Essential internal promoter in the spoIIIA locus of Bacillus subtilis

The Bacillus subtilis spoIII4 locus encodes eight proteins, SpoIIIAA to SpoIIIAH, which are expressed in the mother cell during endospore formation and which are essential for the activation of sigma(G) in the forespore. Complementation studies indicated that this locus may be transcribed from two promoters, one promoter upstream from the first gene and possibly a second unidentified promoter within the locus. Fragments of the spoIII4 locus were expressed at an ectopic site to complement the sporulation-defective phenotype of a spoIIIAH deletion, and we determined that complementation required a fragment of DNA that extended into spoIIIAF. To confirm that there was a promoter located in spoIIIAF, we constructed transcriptional fusions to lacZ and found strong sporulation-induced promoter activity. Primer extension assays were used to determine the transcription start site, and point mutations introduced into the -10 and -35 regions of the promoter reduced its activity. This promoter is transcribed by sigma(E)-RNA polymerase and is repressed by SpoIIID. Therefore, we concluded that the spoIII4 locus is transcribed from two promoters, one at the start of the locus (P1(spoIIIA)) and the other within the locus (P2(spoIIIA)). Based on Campbell integrations and reverse transcription-PCR analysis of the P2(spoIIIA) region, we determined that P2(spoIIIA) is sufficient for transcription of spoIIL4G and spoIIIAH. Inactivation of P2(spoIIIA) blocked spore formation, indicating that P2(spoIIIA) is essential for expression of spoIIIAG and spoIIIAH. The P2(spoIIIA) activity is twice the P1(spoIIIA) activity; therefore, larger amounts of SpoIIIAG and SpoIIIAH than of proteins encoded at the upstream end of the locus may be required.