CodY Regulates Expression of the Bacillus subtilis Extracellular Proteases Vpr and Mpr

Binding of CodY to the vpr regulatory region. (A) Sequence of the vpr insert used to construct the vpr-lacZ fusions. Coordinates are reported with respect to the putative transcription start point (60), indicated by the bent arrow. The core CodY-binding site identified by IDAP-Seq (4) is in boldface. Two overlapping CodY-binding motifs with 4 and 3 mismatches to the consensus are underlined; two more motifs with 5 mismatches to the consensus can be found in the same region and are not shown. The CodY-protected region, detected in DNase I footprinting experiments, is underlined with the dashed line. The directions of translation of vpr and ywcI are indicated by the long arrows. The initiation codons of the two genes are in boldface. (B) Gel mobility shift assay of CodY binding to a radioactively labeled vpr PCR fragment, obtained with oligonucleotides oBB67 and oBB102, in the presence of 10 mM ILV. CodY monomer concentrations are reported below each lane. (C) DNase I footprinting analysis of CodY binding to the vpr fragment. The labeled vpr PCR fragment, obtained with oligonucleotides oGB9 and oBB253, was incubated with increasing amounts of purified CodY in the presence of 10 mM ILV and 2 mM GTP and then with DNase I. Concentrations of CodY monomers are reported above each well. The corresponding A+G sequencing ladder of the bottom DNA strand is on the left. The protected area is shown by the vertical line and the corresponding sequence is reported; the bent arrow indicates the transcription start point and the direction of transcription. The core CodY-binding site identified by IDAP-Seq is in boldface. (D) Gel mobility shift assay of CodY binding to a radioactively labeled vpr_p2 fragment, obtained with oligonucleotides oBB67 and oBB102, in the presence of 10 mM ILV. CodY monomer concentrations are reported below each lane.