Bacillus subtilis CodY Operators Contain Overlapping CodY Binding Sites

ABSTRACT CodY is a global transcriptional regulator that is activated by branched-chain amino acids. A palindromic 15-bp sequence motif, AATTTTCNGAAAATT, is associated with CodY DNA binding. A gel mobility shift assay was used to examine the effect of pH on the binding of Bacillus subtilis CodY to the hutPp and ureAp 3 promoters. CodY at pH 6.0 has higher affinity for DNA, more enhanced activation by isoleucine, and a lower propensity for nonspecific DNA binding than CodY at pH 8.0. DNase I footprinting was used to identify the CodY-protected regions in the hutPp and ureAp 3 promoters. The CodY-protected sequences for both promoters were found to contain multiple copies of the 15-bp motif with 6-bp overlaps. Mutational analysis of the hutPp regulatory region revealed that two overlapping sequence motifs were required for CodY-mediated regulation. The presence of overlapping sequence motifs in the regulatory regions of many B. subtilis CodY-regulated genes suggests that CodY binds to native operators that contain overlapping binding sites.