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

Abstract

Aims: To obtain data concerning gene expression in Thermus thermophilus and demonstrate the use of the b-galactosidase gene from Thermus sp. A4 as a convenient reporter gene. Methods and Results: Thermus thermophilus PPKU was constructed, in which the b-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 b-gal reporter gene derived from Thermus sp. A4 to construct vectors pTEX7, pTEX8 and pTEX9, respectively. The amount of b-galactosidase activity produced by the PdnaK promoter in pTEX7 was substantially above the background level of 0AE3 U mg )1 , and increased from 5AE2 to 10AE4 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 M 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 Thermus and that b-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 b-galactosidase activity in T. thermophilus PPKU can be used to allow reliable screening for b-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 b-gal host-vector system reported here.

Markerless Gene Deletion with Cytosine Deaminase in Thermus thermophilus Strain HB27

“…The ␤-glycosidase assay with crude extracts of T. thermophilus HB27 and LW19 with pNPGal as the substrate revealed reduced ␤-glycosidase activity of the ⌬bglT strain (3 mU mg Ϫ1 of total soluble proteins) compared to the wild type (8 mU mg Ϫ1 ). This result is in agreement with the data published previously (42) and further substantiates the loss of bglT.…”

“…In order to demonstrate the functionality of the new codA counterselection system, we chose bglT as the first target for deletion. BglT is a widely used reporter protein for studying promoter activities in T. thermophilus HB27 (16,42). The additional copy of bglT on the megaplasmid pTT27 of T. thermophilus HB27 interferes with these reporter assays.…”

“…Commonly used reporter genes of expression vectors for T. thermophilus encode thermostable ␣-galactosidase and ␤-galactosidase (42,46,47), ␤-glycosidase (bglT) (16), or phytoene synthase (crtB) (9,48). All the named reporter genes originate from T. thermophilus HB27 or a closely related species whose native enzyme activities interfere with reporter assays.…”

Function, kinetic properties, crystallization, and regulation of microbial malate dehydrogenase

“…In Thermus thermophilus, MDH had a higher expression level when it was grown on malate as the carbon source instead of glucose (Park and Kilbane II, 2004). In addition, mdh from E. coli is expressed at four-fold higher levels with pyruvate than with glucose under aerobic conditions (Park et al, 1995).…”

Evaluation of the Staphylococcus aureus Class C Nonspecific Acid Phosphatase (SapS) as a Reporter for Gene Expression and Protein Secretion in Gram-Negative and Gram-Positive Bacteria

“…Consequently, modification of widely used reporter systems (7,18,54), the optimization of methods used for measuring reporter activity (17,27,51), and the isolation and evaluation of new reporters are continuing. The relevance of the latter is exemplified by several reports regarding the evaluation of new reporters, among others, ␤-galactosidase from Bacillus megaterium (45) and Thermus thermophilus (35), lichenase from Clostridium thermocellum (39), and ␣-galactosidase from Saccharopolyspora erythraea (40).…”