Gene expression studies of Thermus thermophilus promoters PdnaK, Parg and Pscs-mdh.

Abstract

To obtain data concerning gene expression in Thermus thermophilus and demonstrate the use of the beta-galactosidase gene from Thermus sp. A4 as a convenient reporter gene. Thermus thermophilus PPKU was constructed, in which the beta-gal gene was deleted from the chromosome. Two inducible promoters PdnaK (regulating the DnaK heat shock-inducible protein) and Parg (regulating expression of an arginine-inducible protein) and a carbon-regulated promoter, Pscs-mdh (regulating expression of succinyl-coA and malate dehydrogenase) were cloned upstream of the beta-gal reporter gene derived from Thermus sp. A4 to construct vectors pTEX7, pTEX8 and pTEX9, respectively. The amount of beta-galactosidase activity produced by the PdnaK promoter in pTEX7 was substantially above the background level of 0.3 U mg(-1), and increased from 5.2 to 10.4 U mg(-1) after heat-shock induction indicating that significant amounts of DnaK are produced even when T. thermophilus is grown at its optimum temperature. The Parg promoter was found to be maximally induced by 10-30 mm arginine, but was inhibited by higher concentrations. The Pscs-mdh promoter was maximally active in the presence of malate while lower levels of activity were observed in the presence of succinate, pyruvate, glutamate, glucose and the presence of yeast extract or peptone. These results demonstrate that several inducible and regulated promoters are available for genetic studies in Thermus and that beta-galactosidase can be used as a convenient reporter gene for studies of transcriptional regulation in Thermus. The availability of characterized inducible and regulated promoters will facilitate the development of improved gene expression vectors for Thermus. The demonstration that beta-galactosidase activity in T. thermophilus PPKU can be used to allow reliable screening for beta-gal-positive transformant colonies on agar plates will add to the convenience of performing genetic manipulations in T. thermophilus. Future studies of transcriptional regulation in Thermus will benefit from the beta-gal host-vector system reported here.