Screening and analysis of DNA fragments that show promoter activities inThermus thermophilus

We have constructed a Thermus promoter probe vector pPP11 containing the promoterless heat-stable kanamycin resistance (Kmr) gene. Total DNA of T. thermophilus HB27 was randomly sheared into 100-200 bp fragments and inserted into the EcoRI site of pPP11. About 1000 clones which showed Kmr were obtained, and they were classified into three groups according to their Kmr levels. The DNA sequences of the inserted fragments of fifteen clones (Kmr levels were high for seven, medium for three and low for five clones) were determined. Kanamycin nucleotidyl transferase (KNTase) activities of the cell-free extracts of the clones were measured. Transcriptional starting points were determined for ten clones which showed a high or medium level of Kmr. Amounts of Kmr mRNA were also estimated for the ten clones. Amounts of Kmr mRNA synthesized in the clones were in good agreement, and KNTase activities were in fairly good agreement with the Kmr levels of the clones, respectively. From these results, we propose a possible consensus promoter sequence for T. thermophilus.