Surfaces of Spo0A and RNA Polymerase Sigma Factor A That Interact at the spoIIG Promoter in Bacillus subtilis

Spo0A from Bacillus subtilis is a member of the response regulator class of two-component signal transducing systems (reviewed in reference 13). Activation of Spo0A by phosphorylation triggers the onset of endospore development. Active Spo0A binds to DNA at a 7-bp sequence, referred to as the 0A box (2, 26), to positively or negatively regulate the expression of a large number of genes (7). Spo0A activates transcription from two classes of promoters, those used by RNA polymerase containing the primary sigma factor, σA (e.g., spoIIG and spoIIE promoters [22, 30]), and those used by RNA polymerase containing the secondary sigma factor, σH (e.g., the spoIIA promoter [19, 29]). Spo0A may activate the σA-dependent promoters by interacting directly with the σA subunit of the RNA polymerase. Baldus et al. (1) found that spoIIG and spoIIE promoter activity was reduced in mutants of B. subtilis in which σA contained one of two single amino acid substitutions in region 4, with the lysine at position 356 replaced by glutamate (K356E) or the histidine at 359 replaced by arginine (H359R). However, these substitutions did not affect the utilization of σA-dependent, Spo0A-independent promoters or σH-dependent, Spo0A-dependent promoters. Alanine substitutions at these sites in σA produced similar effects (1). Therefore, these substitutions define positions in region 4 of σA that are essential specifically for Spo0A-dependent activation of the spoIIG and spoIIE promoters. A specific region of Spo0A is required to activate the σA-dependent promoters, such as spoIIG. This region has been defined by several single amino acid substitutions (4, 11, 14). For example, substitution of alanine for isoleucine at position 229 of Spo0A (4) or for threonine at position 239 (14) reduces activation of the σA-dependent spoIIG promoter, but does not severely affect activation of the σH-dependent spoIIA promoter. The structure of the Spo0A DNA binding domain has been determined by X-ray crystallography for both free Spo0A and Spo0A in complex with DNA (15, 31). Interestingly, all mutations in Spo0A specifically affecting the ability of σA-dependent RNA polymerase to activate transcription cluster in α-helix E, a flexible helix in the C terminus of the protein that is positioned away from the core structure of the protein. A simple model to account for the requirements for specific amino acids in region 4 of σA and amino acids of α-helix E of Spo0A suggests that these surfaces interact directly during Spo0A-dependent promoter activation. Some support for this model comes from the finding that a single amino acid substitution in α-helix E of Spo0A, with a serine at position 231 replaced by phenylalanine (S231F), partially suppresses the effect of the σA H359R substitution (4). However, the Spo0A S231F mutation also partially suppresses the effects of the H359A and K356E substitutions in σA and the S231F Spo0A mutant efficiently stimulates wild-type σA RNA polymerase. Because the Spo0A suppressor mutation S231F does not display an allele-specific suppression of the sporulation defect caused by the H359R mutation in σA, it is unlikely that the phenylalanine at position 231 in the mutant Spo0A interacts directly with the arginine at 359 in the mutant σA. Perhaps the Spo0A S231F mutant creates a form of Spo0A that interacts with another position of σA or in another subunit of RNA polymerase. Therefore, whether region 4 of σA interacts directly with α-helix E of Spo0A is unknown. Here we report genetic evidence for an interaction between region 4 of σA and Spo0A. We found an intragenic sigA suppressor mutation resulting in substitution of tryptophan for arginine at position 355 (R355W) that almost fully suppresses the sporulation defect due to both the H359R and H359A substitutions in sigA. We used the coordinates of structures for the C-terminal domain of Spo0A (C-Spo0A) (15) and C-Spo0A bound to DNA (31) and for σ region 4 from Thermus aquaticus bound to DNA (5) to model the interactions of Spo0A and σA at the spoIIG promoter. The resulting model predicts that R355 in region 4 of σA interacts with the glutamine at position 221 of Spo0A. The effects of alanine substitutions in σA and Spo0A on spoIIG promoter activity were consistent with this model.