title: Identification and isolation of a regulator protein 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 member of the short-chain dehydrogenase/reductase (SDR) superfamily. It is an inducible and key enzyme in steroid degradation. Elucidating the mechanism of 3,17β-HSD gene (βhsd) regulation may help us to generate prospective C. testosteroni mutants for bioremediation. The genome of C. testosteroni ATCC11996 was sequenced in our previous work. Upon examining the genome with bioinformatics tools, a gene (brp) coding for a regulator protein (BRP) for 3,17β-HSD expression was found upstream of the βhsd gene. A Blast search revealed high identities to a nucleotide binding protein with unknown function in other bacteria. Two potential promoters and two repeat sequences (RS, 16 bp), spaced to each other by 1661 bp, were also found upstream of the βhsd gene C. testosteroni. The brp gene was cloned into plasmid pK18 and pET-15b, expressed in Escherichia coli, and the recombinant BRP protein was purified on a Ni-column. In addition, a brp gene knock-out mutant of C. testosteroni was prepared. These knock-out mutants showed an enhanced expression of both the βhsd gene and the hsdA gene (the latter coding for 3α-HSD/CR) in the presence of testosterone. To characterize the BRP functional DNA domain, different fragments of the βhsd upstream regulatory region were tested in a cotransformation system. Our data reveal that the βhsd gene undergoes complex regulation involving the two promoters, a loop structure via the two repeat sequences, and the steroid testosterone. Furthermore, a proximal repressor gene for βhsd expression, phaR, had been identified in our previous investigations. The exact interplay between all these factors will be determined in future experiments.
authors.affiliation: Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School, Schleswig-Holstein, Kiel, Germany.
authors.forename: Yin
authors.initials: Y
authors.lastname: Wu
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, University 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, University 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*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D020604:Comamonas testosteroni / Q000201:enzymology* / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D064429:Fatty Acid Synthases / Q000235:genetics; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D013256:Steroids / Q000378:metabolism; D013739:Testosterone / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D013256:Steroids; D013739:Testosterone; D004247:DNA; D015067:17-Hydroxysteroid Dehydrogenases; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase; C036543:short chain trans-2-enoyl-CoA reductase; D009247:NADH, NADPH Oxidoreductases; D064429:Fatty Acid Synthases
keywords: 3,17β-Hydroxysteroid dehydrogenase; Comamonas testosteroni; Knock-out mutant; Nucleotide-binding protein; Testosterone induction; βhsd gene