Quantifying promoter activity during the developmental cycle of Chlamydia trachomatis.
Chlamydia trachomatis is an important human pathogen that undergoes a characteristic development cycle correlating with stage-specific gene expression profiles. Taking advantage of recent developments in the genetic transformation in C. trachomatis, we constructed a versatile green fluorescent protein (GFP) reporter system to study the development-dependent function of C. trachomatis promoters in an attempt to elucidate the mechanism that controls C. trachomatis adaptability. We validated the use of the GFP reporter system by visualizing the activity of an early euo gene promoter. Additionally, we uncovered a new ompA promoter, which we named P3, utilizing the GFP reporter system combined with 5' rapid amplification of cDNA ends (RACE), in vitro transcription assays, real-time quantitative RT-PCR (RT-qPCR), and flow cytometry. Mutagenesis of the P3 region verifies that P3 is a new class of C. trachomatis σ(66)-dependent promoter, which requires an extended -10 TGn motif for transcription. These results corroborate complex developmentally controlled ompA expression in C. trachomatis. The exploitation of genetically labeled C. trachomatis organisms with P3-driven GFP allows for the observation of changes in ompA expression in response to developmental signals. The results of this study could be used to complement previous findings and to advance understanding of C. trachomatis genetic expression.
Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.|Department of Microbiology, Third Military Medical University, Chongqing, China, 400038.
Yanguang
Y
Cong
Department of Neonatology, Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing, China, 400014.
Leiqiong
L
Gao
Department of Neonatology, Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing, China, 400014.
Yan
Y
Zhang
Department of Neonatology, Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing, China, 400014.
Yuqi
Y
Xian
Department of Neonatology, Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing, China, 400014.
Ziyu
Z
Hua
Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
Hiba
H
Elaasar
Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
Li
L
Shen
D001426:Bacterial Proteins / Q000235:genetics*; D002692:Chlamydia trachomatis / Q000235:genetics / Q000254:growth & development*; D015964:Gene Expression Regulation, Bacterial; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000235:genetics; D009154:Mutation; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins