In situ investigation of bacterial secretion systems using cryo-electron tomography

PROJECT SUMMARY Bacterial secretion systems encompass a wide range of macromolecular nanomachines which are involved in a variety of functions crucial for cell viability. Despite their significance, our understanding of how the majority of secretion systems known to date are built and function inside a bacterial cell remains limited. In part, the latter is caused by the lack of high-resolution structural information pertaining to intact cells. At our lab, we aim at utilizing cutting-edge microscopy techniques, such as cryo-electron tomography (cryo-ET), and a number of other associated methodologies, to study the assembly, disassembly, and function of bacterial secretion systems. Those techniques enable direct visualization of macromolecular complexes within intact, hydrated- frozen bacterial cells at nanometer resolution. As a start-off, we will focus on the following two specific secretion systems: 1) the bacterial motility nanomachine, the flagellar motor (an example of type III secretion system), and 2) the type IV secretion system (T4SS), the latter being involved in a wide range of processes such as secreting effector proteins and genetic material transfer between bacterial cells (bacterial conjugation). Using cryo-ET, we will unveil the mechanism of how exactly bacteria assemble their seemingly complex flagellar motors in stepwise fashion, and how they disassemble them under harsh environmental conditions to save energy. Jointly utilizing cryo-ET and cryo-light microscopy, we will subsequently decipher the role of the T4SS in the process of intercellular DNA transfer, visualizing the accompanying T4SS structural changes. The results of our studies will deepen the understanding of how, in general, macromolecular complexes assemble and perform their functions under various environmental conditions.