Identification and characterization of a DeoR-specific operator sequence essential for induction of dra-nupC-pdp operon expression in Bacillus subtilis

The deoR gene located just upstream the dra-nupC-pdp operon of Bacillus sub tilis encodes the DeoR repressor protein that negatively regulates the expr ession of the operon at the level of transcription. The control region upst ream of the operon was mapped by the use of transcriptional lacZ fusions, I t was shown that all of the cis-acting elements, which were necessary for f ull DeoR regulation of the operon, were included in a 141-bp sequence just upstream of dra. The increased copy number of this control region resulted in titration of the DeoR molecules of the cell. By using mutagenic PCR and site-directed mutagenesis techniques, a palindromic sequence located from p osition -60 to position -43 relative to the transcription start point was i dentified as a part of the operator site for the binding of DeoR, Furthermo re, it was shown that a direct repeat of five nucleotides, which was identi cal to the 3' half of the palindrome and was located between the -10 and -3 5 regions of the dra promoter, might function as a half binding site involv ed in cooperative binding of DeoR to the regulatory region. Binding of DeoR protein to the operator DNA was confirmed by a gel electrophoresis mobilit y shift assay. Moreover, deoxyribose-5-phosphate was shown to be a likely c andidate for the true inducer of the dra-nupC-pdp expression.