Site of phosphorylation of SpoIIAA, the anti-anti-sigma factor for sporulation-specific sigma F of Bacillus subtilis

Abstract

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 [␥-32 P]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.Early in the sporulation of Bacillus subtilis, the cell divides asymmetrically to form a small compartment, the prespore, and a large compartment, the mother cell. The two compartments embark on separate programs of gene expression, by means of which an endospore is formed. These programs are controlled in part by dedicated sigma factors, which become active in a compartment-specific way at defined times in sporulation (for a review, see reference 4). The first compartmentspecific sigma factor is F , the activity of which is carefully regulated. In the predivisional cell, the F protein is present in latent form, and after septation it becomes activated in the prespore but not in the mother cell; subsequent events in sporulation depend critically on this controlled activation (6). The regulation of F has been studied intensively in recent years, and we now know that regulation involves two proteins that are the products of genes in the operon (spoIIA) that also encodes F (SpoIIAC) itself. SpoIIAB is an inhibitor of F and is thus an anti-sigma factor, while SpoIIAA counteracts the inhibition by SpoIIAB and is thus an anti-anti-sigma factor (1-3, 7, 8, 10). Work in this laboratory has shown that SpoIIAB is also a phosphokinase, which catalyzes the transfer of phosphate from ATP to a serine residue of SpoIIAA (8), and we and our coworkers have recently outlined a model for the involvement of this phosphorylation in the regulation of F (2). We wanted to know which serine residue of SpoIIAA becomes phosphorylated in the phosphotransfer reaction catalyzed by SpoIIAB. Recent experiments have shown that mutagenesis of Ser-58 of SpoIIAA leads to a change in sporulation phenotype, whereas mutagenesis of two other residues (Ser-54 and Ser-84) has no phenotypic effect (2). These results are highly suggestive, but they do not constitute unequivocal proof that Ser-58 is phosphorylated by SpoIIAB or, if it is, that no other residue is phosphorylated. We now describe an experiment that identifies the site of phosphorylation by direct biochemical analysis.SpoIIAA and SpoIIAB were purified (2), and the proteins (20 g of each) were incubated for 30 min at 37ЊC in 100 l of phosphorylation buffer (8) containing 20 Ci of [␥-32 P]ATP with carrier ATP at a final concentration of 20 M. The mixture was subjected to high-pressure liquid chromatography on a size exclusion column, and the fractions containing phosphorylated SpoIIAA were pooled and dialyzed against three 1-liter changes of 100 mM Tris-HCl, pH 8.0. Endoproteinase Glu-C (2 g) was added to the Spo...

Modulator protein RsbR regulates environmental signalling in the general stress pathway of Bacillus subtilis

“…From these results, we conclude that: (i) RsbR is a positive regulator which probably belongs to the upstream, environmental-sensing module; (ii) RsbR augments response to salt and heat stress but is not absolutely required for sensing or transmitting these signals; and (iii) RsbR is unique among the known rsb gene products in that it is differentially involved in transmitting some but not all environmental stress signals. Significantly, genetic and biochemical analysis has established that the phosphorylation state of the RsbS, RsbV, and SpoIIAA antagonist proteins is important for their function, and that a conserved serine residue is the probable site of the phosphorylation event (Alper et al, 1994;Diederich et al, 1994;Dufour and Haldenwang, 1994;Najafi et al, 1995;Duncan et al, 1996;Kang et al, 1996;Yang et al, 1996). As shown in Fig.…”

Compartmentalization of Gene Expression duringBacillus subtilisSpore Formation

“…It was shown that the negative role of SpoIIAB was direct in that it bound F , thus acting as an anti-sigma factor; SpoIIAA antagonized the action of SpoIIAB and so is an anti-anti-sigma factor (51,199). SpoIIAB bore significant similarity to protein kinases, and it was found to phosphorylate SpoIIAA on a serine residue at position 58 (199,207). Mutation of this serine residue to an alanine (mimicking dephosphorylation) resulted in constitutive F activity, and mutation to an aspartic acid (mimicking phosphorylation) blocked F activity in vivo (44), indicating that the phosphorylation state of SpoIIAA is critical for its ability to function as an anti-anti-sigma factor.…”

“…Another area of ongoing research is the nature of the complexes that SpoIIAB forms with its binding partners. The basic model is that SpoIIAB inhibits F activity directly by binding to it and also indirectly by acting as a serine kinase that inactivates SpoIIAA by phosphorylating it (199,207). The phosphatase activity of SpoIIE functions to reverse this reaction (49), enabling SpoIIAA to attack the SpoIIAB-F complex and allowing F to become active.…”

An Essential Role of Glu-243 and His-239 in the Phosphotransfer Reaction Catalyzed by Pyruvate Dehydrogenase Kinase

“…In conclusion, it is interesting to note that bacteria contain another protein of the same lineage, the so-called anti-sigma factor SpoIIAB (29). Its sequence is very similar to the bacterial histidine kinases but, like PDK, contains a properly spaced glutamic acid in the N box.…”