Decrease in srfA expression by H₂O₂ treatment. Cells were grown in modified competence medium, and their β-galactosidase activities were determined as described in Materials and Methods. Growth of the cells monitored with an Klett optical density meter and activities of β-galactosidase are shown in the left and right panels, respectively. The x axis represents the growth time in hours (left panels) and the duration of culture in hours relative to the end of vegetative growth (right panels). Several experiments were performed, and typical results are shown. Arrows in the left panels indicate the time when the indicated concentration of H₂O₂ was added. Closed circles, no addition of H₂O₂; open circles, 0.2 mM H₂O₂; open triangles, 1 mM H₂O₂; open squares, 5 mM H₂O₂. (A) and (B) show the experiments using srfA-lacZ (OAM186) and skfB-lacZ (YBCPd), respectively.

Expression of srfA-lacZ, comK-lacZ, and comE-lacZ in perR disruptant cells. Cells were grown in modified competence medium, and their β-galactosidase activities were determined as described in Materials and Methods. The x axis represents the duration of culture in hours relative to the end of vegetative growth (T0). Several experiments were performed, and typical results are shown. (A) Closed circles, wild-type OAM186; open circles, perR disruptant OAM241. (B) Closed circles, wild-type 8G33; open circles, perR disruptant OAM242. (C) Closed circles, wild-type OAM243; open circles, perR disruptant OAM244.

Amount of ComX in PerR disruptant cells. Various conditioned media were added to wild-type cell cultures carrying the srfA-lacZ fusion (OAM186) grown in modified competence medium at the early logarithmic phase. The conditioned media were prepared as described in Materials and Methods from the wild-type strain OAM186 (lane 2), the perR disruptant OAM241 (lane 3), and the comXΔ mutant OSM102 (lane 4). The effect of adding unconditioned modified competence medium is shown in lane 1. The β-galactosidase activities shown were those measured half an hour after the conditioned medium was added.

Effect of the perR disruption on the expression of srfA-lacZ in rapC and spx mutant cells. The cells were grown in modified competence medium, and their β-galactosidase activities were determined as described in Materials and Methods. The numbers on the x axis represent the duration of culture in hours relative to the end of the vegetative growth (T0). Several experiments were performed, and typical results are shown. (A) Closed circles, wild-type strain OAM186; closed squares, rapC disruptant OAM245; open circles, perR disruptant OAM241; open squares, rapC perR double disruptant OAM246. (B) Closed circles, wild-type strain OAM186; closed squares, spx disruptant OAM233; open circles, perR disruptant OAM241; open squares, spx perR double disruptant OAM247.

Gel mobility shift and footprint assays of the srfA promoter using PerR. The gel shift assay was performed as described in Materials and Methods. A 6% native polyacrylamide gel was used. (A) The katA and srfA probe DNAs span positions −131 to +65 and −237 to +10 relative to the transcription start site, respectively. The rapG probe spans the 180-bp-long promoter region of rapG. These probes were prepared by PCR using the katA-1 and katA-2, srfA-1 and srfA-2, and rapG-1 and rapG-2 primers. The srfA-2 probe DNA spans position −130 to +10 relative to the transcription start site and was amplified by PCR using srfA-3 and srfA-2a. c and f indicate the protein-DNA complex and free probe, respectively. “well” means the start point of the electrophoresis. Reactions contained poly(dI-dC) (0.1 μg/25 μl). The 2 nM probes were incubated with 200 nM His-tagged PerR. − and +, reactions without and with PerR, respectively. Left panel, lanes 1 and 2, katA; lanes 3 and 4, srfA; lanes 5 and 6, rapG. Right panel, lanes 7 and 8, srfA; lanes 9 and 10, srfA-2. (B) The srfA promoter prepared by PCR using srfA-4 and srfA-2 (40 nM) was incubated in separate reactions with the increased amount of His-tagged PerR and subjected to DNase I cleavage. Brackets along the gel indicate the protected regions. Lane 1 shows no PerR, while lanes 2, 3, 4, and 5 show the effect of 0.1 μM, 0.2 μM, 0.4 μM, and 0.6 μM of PerR, respectively. (C) The nucleotide sequence of the srfA promoter region is shown. Brackets over the nucleotide sequence and arrowheads indicate the protected regions and nucleotides, respectively. The numbers at either side of the nucleotide sequence show the nucleotide positions relative to the transcription start site. The PerR and ComA boxes are boxed and underlined, respectively. The bent arrow indicates the 5′ terminus of the srfA-2 probe. The asterisks show the nucleotides matching the consensus sequence of the PerR box (10).

Confirmation that the Per boxes function as cis elements in srfA expression. (A) Various cells were grown in modified competence medium, and sampling was initiated at late logarithmic phase. The β-galactosidase activities from the srfA-lacZ fusions were determined as described in Materials and Methods. The numbers on the x axis represent the duration of culture in hours relative to the end of vegetative growth (T0). The closed and open symbols indicate the wild-type and perR disruptant strains, respectively. Several experiments were performed, and typical results are shown. (B) Schematic representation of the srfA-lacZ fusions. The closed and open boxes indicate the PerR (−153 to −141 and −137 to −123) and ComA (−118 to −103 and −74 to −59) boxes, respectively. The 5′ and 3′ ends of the fusions are indicated as nucleotide positions relative to the transcription start point. The bent arrows show the promoter sequence of srfA. SD indicates the Shine-Dalgarno sequence of lacZ.