Alpha-helix E of Spo0A is required for sigmaA- but not for sigmaH-dependent promoter activation in Bacillus subtilis

Abstract

At the onset of endospore formation in Bacillus subtilis, the DNA binding protein Spo0A activates transcription from two types of promoters. The first type includes the spoIIG and spoIIE promoters, which are used by sigma(A)-RNA polymerase, whereas the second type includes the spoIIA promoter, which is used by RNA polymerase containing the secondary sigma factor sigma(H). Previous genetic analyses have identified specific amino acids in alpha-helix E of Spo0A that are important for activation of Spo0A-dependent, sigma(A)-dependent promoters. However, these amino acids are not required for activation of the sigma(H)-dependent spoIIA promoter. We now report the effects of additional single-amino-acid substitutions and the effects of deletions in alpha-helix E. The effects of alanine substitutions revealed one new position (239) in Spo0A that appears to be specifically required for activation of the sigma(A)-dependent promoters. Based on the effects of a deletion mutation, we suggest that alpha-helix E in Spo0A is not directly involved in interaction with sigma(H)-RNA polymerase.

FIG. 1.

Ribbon diagram of the C-terminal, DNA binding domain of Spo0A. The structure of the Bacillus stearothermophilus Spo0A is based on coordinates from Lewis et al. (7) and is generated by using RasMol version 2.7.1.1. The α-helix E is colored blue, and the rest of the structure is red. Regions of the helix deleted in B. subtilis Spo0A are delineated by the numbered amino acid positions. Amino acid positions R226 and T243 correspond to amino acid positions R218 and S235 in B. stearothermophilus Spo0A, respectively.

FIG. 2.

Effect of substitution T239A on the expression of spoIIA-lacZ, spoIIG-lacZ, and abrB-lacZ transcriptional fusions. DSM cultures of each transduced strain, i.e., EUAKB18 (wild-type-0A •), EUAKB58 (T239A-0A ▪), and EUAKB78 (Null-0A ○), were harvested at hourly intervals beginning at about 1 h before the end of the exponential growth, which is indicated as 0 on the time scale. The collected samples were assayed for β-galactosidase activity indicated in Miller units.

FIG. 3.

Amino acid sequence alignment of the C-terminal domains from Spo0A and helix E deletion derivatives. Shown is an alignment of the amino acid sequences from positions 224 to 267 in wild-type B. subtilis Spo0A and the homologous positions in three deletion-substitution derivatives of Spo0A. Amino acids regions deleted in the three mutants are represented by gaps, and the corresponding positions in wild-type Spo0A are numbered. The two or three glycine residues that were substituted for the deleted amino acids have been aligned arbitrarily. The α-helix E and α-helix F of C-Spo0A are represented by bars above the amino acid sequence.

FIG. 4.

Effect of deletion mutant Spo0A alleles on expression of spoIIA-lacZ, spoIIG-lacZ, and abrB-lacZ transcriptional fusions. DSM cultures of each transduced strain, i.e., EUAKB18 (wild-type-0A •), EUAKB38 (Spo0AΔ1 ▴), EUAKB82 (Spo0AΔ1+V8A ▪), and EUAKB78 (null-0A ○), were harvested at hourly intervals beginning at about 1 h before the end of the exponential growth, which is indicated as 0 on the time scale. The collected samples were assayed for β-galactosidase activity indicated in Miller units.