Genotype, phenotype, and protein structure in a regulator of sporulation: effects of mutations in the spoIIAA gene of Bacillus subtilis

Abstract

SpoIIAA, a phosphorylatable protein, is essential to the regulation of sigmaF, the first sporulation-specific transcription factor of Bacillus subtilis. The solution structure of SpoIIAA has recently been published. Here we examine four mutant SpoIIAA proteins and correlate their properties with the phenotypes of the corresponding B. subtilis mutant strains. Two of the mutations severely disrupted the structure of the protein, a third greatly diminished the rate of its phosphorylation and abolished dephosphorylation, and the fourth left phosphorylation unaffected but reduced the rate of dephosphorylation about 10-fold.

FIG. 1

The structure of SpoIIAA, with the residues whose mutations are discussed in the text indicated.

FIG. 2

Content of wild-type and mutant SpoIIAA proteins detected by immunoblotting in the soluble fraction of cells 2 h after resuspension in sporulation medium. The lanes and the quantities of soluble protein loaded were as follows: lane 1, wild type (1.9 μg); lane 2, strain 42 (0.9 μg); lane 3, strain 69 (1.2 μg); lane 4, strain 565 (1.7 μg); and lane 5, strain 568 (1.6 μg). The arrow shows the position of a SpoIIAA marker.

FIG. 3

Phosphorylation of wild-type and mutant SpoIIAA proteins by SpoIIAB. (A) Wild-type SpoIIAA (squares) and SpoIIAAG95D (triangles). (B) Wild-type SpoIIAA (squares) and SpoIIAAG62D (circles).

FIG. 4

Hydrolysis of phosphorylated wild-type and mutant SpoIIAA proteins by SpoIIE. After electrophoresis in nondenaturing conditions the gels were stained with Coomassie blue and destained with 30% methanol–10% acetic acid. In each panel, lane M contains, as a marker, the relevant nonphosphorylated SpoIIAA protein, which is the expected product of the hydrolysis. (A) Wild-type SpoIIAA-phosphate, with samples taken at the times shown between 0 and 90 min. (B) SpoIIAAG95D-phosphate, with samples taken at the times shown between 0 and 20 h. (C) SpoIIAAG62D-phosphate, with samples taken at the times shown between 0 and 20 h.