Electron Cryotomography of Bacterial Secretion Systems

Abstract

The envelope of bacterial cells consists of at least one‐and often two‐membranes, a cell wall, and possibly a surface layer. This envelope allows cells to differentiate themselves from their environment and handle the resulting osmotic pressure, but it also presents a significant obstacle. Anything a cell wishes to export, from a motility appendage to a plasmid, needs to be ushered across this barrier. To accomplish this, bacteria have evolved a battery of secretion systems. Secretion systems are often constructed from dozens of protein building blocks embedded in the cell's envelope. The size, complexity, and location of these machines make them a particular challenge for structural characterization. High‐resolution structure determination techniques such as X‐ray crystallography and transmission electron microscopy (TEM)‐based single‐particle reconstruction (SPR) require objects to be purified from their cellular environment. This is problematic for membrane‐associated proteins, which embed in the lipid bilayer by means of exposed hydrophobic patches. These patches can be protected during purification by adding detergents to the solvent, but often structural alterations still occur. Secretion systems are also unusually large targets and frequently lose peripheral or loosely‐associated components during purification. In addition, they often cross two membranes and are avidly linked to the cell wall.