Purification of aconitase from Bacillus subtilis and correlation of its N-terminal amino acid sequence with the sequence of the citB gene

Abstract

The DNA sequence for the promoter region of the Bacillus subtilis citB gene has been determined. Presumed "-10" and "-35" regions of the promoter have been identified, and transcriptional and translational start points of citB have been located. To correlate the DNA sequence of citB with the amino acid sequence of its presumed product, aconitase, it was necessary to devise a scheme for purification of this labile enzyme. This procedure relies on the ability to restore enzyme activity at each stage of purification by incubation in a reducing buffer containing a source of ferrous ions. B. subtilis aconitase appears to be a monomer with a molecular weight of approximately 120,000. The amino-terminal amino acids of aconitase fit the sequence predicted by analysis of the citB gene. Thus, citB codes for aconitase.