Transcription of the xyl operon is controlled in Bacillus subtilis by tandem overlapping operators spaced by four base-pairs.

The expression of xylose utilization in Bacillus subtilis is regulated at the level of transcription by xylose dependent Xyl repressor-xyl operator interaction. We have structurally and functionally characterized the binding sites of Xyl repressor in the xyl regulatory region. Methylation and hydroxyl radical protection and ethylation interference of binding suggests tandem overlapping xyl operators spaced by four base-pairs. A mutational inactivation of each and both operators was performed. DNA retardation experiments with these mutants confirmed the existance of two binding sites. They can be simultaneously occupied, despite their overlapping, intertwined organization. In vivo repressor titration and regulation of indicator gene expression by the xylO mutants confirmed that both binding sites contribute to regulation of the xyl operon. The protection and interference patterns of both sites are identical and indicate binding of a repressor oligomer to one side of B-form DNA of each operator. A tandem overlapping arrangement of two operators is also found in the xyl regulatory sequences of Bacillus megaterium, Staphylococcus xylosus and Lactobacillus pentosus. The xyl operon of Bacillus licheniformis contains a similar element in which the second operator is more diverged. This high degree of conservation among bacteria of different genera supports the conclusion that a tandem overlapping arrangement of xyl operators contributes to efficient regulation.