In humans, P. aeruginosa infections are a cause of significant motility in Cystic Fibrosis patients. In P. aeruginosa there are many features of the bacterial cell that localize to the poles, and also contribute to the virulence of this organism. There are four chemotaxis-like systems, three of which are localized to the cell poles. Despite the similarities between these two-component signal transduction systems, they not only control different motility apparatus and virulence factors, they also have distinct sub-cellular addresses. The mechanisms of achieving and maintaining these distinct sub-cellular addresses is unknown, and characterizing this is one of the goals of this research. To this end, I will be studying the soluble methyl accepting chemotaxis proteins (IVICPs), BdIA and IVIcpS, and identifying their cognate chemotaxis systems, as well as their landmark or anchor proteins. This will be achieved using fluorescence microscopy, mutagenesis, and yeast two-hybrid assays. It is known that methylation of MCPs in E. coll affects the localization of some, but not all, MCPs. The mechanism of MCP methylation in P. aeruginosa remains unknown, as does the effect of methylation on MCP localization. These studies will comprise the second aim of this research, and will include mutagenesis, co-purification and methylation assays, along with immunofluorescence. Finally, I will identify and characterize novel proteins that show spatial sequestration within the P. aeruginosa ceil. I have developed a strain that divides asymmetrically, resulting in rods and minicells. The membranes of the these two cell types are being subjected to comparative proteomics using ITRAQ. As histidine kinases have been reported to affect the asymmetry of other bacteria, and P. aeruginosa is predicted to encode 63 histidine kinases, the emphasis of my research will be on histidine kinases with a polar localization pattern. Ultimately, I will be studying how polarity is achieved and maintained within the P. aeruginosa cell, and the effect these polar proteins have on not only the internal organization, but also the lifecycle and virulence of P. aeruginosa.
