DESCRIPTION: Two-Component signaling is the principle form of signal transduction in procaryotes with distinctive examples in eucaryotes. Considerable interest in Bacillus subtilis has been focused on identifying the environmental and physiological signals that initiate the process of sporulation. Nutritional and cell cycle related signals produced during late growth are processed by interconnected regulatory pathways that control which genes are expressed, often culminating in the initiation of sporulation. The PI's characterization of the B. subtilis alkaline phosphatase (APase) multigene family has led to the hypothesis that different environmental signals are received by specific two-component systems that integrate the incoming information to determine the response of B. subtilis to declining phosphate availability. The present proposal is designed to understand how three separate two-component systems (PhoP-PhoR, ResD-ResE, and SpoOA), each receiving their specific environmental signals, communicate to achieve the final Pho response. The PI is in the fortunate position of having the basic architecture of the signal transduction circuitry in place. This provides a framework for testing and refining her hypothesis. Parallel pathways positively regulate the Pho response via PhoR-PhoR. One pathway includes the ResD-ResE system, while the other involves a transition state regulator, AbrB. The SpoO system represses the Pho response by negatively regulating both pathways. Continuing genetic studies will be used to further identify intermediates in the ResD to PhoP-PhoR activation pathway and biochemical studies are planned to probe the interactions between them. The repressor function of SpoOA on the AbrB activation pathway is well udnerstood. The PI will use genetic and biochemical techniques to determine the mechanism of SpoOA repression of the resA promoter which is responsible for the transcription of the resDE genes. She will use biochemical approaches to explore two domains in the PhoR for pssible signaling mechanisms. Promoters known to require PhoP for activation will be analyzed for direct interaction with PhoP and for possible function of conserved sequences within these promoters. This proposal has a number of phases. The first is to employ genetic selections or screenings to identify new components of this complex, interacting regulon. There are four RNA start sites in the phoPR operon promoter region as determined by primer extension, and a detailed quantitative analysis of these in various mutant backgrounds will determine which sites are regulated by which pathway. A deletion analysis is also planned. To characterize PhoP and PhoR themselves, gel retardation and DNA footprinting experiments are all planned to assess direct regulation on various Pho regulated promoters. PhoR contains two regions where interaction with external (the region between two membrane-spanning regions) and internal (the region between the second membrane-spanning region and the histidine kinase domain) signals could occur. These will be mutated or substituted with corresponding domains of other sensor kinases and the effect of the induction upon phosphate starvation assessed. The interacting proteins will be sought using cross-linking.