Bacillus subtilis mutations that alter the pathway of phosphorylation of the anti-anti-sigmaF factor SpoIIAA lead to a Spo- phenotype.
Sigma-F, the first sporulation-specific transcription factor of Bacillus subtilis, is regulated by an anti-sigma factor SpoIIAB, which can also act as a protein kinase that phosphorylates the anti-anti-sigma factor SpoIIAA. The time course of phosphorylation reaction is biphasic, a fact that has been interpreted in terms of a mechanism for sequestering SpoIIAB away from sigmaF and thus allowing activation of sigmaF when needed. Site-directed mutagenesis of SpoIIAA has allowed us to isolate two mutants that cannot activate sigmaF and which are therefore Spo-. The two mutant SpoIIAA proteins, SpoIIAAL61A and SpoIIAAL90A, are phosphorylated with linear kinetics; in addition they are less able to form the stable non-covalent complex that wild-type SpoIIAA makes with SpoIIAB in the presence of ADP. The phosphorylated form of SpoIIAAL90A was hydrolysed by the specific phosphatase SpoIIE at the same rate as wild-type SpoIIAA-P, but the rate of hydrolysis of SpoIIAAL61A-P was much slower. The secondary structure and the global fold of the mutant proteins were unchanged from the wild type. The results are interpreted in terms of a model for the wild type in which SpoIIAB, after phosphorylating SpoIIAA, is released in a form that is tightly bound to ADP and which then makes a ternary complex with an unreacted SpoIIAA. We propose that it is the inability to make this ternary complex that deprives the mutant cells of a means of keeping SpoIIAB from inhibiting sigmaF.
Lee
C S
CS
Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Clarkson
J
J
Shu
J C
JC
Campbell
I D
ID
Yudkin
M D
MD
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010641:Phenotype; D010766:Phosphorylation; D011487:Protein Conformation; D012808:Sigma Factor / Q000037:antagonists & inhibitors*; D020349:Surface Plasmon Resonance; D014157:Transcription Factors
D001426:Bacterial Proteins; C064971:FliA protein, Bacteria; D012808:Sigma Factor; D014157:Transcription Factors; C092468:spoIIR protein, Bacillus subtilis; C030069:spore-specific proteins, Bacillus; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate