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

Aims: To obtain data concerning gene expression in Thermus thermophilus and demonstrate the use of the β-galactosidase gene from Thermus sp. A4 as a convenient reporter gene. Methods and Results: Thermus thermophilus PPKU was constructed, in which the β-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 β-gal reporter gene derived from Thermus sp. A4 to construct vectors pTEX7, pTEX8 and pTEX9, respectively. The amount of β-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. Conclusions: These results demonstrate that several inducible and regulated promoters are available for genetic studies in Thermos and that β-galactosidase can be used as a convenient reporter gene for studies of transcriptional regulation in Thermus. Significance and Impact of the Study: The availability of characterized inducible and regulated promoters will facilitate the development of improved gene expression vectors for Thermus. The demonstration that β-galactosidase activity in T. thermophilus PPKU can be used to allow reliable screening for β-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 Thermos will benefit from the β-gal host-vector system reported here.