Project Summary Type III secretion systems (T3SSs) are nanomachines that are dedicated to protein export in Gram-negative bacteria. T3SSs share the same morphology and overall structure and can be functionally classified into two evolutionary-related classes: the flagellar T3SS, which promotes bacterial locomotion and motility enabled by the flagellum, and the pathogenic T3SS, which uses the injectisome to transport virulence proteins into human or animal host cells. Over the past decade significant progress has been made in understanding the structure, assembly and the mode of operation of T3SS. The principal structural building proteins of the flagellum and the injectisome, from the basal body embedded in the inner and outer bacterial membrane to the tip of the filaments protruding from the cell surface, and the cytosolic components have been extensively characterized. Flagellar proteins and virulence factors (effectors, needle proteins and translocators) form tight complexes with T3S-dedicated chaperones in the cytosol and are subsequently targeted specifically to the export apparatus located at the membrane. Powered by ATP and the proton motive force, the flagellar proteins and bacterial effectors are then translocated through the channel. Fundamental questions about the functional mechanisms underpinning these processes remain unaddressed. We propose to use an integrated approach combining structural, dynamic, thermodynamic, kinetic, biochemical and in vitro and in vivo functional assays to provide insight into the early events of the translocation process that involve the recognition mechanisms by chaperones, targeting mechanisms to the ATPase and the sorting platform, selection mechanisms that control the hierarchical transport of the filament-forming proteins and the effectors and ultimately the assembly of the cytosolic part of the machinery. We have extensively characterized over the last years T3S protein components from the enteropathogenic Escherichia coli (EPEC), the major cause of infantile diarrhea and child mortality worldwide, as well as from Salmonella sp. commonly associated with food poising. We present here novel findings supporting very intriguing hypotheses about the mechanisms used by T3SSs to carry out their function. The specific aims are designed to provide atomic-resolution insight into (i) the mechanisms of specific interactions between and among key T3S proteins, (ii) the mechanistic basis for targeting of T3S proteins to the export gate, (iii) the “recognition” and “secretion” signal, and (iv) the assembly and operation mechanisms of the export gate and ultimately of the entire T3S machinery.