title: Transformation of sexually transmitted infection-causing serovars of chlamydia trachomatis using Blasticidin for selection.
abstract: Plasmid-free Chlamydia trachomatis serovar L2 organisms have been transformed with chlamydial plasmid-based shuttle vectors pGFP::SW2 and pBRCT using β-lactamase as a selectable marker. However, the recommendation of amoxicillin, a β-lactam antibiotics, as one of the choices for treating pregnant women with cervicitis due to C. trachomatis infection has made the existing shuttle vectors unsuitable for transforming sexually transmitted infection (STI)-causing serovars of C. trachomatis. Thus, in the current study, we modified the pGFP::SW2 plasmid by fusing a blasticidin S deaminase gene to the GFP gene to establish blasticidin resistance as a selectable marker and replacing the β-lactamase gene with the Sh ble gene to eliminate the penicillin resistance. The new vector termed pGFPBSD/Z::SW2 was used for transforming plasmid-free C. trachomatis serovar D organisms. Using blasticidin for selection, stable transformants were obtained. The GFP-BSD fusion protein was detected in cultures infected with the pGFPBSD/Z::SW2-trasnformed serovar D organisms. The transformation restored the plasmid property to the plasmid-free serovar D organisms. Thus, we have successfully modified the pGFP::SW2 transformation system for studying the biology and pathogenesis of other STI-causing serovars of C. trachomatis.
authors.affiliation: Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.
authors.forename: Honglei
authors.initials: H
authors.lastname: Ding
authors.forename: Siqi
authors.initials: S
authors.lastname: Gong
authors.forename: Yingxin
authors.initials: Y
authors.lastname: Tian
authors.forename: Zhangsheng
authors.initials: Z
authors.lastname: Yang
authors.forename: Robert
authors.initials: R
authors.lastname: Brunham
authors.forename: Guangming
authors.initials: G
authors.lastname: Zhong
mesh_terms: D002460:Cell Line; D002692:Chlamydia trachomatis / Q000187:drug effects* / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D009705:Nucleosides / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D014169:Transformation, Bacterial; D001618:beta-Lactamases / Q000235:genetics
chemical_list: D009705:Nucleosides; C004500:blasticidin S; D001618:beta-Lactamases