Organization of the divergent yetL and yetM genes and their promoter regions. (A) Organization of the yetL and yetM genes. yetL, yetM, and neighboring genes are indicated by large open arrows, and the two hairpin structures that likely function as ρ-independent transcription terminators are indicated by stem-loops structures. aa, amino acids. (B) Promoter regions of the yetL and yetM genes. The “−35” and “−10” sequences of the two genes are underlined, and the transcription start sites (+1) and the SD sequences are enclosed in boxes. The partial coding regions of yetL and yetM are indicated by lines, and the deleted region in PyetL-ΔE is also indicated by a line. The protected regions in the coding and noncoding strands detected by DNase I footprinting are indicated by bars. The 18 bp of a perfect palindromic sequence in the yetM promoter is indicated by a pair of facing arrows, and the sequences conserved between the YetL binding sites of yetL and yetM are indicated by bold type.

Determination of the transcription start sites of the yetM and yetL genes by primer extension analysis. Total RNAs of strains 168 (wild type) (lane 1) and YETLd (yetL disruptant) (lane 2) used for determination of the transcription start site of yetM (left panel) and total RNAs of FU1035 (yetL⁺) (lane 1) and FU1038 (ΔyetL::tet) (lane 2) used for determination of the transcription start site of yetL (right panel) were prepared and used for the reverse transcription reaction to generate the runoff cDNAs. Lanes G, A, T, and C contained the products of the dideoxy sequencing reactions obtained with the same primer that was used for reverse transcription. The runoff cDNAs are each indicated by an arrow. The partial nucleotide sequences of the coding strands corresponding to the ladders are shown; the “−35” and “−10” sequences are underlined, and the transcription start sites (+1) and the SD sequence are enclosed in boxes.

DNase I footprinting analysis of YetL in the yetL and yetM promoter regions. DNA probes corresponding to the yetL and yetM promoter regions (PyetL and PyetM), 5′ end labeled on either the coding or noncoding strand, were prepared. The 5′-labeled probe (0.8 nM) was incubated in the reaction mixture with the recombinant YetL protein (lanes 2, 5, 6, and 7 for the PyetL probe, 144 nM [a dimer]; lanes 3, 8, 9, and 10 for the PyetL probe, 72 nM; lanes 2, 5, 6, and 7 for the PyetM probe, 36 nM; lanes 3, 8, 9, 10 for the PyetM probe, 18 nM) and without the YetL protein (lanes 1 and 4). A flavonoid solution in DMSO (2 μl) was added to the mixture to obtain a flavonoid concentration of 10 mM (lanes 6 and 9, quercetin; lanes 7 and 10, apigenin), and the same volume of DMSO was added to the mixtures in lanes 5 and 8 before incubation. After partial digestion with DNase I, the resulting mixtures were subjected to urea-PAGE. Lanes G, A, T, and C contained the products of the dideoxy sequencing reactions with the corresponding 5′-labeled primers. Nucleotide sequences protected by YetL are indicated on the right in each panel; the SD sequence and the transcription start site (+1) are enclosed in boxes, the perfect palindrome sequence is indicated by a pair of facing arrows, and the sequences conserved in the protected areas of the PyetL and PyetM probes are indicated by bold type.

In vitro binding affinity of YetL to the yetL and yetM cis sequences. (A) Each DNA probe used for gel retardation is indicated by a bar. The YetL-binding cis sequences in the yetL and yetM promoter regions are indicated by cross-hatched boxes. The results of a comparison of these sequences are shown as motif logos created with the B. subtilis Motif Location Search software (http://dbtbs.hgc.jp/motiflocationsearch.html). (B) The ³²P-labeled DNA probes for the yetL and yetM promoter regions (PyetL and PyetM) and the ³²P-labeled PyetL derivative lacking the cis sequence (PyetL-ΔE) (0.8 nM) were incubated with purified YetL protein, which was followed by PAGE. The YetL protein solution was diluted stepwise twofold, and an aliquot of each dilution was added to the mixture to obtain the concentrations used. The no protein lanes contained no YetL. The arrows indicate lanes for which the K_d was determined. The experiments were repeated at least two times, and the results of representative experiments are shown.

Evaluation of the inhibitory effect of each flavonoid on YetL binding to the yetL and yetM cis sequences by gel retardation. The ³²P-labeled PyetL and PyetM probes (0.8 nM) were incubated with 780 nM and 49 nM YetL, respectively, in the presence of each flavonoid indicated at concentrations of 4.9 μM to 10 mM (12 lanes; twofold dilution with DMSO 11 times before incubation). The DMSO lanes contained no flavonoid, and the no protein lanes contained no YetL. The experiments were repeated at least two times, and the results of representative experiments are shown.

Derepression of the yetL and yetM promoter activities repressed by YetL in response to flavonoids. (A) The yetL and yetM promoter regions were fused to the lacZ reporter gene in opposite orientations and then integrated into the amyE loci of strains 168 and FU1034 (ΔyetL::tet) to monitor the yetL and yetM promoters, respectively. In the resulting strains, FU1036 (yetL⁺) and FU1039 (ΔyetL::tet), the lacZ reporter is under the control of the yetL promoter, whereas in strains FU1037 (yetL⁺) and FU1040 (ΔyetL::tet), the lacZ reporter is controlled by the yetM promoter. (B) lacZ expression during cultivation of strains FU1036 and FU1037 (left panel) and strains FU1039 and FU1040 (right panel). Squares, OD₆₀₀ values for strains without the yetL disruption (strains FU1036 and FU1039); diamonds, OD₆₀₀ values for strains with the yetL disruption (strains FU1039 and FU1040); triangles, β-Gal activities for strains without the yetL disruption (strains FU1036 and FU1037); circles, β-Gal activities for strains with the yetL disruption (strains FU1039 and FU1040). (C) Strain FU1037 was used to monitor the yetM promoter activity in response to addition of each flavonoid. When the OD₆₀₀ reached 0.2, each flavonoid was added to the culture (indicated by an arrow) at a concentration of 200 μg/ml. The squares and triangles indicate the OD₆₀₀ values and β-Gal activities, respectively, and the open and filled symbols indicate cultures without and with flavonoids, respectively. The lacZ fusion experiments were repeated at least two times, and the results of representative experiments are shown.