partitioning.

Plasmid Replication

• Rolling circle replication begins with the nicking of a plasmid strand.
• DNA polymerase III uses the nick as a primer to replicate the plasmid.
• The nicked strand is displaced then rejoins to form a new plasmid strand.
• DNA ligase seals the ends the displaced strand.
• Another round of DNA polymerase III creates a double stranded version of the displaced strand.
• Theta replication shows double-stranded DNA unwinding at replication origin.
• Single-stranded templates are produced for new DNA synthesis, a replication bubble forms with a replication fork at each end.
• The forks proceed around the circle, eventually yielding two circular DNA molecules.

Control of Plasmid Replication and Copy Number

• Plasmid copy number is determined by regulating the initiation of plasmid replication.
• This is achieved by regulating the amount of available primer for initiation, the amount of essential replication proteins, and the function of these proteins.

F Factor/Plasmid (Fertility Factor/Plasmid)

• Contains OriT (origin of transfer), OriV (origin of replication), tra-region (transfer genes) and IS (insertion elements).

States of the F Plasmid:

• F+ is a plasmid independent of the bacterial genome and contains only F factor DNA.
• Hfr (High frequency of recombination) is when the F factor is integrated into the bacterial genome.
• F' is when the F factor exists as an extrachromosomal element but contains chromosomal DNA sections.
• F- is a strain with no F factor.

Mobile Genetic Elements

• Provide insights into molecular biology and evolution.
• Mobile genetic elements serve as useful tools.
• Three groups of MGEs exist:
  • Insertion sequences (encode gene products required for transposase synthesis and regulation).
  • Composite transposons (flanked by IS elements on the left and right ends).
  • Non-composite transposons (lack flanking IS elements).

Insertion Sequences (IS)

• Designated by IS followed by an internationally recognized number, that roughly represents order of discovery.
• Transposons are designated by Tn, followed by a number, plus a capital letter and lower case to show phenotypic indications.
• Presence of insertion elements is shown using ::.

IS Elements Physical Properties

• Have sizes typically between 0.7 and 1.8 kb.
• Termini tend to be 10 to 40 base pairs with perfect/nearly perfect repeats.
• Encode one large open reading frame of 300 to 400 aa involved in the transposition event.
• Are found in a small number of copies in the chromosome, for example E. coli contains 6–10 IS1, and 5 IS2 and IS3 copies.

IS Elements Genetic Effects

• Act as a mutagen by causing spontaneous mutations.
• Excision of IS elements restores gene function.
• IS elements generate small, directly repeated duplications of target DNA.
• Duplications are characteristic to each element and are typically 2-13 bp.
• IS can cause deletion in neighboring genes, at a rate of 10^-3 to 10^-4 per generation, and sometimes deletion of themselves.
• IS can invert a neighboring segment of DNA.
• Cointegrate formation fuses two replicons.
• Cause Homologous recombination effects including Hfr and F' formation, amplification, and inversion.