Site-specific, Insertional Inactivation of incA in Chlamydia Trachomatis Using a Group II Intron

PLoS One. 2013 Dec 31;8(12):e83989. doi: 10.1371/journal.pone.0083989. eCollection 2013.

Abstract

Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that has until more recently remained recalcitrant to genetic manipulation. However, the field still remains hindered by the absence of tools to create selectable, targeted chromosomal mutations. Previous work with mobile group II introns demonstrated that they can be retargeted by altering DNA sequences within the intron's substrate recognition region to create site-specific gene insertions. This platform (marketed as TargeTron™, Sigma) has been successfully employed in a variety of bacteria. We subsequently modified TargeTron™ for use in C. trachomatis and as proof of principle used our system to insertionally inactivate incA, a chromosomal gene encoding a protein required for homotypic fusion of chlamydial inclusions. C. trachomatis incA::GII(bla) mutants were selected with ampicillin and plaque purified clones were then isolated for genotypic and phenotypic analysis. PCR, Southern blotting, and DNA sequencing verified proper GII(bla) insertion, while continuous passaging in the absence of selection demonstrated that the insertion was stable. As seen with naturally occurring IncA(-) mutants, light and immunofluorescence microscopy confirmed the presence of non-fusogenic inclusions in cells infected with the incA::GII(bla) mutants at a multiplicity of infection greater than one. Lack of IncA production by mutant clones was further confirmed by Western blotting. Ultimately, the ease of retargeting the intron, ability to select for mutants, and intron stability in the absence of selection makes this method a powerful addition to the growing chlamydial molecular toolbox.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins / genetics*
  • Blotting, Southern
  • Blotting, Western
  • Cells, Cultured
  • Chlamydia Infections / genetics
  • Chlamydia Infections / microbiology*
  • Chlamydia trachomatis / genetics*
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Inclusion Bodies
  • Introns / genetics*
  • Membrane Proteins / genetics*
  • Mice
  • Microscopy, Fluorescence
  • Mutagenesis, Insertional / genetics*
  • Mutagenesis, Site-Directed
  • Mutation / genetics*
  • Plasmids / genetics
  • Polymerase Chain Reaction

Substances

  • Bacterial Proteins
  • IncA protein, Chlamydia trachomatis
  • Membrane Proteins

Grant support

Research was supported by internal funds to DJF and an SIU Master’s Fellowship to CMJ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.