Synergistic kinetic interactions between components of the phosphorelay controlling sporulation in Bacillus subtilis.

The four individual phosphotransfer steps in the multicomponent phosphorelay system controlling sporulation in Bacillus subtilis have been characterized kinetically using highly purified samples of the individual protein components in vitro. The autophosphorylation of KinA is the initial occurrence, and a divalent metal ion is required. KinA-mediated phosphotransfer, which displays a 57,000-fold preference (kcat/Km) for catalysis of Spo0F-P formation relative to Spo0A-P formation, is shown to proceed via a hybrid ping-pong/sequential mechanism with pronounced (> or = 40-fold) substrate synergism by Spo0F of KinA autophosphorylation. In addition, evidence is presented for formation of an abortive KinA.Spo0F complex. Kinetic parameters derived for Spo0F-P and Spo0A as substrates for Spo0B, the second phosphotransferase in the phosphorelay chain, indicate that Spo0B-mediated production of Spo0A-P is 1.1-million-fold more efficient (kcat/KSpo0A) than the direct KinA-mediated process. A rationale is presented for a four component cascade as the means for controlling sporulation, which focuses on the utility of synergistic interactions among the phosphorelay components that may be modulated by environmental stimuli.

Multiple protein-aspartate phosphatases provide a mechanism for the integration of diverse signals in the control of development in B. subtilis.

The initiation of sporulation in B. subtilis is regulated by the Spo0A transcription factor, which is activated by phosphorylation to control developmental switching from the vegetative to the sporulation state. The level of phosphorylation of Spo0A is regulated by the phosphorelay, a signal transduction system based on the protein-histidine kinase-response regulator two-component paradigm. To initiate sporulation, the cell must recognize and interpret a large variety of environmental, metabolic, and cell cycle signals that influence the phosphorylation level of Spo0A. We describe here a family of protein-aspartate phosphatases with activity on Spo0F approximately P, a response regulator component of the phosphorelay, that provide a mechanism for signal recognition and interpretation. These phosphatases function to drain the phosphorelay, lower Spo0A approximately P levels, and prevent sporulation. The integration of diverse environmental signals that affect the initiation of sporulation likely occurs through the competition between opposing activities of protein kinases and protein phosphatases.

Control of the initiation of sporulation in Bacillus subtilis by a phosphorelay.

Sporulation in Bacillus subtilis is a developmental process induced as a response to nutritional stress. Activation of sporulation-specific gene transcription is under the control of the spoOA gene product. The SpoOA protein and the SpoOF protein are both homologous to response regulator proteins of two-component regulatory systems which control bacterial responses to a variety of environmental challenges. Response regulators are activated by specific kinases which phosphorylate them. In this study, it was shown that phosphorylation of SpoOA occurs via a phosphotransferase which is the product of the spoOB locus. The phosphodonor in this reaction is the phosphorylated form of SpoOF. It is postulated that SpoOF acts as a secondary messenger that can be phosphorylated by a variety of kinases depending on the particular environmental stress. The series of phosphate transfer reactions in this system is called a phosphorelay. The end product of this series of reactions is SpoOA approximately P which is shown to have greater affinity for the DNA target, the OA box, of SpoOA on the abrB promoter than the unphosphorylated form. SpoOA approximately P, but not SpoOA, was shown to be an activator of transcription of the spoIIA operon which codes for the sporulation-specific sigma factor sigma F. Thus, the initiation of sporulation is dependent on SpoOA approximately P which arises through the phosphorelay and which acts as a transcription factor to repress certain genes, e.g. abrB, and activate others, e.g. spoIIA.

Detection of actin and localization of phytochrome in the green alga Mougeotia by monoclonal antibodies.

In order to get insight into the topological relationship of phytochrome and the actin cytoskeleton in Mougeotia, phytochrome was localized by indirect immunofluorescence in fixed protoplasts of Mougeotia with the monoclonal antibody Z-3B1, raised against purified Zea mays phytochrome (Schneider-Poetsch et al 1988, Planta 173, 61-72). So far no detection of phytochrome in the immunoblot was possible by this antibody, in contrast to the detection of actin by the monoclonal anti-actin C4 (Lessard 1988, Cell Motil. Cytoskeleton 10, 349-362). Preliminary results are presented on attempts to enrich plant factors which interfere with the G-/F-actin equilibrium, as probed by the viscometric falling ball assay.