title: Conditional gene expression in Chlamydia trachomatis using the tet system.
abstract: Chlamydia trachomatis is maintained through a complex bi-phasic developmental cycle that incorporates numerous processes that are poorly understood. This is reflective of the previous paucity of genetic tools available. The recent advent of a method for transforming Chlamydia has enabled the development of essential molecular tools to better study these medically important bacteria. Critical for the study of Chlamydia biology and pathogenesis, is a system for tightly controlled inducible gene expression. To accomplish this, a new shuttle vector was generated with gene expression controlled by the Tetracycline repressor and anhydryotetracycline. Evaluation of GFP expression by this system demonstrated tightly controlled gene regulation with rapid protein expression upon induction and restoration of transcription repression following inducer removal. Additionally, induction of expression could be detected relatively early during the developmental cycle and concomitant with conversion into the metabolically active form of Chlamydia. Uniform and strong GFP induction was observed during middle stages of the developmental cycle. Interestingly, variable induced GFP expression by individual organisms within shared inclusions during later stages of development suggesting metabolic diversity is affecting induction and/or expression. These observations support the strong potential of this molecular tool to enable numerous experimental analyses for a better understanding of the biology and pathogenesis of Chlamydia.
authors.affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, United States of America.
authors.forename: Jason
authors.initials: J
authors.lastname: Wickstrum
authors.forename: Lindsay R
authors.initials: LR
authors.lastname: Sammons
authors.forename: Keasha N
authors.initials: KN
authors.lastname: Restivo
authors.forename: P Scott
authors.initials: PS
authors.lastname: Hefty
mesh_terms: D000818:Animals; D017920:Antiporters / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D045744:Cell Line, Tumor; D002692:Chlamydia trachomatis / Q000235:genetics* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013753:Tetracycline Resistance / Q000235:genetics; D013754:Tetracyclines / Q000494:pharmacology*
chemical_list: D017920:Antiporters; D001426:Bacterial Proteins; D012097:Repressor Proteins; D013754:Tetracyclines; C079051:tetA protein, Bacteria; C034811:tetracycline resistance-encoding transposon repressor protein; D049452:Green Fluorescent Proteins; C016229:4-epianhydrotetracycline