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Essential Internal Promoter in the spoIIIA Locus of Bacillus subtilis

Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322

Received 12 June 2007/ Accepted 31 July 2007

The Bacillus subtilis spoIIIA 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 ^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 spoIIIA 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 ^E-RNA polymerase and is repressed by SpoIIID. Therefore, we concluded that the spoIIIA 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 spoIIIAG 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.

Published ahead of print on 10 August 2007.