title: Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis.
abstract: Although progress in Chlamydia genetics has been rapid, genomic modification has previously been limited to point mutations and group II intron insertions which truncate protein products. The bacterium has thus far been intractable to gene deletion or more-complex genomic integrations such as allelic exchange. Herein, we present a novel suicide vector dependent on inducible expression of a chlamydial gene that renders Chlamydia trachomatis fully genetically tractable and permits rapid reverse genetics by fluorescence-reported allelic exchange mutagenesis (FRAEM). We describe the first available system of targeting chlamydial genes for deletion or allelic exchange as well as curing plasmids from C. trachomatis serovar L2. Furthermore, this approach permits the monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms. As proof of principle, trpA was successfully deleted and replaced with a sequence encoding both green fluorescent protein (GFP) and β-lactamase. The trpA-deficient strain was unable to grow in indole-containing medium, and this phenotype was reversed by complementation with trpA expressed in trans. To assess reproducibility at alternate sites, FRAEM was repeated for genes encoding type III secretion effectors CTL0063, CTL0064, and CTL0065. In all four cases, stable mutants were recovered one passage after the observation of transformants, and allelic exchange was limited to the specific target gene, as confirmed by whole-genome sequencing. Deleted sequences were not detected by quantitative real-time PCR (qPCR) from isogenic mutant populations. We demonstrate that utilization of the chlamydial suicide vector with FRAEM renders C. trachomatis highly amenable to versatile and efficient genetic manipulation.
  
  
  OBJECTIVE
  The obligate intracellular nature of a variety of infectious bacteria presents a significant obstacle to the development of molecular genetic tools for dissecting pathogenicity. Although progress in chlamydial genetics has been rapid, genomic modification has previously been limited to point mutations and group II intron insertions which truncate protein products. The bacterium has thus far been intractable to gene deletion or more-complex genomic integrations such as allelic exchange. Here, we present a novel suicide vector dependent on inducible expression of a chlamydial gene that renders Chlamydia trachomatis fully genetically tractable and permits rapid reverse genetics by fluorescence-reported allelic exchange mutagenesis (FRAEM). We describe the first available system of targeting chlamydial genes for deletion or allelic exchange as well as curing plasmids from C. trachomatis L2. Furthermore, this approach permits monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms.
authors.affiliation: Department of Microbiology, Immunology & Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky, USA.
authors.forename: Konrad E
authors.initials: KE
authors.lastname: Mueller
authors.affiliation: Department of Microbiology, Immunology & Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky, USA.
authors.forename: Katerina
authors.initials: K
authors.lastname: Wolf
authors.affiliation: Department of Microbiology, Immunology & Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky, USA Ken.fields@uky.edu.
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Fields
mesh_terms: D002692:Chlamydia trachomatis / Q000235:genetics*; D002876:Chromosomes, Bacterial; D005453:Fluorescence; D017353:Gene Deletion; D055786:Gene Knockout Techniques / Q000379:methods*; D017930:Genes, Reporter; D005827:Genetics, Microbial / Q000379:methods*; D008856:Microscopy, Fluorescence; D010957:Plasmids