| Summary: | <i>Bacillus thuringiensis</i> produces insecticidal crystal proteins encoded by <i>cry</i> or <i>cyt</i> genes and targets a variety of insect pests. We previously found that a strong promoter of a DeoR family transcriptional regulator (HD73_5014) can efficiently drive <i>cry1Ac</i> expression in <i>B. thuringiensis</i> HD73. Here, we investigated the regulation of neighbor genes by HD73_5014. The HD73_5014 homologs are widely distributed in Gram-positive bacterial species. Its neighbor genes include <i>pepV</i>, <i>rsuA</i>, and <i>ytgP</i>, which encode dipeptidase, rRNA pseudouridine synthase and polysaccharide biosynthesis protein, respectively. The four open reading frames (ORFs) are organized to be a <i>pepR</i> gene cluster in HD73. RT-PCR analysis revealed that the <i>rsuA</i> and <i>ytgP</i> genes formed a transcriptional unit (<i>rsuA</i>-<i>ytgP</i> operon), while <i>pepV</i> formed a transcriptional unit in HD73. Promoter-<i>lacZ</i> fusion assays showed that the <i>pepV</i> and <i>rsuA</i>-<i>ytgP</i> promoters are regulated by HD73_5014. EMSA experiments showed that HD73_5014 directly binds to the <i>pepV</i> promoter region but not to the <i>rusA</i>-<i>ytgP</i> promoter region. Thus, the HD73_5014 transcriptional regulator, which controls the expression of the dipeptidase <i>pepV</i>, was named PepR (dipeptidase regulator). We also confirmed the direct regulation between PepR and PepV by the increased sensitivity to vancomycin in Δ<i>pepV</i> and Δ<i>pepR</i> mutants compared to HD73.
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