A reporter gene system for the precise measurement of promoter activity in Thermus thermophilus HB27

• Published in: Extremophiles : life under extreme conditions Vol. 19; no. 6; pp. 1193 - 1201
• Main Authors: Fujita, Atsushi, Sato, Takaaki, Koyama, Yoshinori, Misumi, Yoshio
• Format: Journal Article
• Language: English
• Published: Tokyo Springer Japan 01-11-2015; Springer Nature B.V
• Subjects: Antibiotic resistance; Bacterial Proteins - genetics; beta-Galactosidase - genetics; Biochemistry; Biomedical and Life Sciences; Biotechnology; Carotenoids; Carotenoids - genetics; Genes; Genes, Bacterial; Genes, Reporter; Genetic Techniques; Life Sciences; Measurement techniques; Method Paper; Microbial Ecology; Microbiology; Multienzyme Complexes - genetics; Promoter Regions, Genetic; Space life sciences; Thermal energy; Thermus; Thermus thermophilus; Thermus thermophilus - genetics; Reporter gene system; Thermophile; Carotenoid; β-Galactosidase; Thermus thermophilus
• ISSN: 1431-0651; 1433-4909
• DOI: 10.1007/s00792-015-0789-3

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.