SPO0A BINDS TO A PROMOTER USED BY SIGMA-A RNA-POLYMERASE DURING SPORULATION IN BACILLUS-SUBTILIS

Examination of the effects of 56 single-base-pair substitutions in the spoIIG promoter and studies of the interaction of the spo0A product (Spo0A) with this promoter in vitro demonstrated that Spo0A acts directly to enable this promoter to be used by sigma-A-associated RNA polymerase (EC 2.7.7.6). The spoIIG operon from Bacillus subtilis is transcribed during sporulation by a form of RNA polymerase containing sigma-A, the primary sigma-factor in vegetative cells. The spoIIG promoter is unusual in that it contains sequences that are similar to those found at the -10 and -35 regions of promoters that are used by sigma-A-associated RNA polymerase, but these sigma-A-like recognition sequences are separated by 22 base pairs rather than the typical 17 or 18 base pairs. We found that single-base-pair substitutions in and around the -35-like sequence, and substitutions in a region upstream from this position, around position -87, reduced promoter activity. DNase I protection and electrophoretic gel mobility shift assays were used to demonstrate that Spo0A binds specifically to these regions in vitro. Evidently, the -35-like sequence is part of a Spo0A binding site and therefore is possibly not a sigma-A-recognition sequence. These results support a model in which Spo0A activates the spoIIG promoter after the onset of endospore formation.