title: Transformation of a plasmid-free, genital tract isolate of Chlamydia trachomatis with a plasmid vector carrying a deletion in CDS6 revealed that this gene regulates inclusion phenotype.
abstract: The development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis provides the basis for the detailed investigation of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In this study we constructed a plasmid vector with CDS6 deleted (pCDS6KO) from the original Escherichia coli/C. trachomatis shuttle vector pGFP::SW2. pCDS6KO was transformed into a clinical isolate of C. trachomatis from Sweden that is plasmid-free (C. trachomatis SWFP-). Penicillin-resistant transformants expressing the green fluorescent protein were selected. These transformants did not stain with iodine, indicating that this property is regulated by CDS6 or its gene product. In addition, mature inclusions of C. trachomatis SWFP- transformed by pCDS6KO displayed an identical morphological phenotype to the untransformed plasmid-free recipient host. In this phenotype the morphology of inclusions was altered with the chlamydiae lining the periphery of the inclusion leaving a 'hole' in the centre. These green fluorescent inclusions appear 'doughnut-shaped' with an empty centre when examined under blue light, giving rise to a characteristic 'black hole' phenotype. Our study demonstrates the power of the new genetic system for investigating chlamydial gene function using gene deletion technology.
authors.affiliation: Faculty of Medicine, Molecular Microbiology Group, University of Southampton, Southampton General Hospital, Southampton, UK.
authors.forename: Yibing
authors.initials: Y
authors.lastname: Wang
authors.forename: Lesley T
authors.initials: LT
authors.lastname: Cutcliffe
authors.forename: Rachel J
authors.initials: RJ
authors.lastname: Skilton
authors.forename: Kenneth
authors.initials: K
authors.lastname: Persson
authors.forename: Carina
authors.initials: C
authors.lastname: Bjartling
authors.forename: Ian N
authors.initials: IN
authors.lastname: Clarke
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002690:Chlamydia Infections / Q000382:microbiology; D002692:Chlamydia trachomatis / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D005835:Genitalia / Q000382:microbiology*; D006003:Glycogen / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000235:genetics*; D014169:Transformation, Bacterial
chemical_list: D001426:Bacterial Proteins; D006003:Glycogen