Inducible and constitutive expression using new plasmid and integrative expression vectors for Thermus sp.

Aims: To develop molecular teals and examine inducible and constitutive gen e expression in Thermus thermophilus. Methods and Results: Two plasmid promoter probe vectors and an integrative promoter probe vector were constructed using a promoterless thermostable ka namycin nucleotidyl-transferase (Km(R)) cassette. Three expression vectors were constructed based on a constitutive promoter J17, that functions in bo th Thermus and Escherichia coli. An inducible expression vector was constru cted using the heat-shock inducible promoter (70 to 85 degreesC) from the d naK gene of T. flavus, and the malate dehydrogenase gene (mdh) from T. flav us was cloned and expressed in both E. coli and T. thermaphilus HB27. Conclusions: This report describes the construction and use of improved pro moter probe and expression vectors for use in Thermus species. The mdh gene can be used as a high temperature (85 degreesC) reporter gene for Thermus sp. The dnaK promoter is thermo-inducible. Significance and Impact of the Study: The expression vectors and molecular tools described here are significant improvements over previously reported vectors for Thermus sp. The mdh gene and the thermo-inducible dnaK promoter will facilitate high temperature studies employing Thermus species.