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Homemade Site Directed Mutagenesis of Whole Plasmids
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Homemade Site Directed Mutagenesis of Whole Plasmids

Published on: May 11, 2009

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Mutation in ESBL Plasmid from Escherichia coli O104:H4 Leads Autoagglutination and Enhanced Plasmid Dissemination

Mickaël Poidevin1, Mari Sato2, Ipek Altinoglu1,3

  • 1Institute for Integrative Biology of the Cell, Université Paris-Saclay, CEA, CNRS, Université Paris-Sud, Gif-sur-Yvette, France.

Frontiers in Microbiology
|February 20, 2018
PubMed

Insights

Genetic mutations can enhance the spread of multidrug resistance (MDR) plasmids. This study reveals how specific mutations in the Hft region of IncI plasmids lead to increased TraA protein expression, boosting plasmid transfer and bacterial spread.

Area of Science:

  • Microbiology
  • Genetics
  • Molecular Biology

Background:

  • Conjugative plasmids, particularly IncI incompatibility plasmids, are key drivers of multidrug resistance (MDR) dissemination in clinical and environmental settings.
  • The pESBL-EA11 plasmid, isolated from an *Escherichia coli* outbreak strain, has shown enhanced transfer ability due to mutations in a specific DNA region (Hft).

Purpose of the Study:

  • To investigate the regulatory elements governing the conjugative transfer of the pESBL-EA11 IncI plasmid.
  • To understand how genetic modifications, specifically in the Hft region, enhance plasmid transmissibility.

Main Methods:

  • Transposon mutagenesis and nucleotide substitution analysis to identify key genetic elements.
  • Gene expression analysis to quantify TraA and TraB levels.
  • Atmospheric Scanning Electron Microscopy (A-SEM) to visualize pili structure and distribution.
  • Conjugation assays to measure plasmid transfer efficiency.

Main Results:

  • Overexpression of the TraA protein significantly elevated the transfer rate of the pESBL-EA11 plasmid.
  • Mutations in the Hft region resulted in strong TraA overexpression, activating the TraB regulator and likely leading to increased conjugative pili production.
  • IncI pili were observed to be expressed throughout the cell surface, distinct from other pilus types.
  • Hyperpiliation in mutant strains facilitated enhanced cell-to-cell adhesion and autoagglutination, contributing to high transfer efficiency.

Conclusions:

  • Genetic evolution, including 'superspreader' mutations, can dramatically enhance plasmid transmissibility, posing a significant threat to controlling MDR spread.
  • The findings highlight the potential for plasmids to evolve increased infectivity and transmissibility, with possible implications for host pathogenicity.
Keywords:
ESBLatmospheric scanning electron microscopyconjugationhorizontal gene transfermultidrug resistant

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