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 Bacillus stearothermophilus and the characterization and purification to homogeneity of its product. The deduced amino acid sequence of the 16.8-kDa ArgR subunit shares 72% identity with its mesophilic homologue AhrC of Bacilus subtilis. Sequence analysis of B. stearothermophilus ArgR and comparisons with mesophilic arginine repressors suggest that the thermostable repressor comprises an N-terminal DNA-binding and a C-terminal oligomerization and arginine-binding region. B. stearothermophilus ArgR has been overexpressed in E. coli and purified as a 48.0-kDa trimeric protein. The repressor inhibits the expression of a B. stearothermophilus argC-lacZ fusion in E. coli cells. In the presence of arginine, the purified protein binds tightly and specifically to the argC operator, which largely overlaps the argC promoter. The purified B. stearothermophilus repressor proved to be very thermostable with a half-life of approximately 30 min at 90 degrees C, whereas B. subtilis AhrC was largely inactivated at 65 degrees C. Moreover, ArgR operator complexes were found to be remarkably thermostable and could be formed efficiently at up to 85 degrees C, well above the optimal growth temperature of the moderate thermophile B. stearothermophilus. This pronounced resistance of the repressor-operator complexes to heat treatment suggests that the same type of regulatory mechanism could operate in extreme thermophiles.
Dion
M
M
Laboratoire de Biotechnologie, Faculté des Sciences et Techniques, Université de Nantes, France.
Charlier
D
D
Wang
H
H
Gigot
D
D
Savchenko
A
A
Hallet
J N
JN
Glansdorff
N
N
Sakanyan
V
V
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000235:genetics*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D012097:Repressor Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA
C087018:ArgR protein, Bacteria; D001426:Bacterial Proteins; D012097:Repressor Proteins