Molecular characterization of the transition state regulator AbrB from Bacillus stearothermophilus

The Bacillus subtilis transition state regulator AbrB(su) is a DNA-binding protein that acts on several genes either as activator, repressor, or preve nter. However, among genes under its control, neither common binding sites could be identified nor could the structural features of this broad and spe cific interaction be elucidated. Attempts to elucidate these interesting fe atures by crystallizing AbrB(su) have failed so far. Therefore, to solve th is problem, we focused in this work on identifying an AbrB(su) homologue fr om Bacillus stearothermophilus. Using a novel method. the entire abrB(st) g ene of B. stearothermophilus was cloned and sequenced. The gene encodes a 9 5 amino acid protein that shows 77% identity and 85% similarity to the meso philic B. subtilis protein. A calmodulin binding peptide-tagged fusion of t he thermophilic gene was constructed for overexpression and efficient affin ity column purification of the AbrB(st) protein. The purified protein showe d, after removal of the tag, an oligomerization behavior through hexamer fo rmation that is essential for its DNA binding activity. (C) 2000 Elsevier S cience B.V. All rights reserved.