title: A reporter gene system for the precise measurement of promoter activity in Thermus thermophilus HB27.
abstract: We developed a reporter gene system that enables precise analysis of promoter activity in Thermus thermophilus HB27. The reporter vector employs a promoterless β-galactosidase gene of Thermus spp. strain T2. However, T. thermophilus HB27 strain has three genes (TTP0042, TTP0220 and TTP0222) whose products have β-galactosidase activity, which would interfere with correct measurements of promoter activities. Thus, to eliminate this background activity, we disrupted all three of these genes to generate a host strain for measuring promoter expression as β-galactosidase activity. In addition, T. thermophilus strains also produce carotenoids called thermoxanthins that are yellow pigments. To avoid the influence of these carotenoids on the β-galactosidase assay, we also disrupted the phytoene synthase gene (crtB). The reporter gene system developed here is a powerful tool for studying transcriptional activity and the mechanisms that regulate gene expression in T. thermophilus HB27. We also showed that the crtB gene cassette could be used in repeated gene-disruption experiments to screen transformants by colony colour, thus eliminating the need for antibiotic resistance markers.
authors.affiliation: Biomedical Research Institute, Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka, 563-8577, Japan. a-fujita@aist.go.jp.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Fujita
authors.affiliation: Biomedical Research Institute, Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka, 563-8577, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Sato
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Koyama
authors.affiliation: Department of Cell Biology, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka, 814-0180, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Misumi
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D002338:Carotenoids / Q000235:genetics; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005821:Genetic Techniques; D009097:Multienzyme Complexes / Q000235:genetics; D011401:Promoter Regions, Genetic; D016963:Thermus thermophilus / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D009097:Multienzyme Complexes; C022434:prelycopersene pyrophosphate synthetase; D002338:Carotenoids; D001616:beta-Galactosidase
keywords: Carotenoid; Reporter gene system; Thermophile; Thermus thermophilus; β-Galactosidase