Essential internal promoter in the spoIIIA locus of Bacillus subtilis

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

^* To whom correspondence should be addressed. Email: moran{at}microbio.emory.edu.

	Abstract

The Bacillus subtilis spoIIIA locus encodes eight proteins, SpoIIIAA-AH, that are expressed in the mother cell during endospore formation but which are essential for the activation of ^G in the forespore. Complementation studies indicated that the locus may be transcribed from two promoters, one 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 RT-PCR analysis of the P2_spoIIIA region, we determined that P2_spoIIIA is sufficient for the transcription of spoIIIAG and spoIIIAH. Inactivation of P2_spoIIIA blocked spore formation, indicating that P2_spoIIIA is essential for expression of spoIIIAG and spoIIIAH. P2_spoIIIA activity is twice that of P1_spoIIIA; therefore, larger amounts of SpoIIIAG and SpoIIIAH may be required than proteins encoded at the upstream end of the locus.