A region in the Bacillus subtilis transcription factor Spo0A that is important for spoIIG promoter activation

Abstract

Spo0A is a DNA binding protein in Bacillus subtilis required for the activation of spoIIG and other promoters at the onset of endospore formation. Activation of some of these promoters may involve interaction of Spo0A and the sigmaA subunit of RNA polymerase. Previous studies identified two single-amino-acid substitutions in sigmaA, K356E and H359R, that specifically impaired Spo0A-dependent transcription in vivo. Here we report the identification of an amino acid substitution in Spo0A (S231F) that suppressed the sporulation deficiency due to the H359R substitution in sigmaA. We also found that the S231F substitution partially restored use of the spoIIG promoter by the sigmaA H359R RNA polymerase in vitro. Alanine substitutions in the 231 region of Spo0A revealed an additional amino acid residue important for spoIIG promoter activation, I229. This amino acid substitution in Spo0A did not affect repression of abrB transcription, indicating that the alanine-substituted Spo0A was not defective in DNA binding. Moreover, the alanine-substituted Spo0A protein activated the spoIIA promoter; therefore, this region of Spo0A is probably not required for Spo0A-dependent, sigmaH-directed transcription. These and other results suggest that the region of Spo0A near position 229 is involved in sigmaA-dependent promoter activation.

FIG. 1

Domain structure of Spo0A. Spo0A consists of two domains, an N-terminal domain, which undergoes specific aspartate phosphorylation at D56, indicated by the arrow, and a CTD, which is responsible for DNA binding. The position number for the identifying amino acid of each domain is indicated. A magnification of the 5-amino-acid region of Spo0A discussed in this study is shown, and the amino acids are numbered. The S231F mutation was found to suppress the sporulation defect of the ς^A mutation H359R.

FIG. 2

Spo0A S231F suppresses the sporulation phenotypes of sigA mutants. Shown are the numbers of heat-resistant spores (expressed as logs of the numbers shown along the y axis) produced by strains containing wild-type (wt) or mutant sigA alleles (shown along the x axis) in combination with wild-type or mutant (S231F) spo0A alleles (solid or shaded bars, respectively).

FIG. 3

Spo0A S231F partially restores spoIIG promoter activation in vitro in the presence of RNA polymerase containing ς^A H359R. In vitro transcription experiments were performed at the spoIIG promoter by using B. subtilis RNA polymerase containing either wild-type (wt) ς^A (lanes 1 to 6) or ς^A H359R (lanes 7 to 12) in the presence of the wild-type Spo0A CTD (lanes 1 to 3 and 7 to 9) or the Spo0A S231F CTD (lanes 4 to 6 and 10 to 12). Increasing concentrations of the Spo0A CTD were used for activation: none (lanes 1, 4, 7, and 10), 250 nM (lanes 2, 5, 8, and 11), and 500 nM (lanes 3, 6, 9, and 12). The arrow indicates transcripts from the spoIIG promoter.

FIG. 4

Effects of amino acid substitutions in Spo0A on spoIIG, spoIIA, and abrB promoter activities. B. subtilis EUC9762 (wild-type spo0A) (circles), EUC9763 (spo0AI229A) (squares), and EUC9790 (spo0A195) (triangles) containing the spoIIG (a), spoIIA (b), or abrB (c) promoter-lacZ fusion were grown in DSM medium. Samples were taken from cultures growing at mid-log phase (T₋₁), at the end of exponential growth (T₀), and at 1-h intervals after the onset of stationary phase (T₁ to T₄) and were then assayed for β-galactosidase activity (22). Independent transductants of each of the above strains were assayed for β-galactosidase activity and were found to show essentially the same results (data not shown).

FIG. 4

Effects of amino acid substitutions in Spo0A on spoIIG, spoIIA, and abrB promoter activities. B. subtilis EUC9762 (wild-type spo0A) (circles), EUC9763 (spo0AI229A) (squares), and EUC9790 (spo0A195) (triangles) containing the spoIIG (a), spoIIA (b), or abrB (c) promoter-lacZ fusion were grown in DSM medium. Samples were taken from cultures growing at mid-log phase (T₋₁), at the end of exponential growth (T₀), and at 1-h intervals after the onset of stationary phase (T₁ to T₄) and were then assayed for β-galactosidase activity (22). Independent transductants of each of the above strains were assayed for β-galactosidase activity and were found to show essentially the same results (data not shown).

FIG. 4

Effects of amino acid substitutions in Spo0A on spoIIG, spoIIA, and abrB promoter activities. B. subtilis EUC9762 (wild-type spo0A) (circles), EUC9763 (spo0AI229A) (squares), and EUC9790 (spo0A195) (triangles) containing the spoIIG (a), spoIIA (b), or abrB (c) promoter-lacZ fusion were grown in DSM medium. Samples were taken from cultures growing at mid-log phase (T₋₁), at the end of exponential growth (T₀), and at 1-h intervals after the onset of stationary phase (T₁ to T₄) and were then assayed for β-galactosidase activity (22). Independent transductants of each of the above strains were assayed for β-galactosidase activity and were found to show essentially the same results (data not shown).