THE HIGHLY THERMOSTABLE ARGININE REPRESSOR OF BACILLUS-STEAROTHERMOPHILUS - GENE CLONING AND REPRESSOR-OPERATOR INTERACTIONS

We report here the cloning of the arginine repressor gene argR of Baci llus stearothermophilus and the characterization and purification to h omogeneity of its product, The deduced amino acid sequence of the 16.8 -kDa ArgR subunit shares 72% identity with its mesophilic homologue Ah rC of Bacilus subtilis. Sequence analysis of B. stearothermophilus Arg R and comparisons with mesophilic arginine repressors suggest that the thermostable repressor comprises an N-terminal DNA-binding and a C-te rminal oligomerization and arginine-binding region. B. stearothermophi lus ArgR has been overexpressed in E. coli and purified as a 48.0-kDa trimeric protein. The repressor inhibits the expression of a B. stearo thermophilus argC-lacZ fusion in E. coli cells. in the presence of arg inine, the purified protein binds tightly and specifically to the argC operator, which largely overlaps the argC promoter. The purified B. s tearothermophilus repressor proved to be very thermostable with a half -life of approximately 30 min at 90 degrees C, whereas B. subtilis Ahr C was largely inactivated at 65 degrees C. Moreover, ArgR operator com plexes were found to be remarkably thermostable and could be formed ef ficiently at up to 85 degrees C, well above the optimal growth tempera ture of the moderate thermophile B. stearothermophilus. This pronounce d resistance of the repressor-operator complexes to heat treatment sug gests that the same type of regulatory mechanism could operate in extr eme thermophiles.