KapB is a lipoprotein required for KinB signal transduction and activation of the phosphorelay to sporulation in Bacillus subtilis.

KinB is one of the two major histidine kinases that provide phosphate input in the phosphorelay to produce SpoOA approximately P, the key transcription factor controlling the initiation of sporulation. A search for insertion mutants affected in activation of KinB-dependent sporulation led to the identification of the lgt locus encoding the lipoprotein glyceryltransferase required for the lipid modification of prolipoproteins before their cleavage and translocation across the cytoplasmic membrane. In parallel, a putative lipoprotein signal peptide cleavage site was detected in KapB, known to be strictly required for KinB-mediated sporulation and located downstream of KinB in a single transcription unit. Using PhoA peptide fusions, we have shown that KapB signal-peptide can direct active alkaline phosphatase to the outer surface of the cytoplasmic membrane in an LGT-dependent manner, strongly suggesting that KapB is a lipoprotein tethered to the outer face of the cytoplasmic membrane via a lipid anchor. As KapB proved to be dispensable for expression of the kinBkapB operon, a chimeric kinase was built consisting of KinA sensor domain fused to KinB kinase domain (KinA'-'B) to assess (i) the involvement of KapB in catalysis of the kinase reaction, and (ii) the ability of KinB to phosphorylate SpoOF in vitro. It was shown that KapB is dispensable for both in vivo and in vitro activation of the phosphorelay by the KinA'-'B chimera and that KinA'-'B phosphorylates SpoOF directly in vitro. Models for the role of KapB in regulating KinB activity are discussed.

Identification of a membrane protein involved in activation of the KinB pathway to sporulation in Bacillus subtilis.

The initiation of sporulation in Bacillus subtilis is dependent on the phosphorylation of the Spo0A transcription factor mediated by the phosphorelay and by two major kinases, KinA and KinB. Temporal expression of these kinases was analyzed, and an assessment of their respective contributions to the production of Spo0A-P was undertaken. The results show that KinB is expressed and activated prior to KinA; i.e., the two kinases are solicited sequentially in the sporulation process and are thought to be activated by different signaling pathways. A strategy was developed to isolate mutations specifically affecting the KinB pathway, using the newly improved mini-Tn10 delivery vector pIC333. Several mutants were obtained, one of which carried a transposon in a gene coding for a small integral membrane protein, named KbaA. Inactivation of the kbaA gene appeared to affect KinB activity but not transcription of kinB. A Spo+ suppressor (kinB45) of the kbaA null mutation was isolated in the promoter region of kinB. An eightfold increase of kinB expression levels over wild-type levels was observed in the kinB45 mutant. Thus, overexpression of the kinB-kapB operon was sufficient to overcome the sporulation defect caused by inactivation of kbaA in a KinA- strain. Transcription of kinB was found to be repressed by SinR, while the kinB45 mutant was no longer sensitive to SinR regulation. Implications of these observations on the transcriptional regulation of kinB and the role of KbaA in KinB activation are discussed.

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.