title: Isolation and identification of a repressor TetR for 3,17β-HSD expressional regulation in Comamonas testosteroni.
abstract: Comamonas testosteroni (C. testosteroni) is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons. 3,17β-Hydroxysteroid dehydrogenase (3,17β-HSD) from C. testosteroni is a key enzyme in steroid degradation. Understanding the mechanism of 3,17β-HSD gene (βhsd) induction may help us to elucidate its complete molecular regulation. Sequencing the C. testosteroni ATCC11996 genome lead us to identify the tetR (522 bp) downstream of βhsd. Two repeat sequences (RS; 13 bp), that are separated to each other by 1661 bp, were found upstream of βhsd. A bioinformatic analysis revealed that TetR family proteins act as transcriptional repressors which are sensitive to environmental signals. Since, C. testosteroni responds to environmental steroid induction and upregulates steroid catabolic genes, we hypothesized that TetR might act in C. testosteroni as repressor for βhsd expression. The tetR was cloned into different plasmids, including an EGFP reporter system, for functional characterization and/or overexpression. The data indicate that, indeed, TetR acts as a repressor for 3,17β-HSD expression. Testosterone in turn, which is known to induce βhsd expression, could not resolve TetR repression. To further substantiate TetR as repressor for βhsd expression, a tetR gene knock-out mutant of C. testosteroni was generated. TetR gene knock-out mutants showed the same basal low level of βhsd expression as the C. testosteroni wild type cells. Interestingly, testosterone induction leads to a strong increase in βhsd expression, especially in the tetR gene knock-out mutants. The result with the knock-out mutant, in principle, supports our hypothesis that TetR is a repressor for βhsd expression, but the exact role of testosterone in this context remains unknown. Finally, it turned out that TetR is obviously also involved in the regulation of the hsdA gene.
authors.affiliation: Institute of Toxicology and Pharmacology for Natural Scientists, Medical School Schleswig-Holstein, Kiel, Germany; Department of Family Medicine, The First Affiliated Hospital, School of Medicine, Zhejiang University, 310000 Hangzhou, China.
authors.forename: Tianyuan
authors.initials: T
authors.lastname: Pan
authors.affiliation: Department of Biochemistry and Genetics, School of Medicine, Zhejiang University, 310058 Hangzhou, China.
authors.forename: Pu
authors.initials: P
authors.lastname: Huang
authors.affiliation: Institute of Toxicology and Pharmacology for Natural Scientists, Medical School Schleswig-Holstein, Kiel, Germany.
authors.forename: Guangming
authors.initials: G
authors.lastname: Xiong
authors.affiliation: Institute of Toxicology and Pharmacology for Natural Scientists, Medical School Schleswig-Holstein, Kiel, Germany. Electronic address: maser@toxi.uni-kiel.de.
authors.forename: Edmund
authors.initials: E
authors.lastname: Maser
mesh_terms: D015067:17-Hydroxysteroid Dehydrogenases / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D020604:Comamonas testosteroni / Q000201:enzymology* / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D009154:Mutation / Q000235:genetics; D012097:Repressor Proteins; D013256:Steroids / Q000378:metabolism; D013739:Testosterone / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; D013256:Steroids; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013739:Testosterone; D015067:17-Hydroxysteroid Dehydrogenases; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase
keywords: 3,17β-Hydroxysteroid dehydrogenase; Comamonas testosteroni; Gene regulation; Testosterone induction; TetR family repressor; βhsd