A Region of ς(K) Involved in Promoter Activation by GerE in Bacillus subtilis

During endospore formation in Bacillus subtilis, the DNA binding protein GerE stimulates transcription from several promoters that are used by RNA polymerase containing ς(K). GerE binds to a site on one of these promoters, cotX, that overlaps its −35 region. We tested the model that GerE interacts with ς(K) at the cotX promoter by seeking amino acid substitutions in ς(K) that interfered with GerE-dependent activation of the cotX promoter but which did not affect utilization of the ς(K)-dependent, GerE-independent promoter gerE. We identified two amino acid substitutions in ς(K), E216K and H225Y, that decrease cotX promoter utilization but do not affect gerE promoter activity. Alanine substitutions at these positions had similar effects. We also examined the effects of the E216A and H225Y substitutions in ς(K) on transcription in vitro. We found that these substitutions specifically reduced utilization of the cotX promoter. These and other results suggest that the amino acid residues at positions 216 and 225 are required for GerE-dependent cotX promoter activity, that the histidine at position 225 of ς(K) may interact with GerE at the cotX promoter, and that this interaction may facilitate the initial binding of ς(K) RNA polymerase to the cotX promoter. We also found that the alanine substitutions at positions 216 and 225 of ς(K) had no effect on utilization of the GerE-dependent promoter cotD, which contains GerE binding sites that do not overlap with its −35 region.