Mother cell-specific expression of <i>sprB</i> during sporulation.

<p>(A) Genetic organization of the <i>sprB</i> region. The black and red promoter symbols indicate the promoter upstream of <i>yosX</i> and the mother cell-specific promoter directly upstream of <i>sprB</i>, respectively. The thick black line indicates the <i>sprB</i> probe for Northern blotting. The wavy lines indicate the <i>sprB</i> transcripts with their respective lengths (kb). The red and black arrows indicate the <i>sprB</i>-specific primer for reverse transcription (RT primer) and the <i>yosX</i>-, <i>yotB</i>C<i>D</i>-, and <i>sprB</i>-specific primers for the PCR reactions, respectively. The gray lines show the products of RT followed by PCR amplification. (B) Nucleotide sequence of the <i>sprB</i> promoter region. The transcriptional start site (TSS) of <i>sprB</i> is indicated by the red arrow. Boxes indicate −35 and −10 elements of the <i>sprB</i> promoter. The consensus sequences for σ^E and σ^K binding are shown below (K = G or T; N = A, T, G, or T). (C) Northern blotting. Total RNA was isolated from <i>B. subtilis</i> 168 vegetative cells treated with (+) or without (−) 0.5 µg/ml MMC at 37°C for 60 min and from sporulating cells 4 hours after onset of the sporulation (T₄). The RNA samples were subjected to Northern blotting using the <i>sprB</i> probe. The bottom panel shows methylene blue-stained 16S rRNA as a loading control. (D) RT-PCR. The <i>sprB</i> cDNA was synthesized using the <i>sprB</i>-specific primer (Figure 5A, the red arrow RT primer) and total RNA from the <i>B. subtilis</i> 168 vegetative cells treated with (+) or without (−) MMC and from sporulating cells (T₄). Internal regions of the cDNA were amplified with the <i>yosX</i>-, <i>yotB</i>C<i>D</i>-, and <i>sprB</i>-specific primer sets. The PCR product was analyzed by 2% agarose gel electrophoresis. (E) Compartmentalization of SprB–GFP expression. BsSPRBG, carrying the <i>sprB</i>–<i>gfp</i> fusion gene under the control of the mother cell specific <i>sprB</i> promoter, was cultured at 37°C in liquid DSM containing FM4-64 (0.25 µg/ml) and kanamycin (10 µg/ml). Sporulating cells at T₄ were observed using fluorescence microscopy. PC, phase contrast; Membrane, cell membranes stained with FM4-64; GFP, GFP fluorescence; Merge, merged images of Membrane and GFP. Scale bar, 2 µm.