Study Notes on Conjugation and Plasmid Transfer

Conjugation and Plasmid Transfer

Overview of Plasmids

  • A plasmid is a small, circular piece of DNA found in bacteria that can replicate independently of chromosomal DNA.

  • Self-transmissible plasmids: Capable of transferring themselves during conjugation.

    • F plasmid: A specific type of self-transmissible plasmid involved in bacterial conjugation.

    • The cell that receives the plasmid is called the transconjugant.

Characteristics of Plasmids

  • Self-transmissible plasmids encode all proteins necessary for the transfer process, while mobilizable plasmids require self-transmissible factors to facilitate transfer.

  • In Gram-negative bacteria, the F plasmid encodes for the sex pilus involved in mating.

  • Donor strains that contain the F plasmid are referred to as male strains.

F Plasmid Structure

  • Partial genetic map of the F plasmid is approximately 100 kbp.

    • Contains insertion elements IS3 and IS2, and transposon Tn1000.

    • oriV: Origin of replication.

    • oriT: Origin of conjugative transfer.

    • tra genes (~1/3 of the F plasmid) are essential for the transfer of the plasmid and are divided into two components:

    • Dtr: DNA transfer and conjugal replication.

    • MpF: Mating pair formation.

  • There are systems in place to ensure daughter cells receive a copy of the plasmid, which includes:

    • A partitioning system.

    • A killing system for cells that do not receive the plasmid.

    • Mechanisms to block the replication of T7 bacteriophage and to repress the SOS DNA repair response during conjugation.

Tra Genes and Pilus Assembly

  • Pilus is composed of pilin (TraA) which has a long signal peptide that is cleaved during membrane crossing, then it assembles on the cell surface through cyclization.

  • Coupling proteins enable communication between the Mpf and Dtr systems, with TraD acting as a DNA translocase.

DNA Transfer Mechanism

  • The conjugation process involves the following steps:

    • The donor cell produces a pilus that contacts a recipient cell.

    • A pore is created in the membranes of both cells.

    • The coupling protein signals the relaxase from the Dtr component (TraI) to initiate transfer by nicking the plasmid at oriT, remaining attached to the plasmid.

    • Helicase separates the DNA strands.

    • After transfer, the plasmid is re-circularized by the relaxase in the recipient cell.

    • Primase starts replication in the recipient cell.

Relaxase Functionality

  • The role of relaxase at oriT includes:

    • (A) Nicking the DNA, transferring the 5’PO4 to tyrosine on the relaxase via a transesterification reaction.

    • (B) Transferring of the relaxase to the recipient cell, bringing along the DNA.

    • (C) Reversal of the transesterification for re-circularization of the plasmid.

Fertility Inhibition

  • The genetic organization of the tra region includes:

    • TraJ: A transcriptional activator required for activating other tra genes such as traY and traX, and for stabilizing the FinO.

    • The FinP RNA hybridizes with traJ mRNA, blocking its translation. RNAseE cleaves the traJ mRNA, thus regulating gene expression for the transfer process.

Mobilization of Plasmids

  • Some plasmids depend on self-transmissible plasmids for transfer (not self-transmissible):

    • Identification of oriT site is crucial for transfer.

    • Random cloning of plasmid fragments into a non-mobilizable vector.

    • Transformed mixture into cells with self-transmissible plasmid and combination with potential recipient cells to identify mobilizable pieces.

Mechanism of Plasmid Mobilization

  • The mobilizable plasmid is transferred via a self-transmissible donor cell, which has:

    • A single-stranded nick made at the oriT in the mob region.

    • Transfer and replication of mobilizable plasmid occurs alongside the self-transmissible plasmid transfer.

    • Coupling proteins must recognize the mobilizable plasmid for successful transfer.

Triparental Matings

  • Describes the transfer of plasmids via three parental cells:

    1. A self-transmissible plasmid from parent I transfers to parent II.

    2. The self-transmissible plasmid then transfers the mobilizable plasmid to parent III.

    • This method operates even if the plasmids belong to the same Inc group and cannot replicate in certain parent cells.

Formation of Hfr Strains

  • Integration of the F plasmid into the bacterial chromosome creates Hfr (high-frequency recombination) strains through:

    • Recombination between IS2 sequences in both the plasmid and chromosome, leading to chromosomal DNA transfer during conjugation.

Prime Factors and Recombination

  • Conjugal plasmids (F’) can carry chromosomal DNA due to:

    • Recombination generating a prime factor through excision that includes chromosomal DNA.

    • Note: Deletion in chromosome is not lethal as genes are located on the F plasmid.

Transfer Systems in Gram+ Bacteria

  • Differences in Mpf systems between Gram-negative and Gram-positive bacteria due to the absence of an outer membrane in Gram-positive organisms:

    • Pheromones play a role in plasmid transfer in Gram-positive bacteria:

    • Recipient cells produce propheromones, which are processed when exported.

    • The donor cell represses transcription of most tra genes through TraA, except for traC, which encodes a surface receptor for pheromones.

    • When pheromones bind to TraC, it facilitates plasmid transfer by inducing further expression of conjugative genes and blocks auto-induction.

Integrative Conjugative Elements (ICE)

  • ICEs integrate into the bacterial chromosome and:

    • Code Tra functions to transfer themselves or mobilizable plasmids.

    • Must excise from the chromosome to facilitate transfer to the recipient cell.