Abstract
A specific expression system for Thermus spp. is described. Plasmid pMKE1 contains replicative origins for Escherichia coli and Thermus spp., a selection gene encoding a thermostable resistance to kanamycin, and a 720 bp DNA region containing the promoter ( Pnar), and the regulatory sequences of the respiratory nitrate reductase operon of Thermus thermophilus HB8. Two genes, encoding a thermophilic β-galactosidase and an alkaline phosphatase were cloned in pMKE1 as cytoplasmic and periplasmic reporters, respectively. The expression of the reporters was specifically induced by the combined action of nitrate and anoxia in facultative anaerobic derivatives of T. thermophilus HB27 to which the gene cluster for nitrate respiration was transferred by conjugation. Overexpressions in the range of ∼200-fold were obtained for the cytoplasmic reporter, whereas that of the periplasmic reporter was limited to ∼20-fold, with respect to their intrinsic respective activities.
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