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We constructed a new Thermus thermophilus cloning vector which enables the colour selection of cloned DNA inserts in the T. thermophilus HB27 host strain (β-gal) on growth plates containing 3,4-cyclohexenoesculetin β- d-galactopyranoside (S-gal) in the medium. This vector harbors a modified β-galactosidase gene (TTP0042 of T. thermophilus HB27) with 12 unique restriction enzyme sites ( Acc65I, AvrII, BlpI, BssHII, EcoRI, EcoRV, HindIII, NruI, SalI, SpeI, SphI and XbaI) as multiple cloning sites under the control of the T. thermophilus slpA promoter. This host-vector system facilitates cloning procedures in T. thermophilus HB27. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]