Genetic and Biochemical Studies of Phob Activation

McCleary, William

Abstract

The objective of the proposed research is to understand how signals are transmitted through the PhoB-PhoR two-component signal transduction pathway in Escherichia coli. Two-component signal transduction systems control many aspects of the physiology of microorganisms. The phosphate signaling system is present in many bacteria and provides a model for understanding other phosphorylation-based signaling cascades. The phosphate signaling pathway is comprised of a phosphate transporter, PstSCAB; an auxiliary protein of unknown biochemical function, PhoU; the histidine kinase, PhoR, which receives and processes environmental signals; and the response regulator, PhoB, which functions as a transcriptional activator. PhoB is a multi-domain protein whose N-terminal domain becomes phosphorylated on an aspartate residue and whose C-terminal domain binds DNA and interacts with RNA polymerase to activate transcription. The proposed experiments will employ genetic and biochemical techniques to identify amino acid residues that are involved in the propagation of the aspartyl phosphorylation-based signal from the receiver domain to the output domain. In addition, the research will also investigate the 6-7 amino acid linker segment that connects the two domains by conducting site-directed mutagenesis experiments and analyzing the phenotypes of the mutants. Attempts will be made to understand the role of each residue in the linker region. Very little is known about the PhoU protein. Its domain structure will be investigated by using partial proteolysis experiments and through a genetic deletion analysis. PhoU's cellular localization will be determined using GFP-PhoU fusion proteins and/or through immunoelectron microscopy. In addition, the amounts of PhoU, PhoR, PhoB, and the PstSCAB transporter under different growth conditions will be determined through quantitative Western blot analysis. The effects of under- and over-expression of PhoU will also be determined in a phoU minus genetic background. The proposed work is important because these signaling proteins are essential for bacteria to survive changing environments. This feature, combined with their absence in higher eukaryotes, makes them targets for the development of new antibiotics. An increased understanding of these signal transduction proteins will assist in the rational design of drugs to combat pathogens.