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 similar to P, the key trans cription factor controlling the initiation of sporulation. A search fo r insertion mutants affected in activation of KinB-dependent sporulati on led to the identification of the Igt locus encoding the lipoprotein glyceryltransferase required for the lipid modification of prolipopro teins before their cleavage and translocation across the cytoplasmic m embrane. In parallel, a putative lipoprotein signal peptide cleavage s ite was detected in KapB, known to be strictly required for KinB-media ted sporulation and located downstream of KinB in a single transcripti on unit. Using PhoA peptide fusions, we have shown that KapB signal-pe ptide can direct active alkaline phosphatase to the outer surface of t he cytoplasmic membrane in an LGT-dependent manner, strongly suggestin g that KapB is a lipoprotein tethered to the outer face of the cytopla smic membrane via a lipid anchor. As KapB proved to be dispensable for expression of the kinBkapB operon, a chimeric kinase was built consis ting of KinA sensor domain fused to KinB kinase domain (KinA'-'B) to a ssess (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 activatio n of the phosphorelay by the KinA'-'B chimera and that KinA'-'B phosph orylates SpoOF directly in vitro, Models for the role of KapB in regul ating KinB activity are discussed.