2-component signaling is the principal form of signal transduction in prokaryotes with distinctive examples in eukaryotes. At the end of logarithmic growth, the "transition state" Bacillus cell receives multiple signals from the environment that are simultaneously reporting conditions such as temperature, cell density, nutrition availability and oxygen tension. These signals are processed to determine the most appropriate gene expression and metabolic response for survival. The hypothesis we are testing is that "The processing of the multiple signals is accomplished by regulatory networks involving multiple 2-component systems that function to establish dependencies or hierarchies between systems. Cross-system interaction between regulons provides a mechanism for signal integration and amplification, a mechanism which results in fine tuning of a given response that accommodates the entire signal input experienced by the organism at any 1 time." The Pho signal transduction network is comprised of at least 3 2-component systems: PhoP-PhoR, ResD-ResE and Spo0A, and global stress, catabolite, transition state and developmental regulators. We will examine the role of the Spo0A~P-AbrB pathway in the Pho signal transduction network by determining which of 6 phoPR promoter(s) respond to direct AbrB binding and if AbrB also has an indirect role via ScoC. We will determine what unknown regulatory circuits control phoPR P5 promoter regulation in the absence of a second Pi starvation global regulator, SigB. We will examine the positive feedback loop that is essential for resABCDE transcription (ResD and ResE production) during phosphate starvation by analyzing mutations that bypass the requirement for PhoP in resA transcription and via reconstruction of resA transcription in vitro to understand the essential but insufficient role of both PhoP and ResD. We will examine the cross-system interaction via the positive feedback loop that is essential for full induction of the Pho regulon by determining the role of upstream regulator, ResD, in controlling the Pi deficiency signal and/or modulation of that signal. We will determine how reduced menaquinones that inhibit the autophosphorylation of PhoR in vitro, modulate the phosphate deficiency signal in vivo and ask if redox-reactive cysteines play a role. These studies will contribute to the knowledge base of two-component signal transduction systems of gram-positive bacteria as targets for antimicrobial therapy.