This is a competitive revision proposal in response to Notice Number NOT-OD-09-058. The Notice Title is: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. The long-term objective of the parent grant is to elucidate the molecular signaling mechanisms of the chemoreceptors that mediate chemotactic behavior in E. coli. These transmembrane receptors form stable ternary complexes with two cytoplasmic proteins - CheA, a histidine autokinase, and CheW, which couples CheA to chemoreceptor control. The receptor signaling complexes form large clusters at the cell poles and operate in a highly cooperative manner to amplify small chemical stimuli into large motility-controlling signals. Our studies address two key mechanistic questions: How do receptor molecules control and amplify CheA output signals? How do receptor molecules couple input stimulus information to output signal control? The main subject of these studies is the serine chemoreceptor, Tsr. Our principal approach has been to isolate or construct mutant Tsr proteins and to characterize their signaling properties. Our understanding of the signaling defects in mutant receptors is limited by the measurement tools at our disposal. The objective of this competitive revision is to implement a recently-developed in vivo method for measuring receptor-regulated kinase activity. The method involves FRET (F"rster or fluorescence resonance energy transfer) microscopy on populations of several hundred cells, which are subjected to attractant or repellent stimuli to gauge their signaling responses. The technology is reasonably simple and will allow precise analysis of signaling behaviors in the large collection of mutant receptors generated by the parent project. PUBLIC HEALTH RELEVANCE: Bacterial chemotaxis, the movement of cells toward or away from chemicals, plays important roles in the formation of microbial communities such as biofilms and in the establishment of beneficial symbioses and harmful infections. Better understanding of the molecular mechanisms of stimulus detection and motor response during bacterial chemotaxis should lead to new strategies for augmenting the beneficial behaviors of bacteria and for hindering or reversing their deleterious activities.