Identification of proteins phosphorylated by ATP during sporulation of Bacillus subtilis

Abstract

Protein phosphorylation in BaciUus subtilis was assayed in vitro by using extracts prepared from cells at various times during'growth and sporulation. At least six proteins were labeled in vitro by using [_y-32PATP and extracts of ve'getative cells. In extracts prepared at the end of exponential growth and during'stationary phase, 12 to 13 proteins were labeled. Seven of the phosphoproteins were purified' by fast-performance liquid chromatography and polyacrylamide gel electrophoresis, blotted to Immobilon membranes, and subjected to partial protein sequencing. One of the sequences had sequence homology (>45%) to elongation factor G from several bacterial species, and four seqwences matched the predicted amino-terminal sequences of the outB, orJY-tsr, oiffl, and ptsH genes.Protein phosphorylation, an established regulatory mechanism in eukaryotes (4), has been observed for many bacterial species (3, 16). One example of prokaryotic protein phosphorylation occurs in the sensor-kinase and responseregulator systems, in which one of the proteins is an environment-sensing kinase and the other is a phosphorylation substrate and transcriptional regulator. Four pairs of these proteins have been identified in Bacillus subtilis (20); they are involved in genetic competence (ComnP-ComA), phosphorus utilization (PhoR-PhoB), expression of secreted proteases (DegS-DegU), and sporulation (KinA-SpoOA). The latter involves the autophosphorylation of a histidine residue on KinA and subsequent transfer of the phosphate group to aspartate residues of proteins SpoOF, SpoOB, and SpoOA, a transcriptional regulator (2). Another example of protein phosphorylation is the phosphoenolpyruvate:sugar phosphotransferase system for sugar transport, in which the phosphate from phosphoenolpyruvate is' transferred, via specific histidine residues of several proteins (HPr, enzymes I and II/III), to a sugar (16). In this system, the activity of one protein, HPr, is also regulated by ATP-dependent phosphorylation of a serine residue (14,16). Changes in protein phosphorylation during sporulation, germination, and outgrowth have also been described for B. subtilis by using in vivo and in vitro assays (9), but nQne of the modified proteins were identified. Previously, we reported changes in phosphloproteins during sporulation of Bacillus megaterium (12a) and B. subtili. '(12). In this study, we have identified the N-termipal amino acid seqpences of several proteins that wfere phosphorylated in B. subtilis.

The PrpC Serine-Threonine Phosphatase and PrkC Kinase Have Opposing Physiological Roles in Stationary-Phase Bacillus subtilis Cells

“…Moreover, E. coli EF-G is known to be phosphorylated during phage T7 infection (20), and it was suggested earlier that a B. subtilis EF-G homologue becomes phosphorylated during the sporulation process (16). It is therefore an attractive possibility that the dynamic control of the phosphorylation state of EF-G serves a regulatory role in stationary-phase B. subtilis cells, and that disruption of this control contributes to the observed phenotypes of the prpC and prkC mutants.…”

Cold shock stress-induced proteins in Bacillus subtilis

“…It is interesting that trigger factor in E. coli, which possesses prolyl isomerase activity, is a CIP (23), which leads to the assumption that isomerase activity is important at low temperatures. The Csi12P protein, which was identified as a main CIP, had previously been identified as a protein phosphorylated by ATP during the sporulation of B. subtilis (35). The encoding gene, orfU, is cotranscribed with tsr, which lies downstream of spo0F (52).…”

Nitrogen control in bacteria.

“…The sequence of the outB product shows 52% identity with that of the E. coli ammoniadependent NAD synthetase encoded by nadE (2,263), and while the mechanism of action of OutB is not known, the protein has some similarity to the receiver domains of twocomponent regulatory systems. It has also been shown to be phosphorylated when stationary-phase cell extracts are incubated with [␥-32 P]ATP (177), suggesting that this protein might respond to an environmental signal.…”