The fundamental problem of protein transport across biological membranes was solved by the evolution of sophisticated nanomachines that physically puncture the membranes by sharp cargo-loaded needles. These nanomachines are used by bacteria to deliver toxins into their competitors or host cells. Our project aims at mechanistic understanding of the nanomachine’s mode of action. Type VI Secretion System (T6SS) is a poorly understood nanomachine that many Gram-negative bacteria use to kill both bacterial and eukaryotic cells. The main goal of this proposal is to address a fundamental question about T6SS: where does the force needed to secrete toxins into target cells come from? In frame of this proposal, we will use state-of-the-art electron microscopy techniques to obtain high resolution structures of diverse T6SS components from different organisms. We will test hypotheses about mechanisms of assembly by mutagenesis of T6SS components in model organisms and evaluation of the efficiency of substrate secretion and target cell killing. Overall, this project will provide a detailed understanding of how T6SS delivers proteins into target cells. Understanding of bacterial cell biology opens avenues for new approaches to inhibit bacterial pathogenesis. This proposal is aimed at one of the fundamental processes that bacteria use to influence their environment and thus has a potential to contribute to an ongoing effort to combat bacterial infections.