ABSTRACT
Phosphorylation of SpoIIAA on Ser-58 catalyzed by SpoIIAB is important in the regulation of sporulation of Bacillus subtilis. Nucleotide binding experiments showed that the affinity of SpoIIAB for ATP was greatly increased in the presence of SpoIIAA or a mutant SpoIIAA in which Ser-58 had been changed to alanine. Study of the phosphorylation reaction showed that the Km for ATP and the Ki for ADP were both about 1 microM. The kinetics of phosphorylation of SpoIIAA by SpoIIAB were biphasic, comprising a rapid phase (leading to phosphorylation of 1 mol of SpoIIAA/mol of SpoIIAB) followed by a slower, steady-state phase. In the steady state, the rate-determining step proved to be the dissociation of a SpoIIAB-ADP complex. The rate of this dissociation was not affected significantly by changes in the concentration of ATP.
Subject(s)
Bacillus subtilis/physiology , Bacterial Proteins/metabolism , Sigma Factor , Spores, Fungal/physiology , Transcription Factors , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Kinetics , Models, Chemical , PhosphorylationABSTRACT
SpoIIAA is the first protein of the spoIIA operon. Here we show that SpoIIAA can bind and hydrolyze GTP. The protein also accepts ATP, but with lower affinity. GDP competes poorly for binding of GTP. The GTPase activity of SpoIIAA is within the range found for other GTP-binding proteins.
Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/metabolism , Guanosine Triphosphate/metabolism , Sigma Factor , Transcription Factors , Bacterial Proteins/classification , Bacterial Proteins/drug effects , Bacterial Proteins/radiation effects , Dose-Response Relationship, Drug , GTP Phosphohydrolases/classification , GTP Phosphohydrolases/drug effects , GTP Phosphohydrolases/radiation effects , GTP-Binding Proteins/classification , GTP-Binding Proteins/drug effects , GTP-Binding Proteins/radiation effects , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/radiation effects , Magnesium/pharmacology , Ultraviolet RaysABSTRACT
Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two cell types that are formed by an asymmetric cell division. Major changes in the pattern of transcription during sporulation are brought about by the synthesis of new sigma factors (sigma), which are subunits of RNA polymerase that determine promoter specificity. Transcription in the smaller prespore cell type is initiated by a sigma factor called sigma F, the activity of which is subject to tight spatial and temporal control. It is negatively regulated by an anti-sigma factor, SpoIIAB, which is in turn controlled by an anti-anti-sigma factor, SpoIIAA. SpoIIAA and SpoIIAB participate in two contrasting reactions in vitro. In the presence of ATP, the proteins interact transiently and SpoIIAA is inactivated by phosphorylation on a specific serine residue; SpoIIAA then remains free to inhibit sigma F. In the presence of ADP, SpoIIAA binds tightly to SpoIIAB, and sigma F is set free. Release of sigma F activity in vivo might thus be effected by a prespore-specific reduction in the ATP/ADP ratio. Genetic experiments have implicated a fourth protein, called SpoIIE, in this system. It now appears that SpoIIE has two important and independent functions in the establishment of the prespore-specific transcription by sigma F. First it regulates sigma F activity, probably acting as a phosphatase to regenerate the active, non-phosphorylated form of SpoIIAA. Second it controls the formation of the septum that generates the prespore compartment. Combination of these two functions in a single polypeptide may provide a means of coupling gene expression with morphogenesis.
Subject(s)
Bacillus subtilis/physiology , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Sigma Factor , Transcription Factors , Amino Acid Sequence , Bacillus subtilis/genetics , DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/metabolism , Gene Expression Regulation, Bacterial , Genes, Bacterial , Information Systems , Molecular Sequence Data , Promoter Regions, Genetic , Sequence Homology, Amino Acid , Spores, Bacterial , Transcription, GeneticABSTRACT
Sigma F is regulated by an anti-sigma factor, SpoIIAB, and an anti-anti-sigma factor, SpoIIAA. SpoIIAB also functions as a phosphokinase which transfers phosphate from ATP to SpoIIAA; this phosphorylation is thought to be involved in the regulatory mechanism. By using [gamma-32P]ATP to phosphorylate SpoIIAA, cleaving the protein proteolytically, and analyzing the one resulting radiolabelled peptide by the Edman degradation procedure, we show that the site of phosphorylation in SpoIIAA is Ser-58.
