Abstract
Bacteria have evolved macromolecular machineries that secrete effectors and toxins to survive and thrive in diverse environments. The type VI secretion system (T6SS) is a contractile machine that is related to Myoviridae phages. The T6SS is composed of a baseplate that contains a spike onto which an inner tube is built, surrounded by a contractile sheath. Unlike phages that are released to and act in the extracellular medium, the T6SS is an intracellular machine inserted in the bacterial membranes by a trans-envelope complex. This membrane complex (MC) comprises three proteins: TssJ, TssL and TssM. We previously reported the low-resolution negative stain electron microscopy structure of the enteroaggregative Escherichia coli MC and proposed a rotational 5-fold symmetry with a TssJ:TssL:TssM stoichiometry of 2:2:2. Here, cryo-electron tomography analysis of the T6SS MC confirmed the 5-fold symmetry in situ and identified the regions of the structure that insert into the bacterial membranes. A high resolution model obtained by single particle cryo-electron microscopy reveals its global architecture and highlights new features: five additional copies of TssJ, yielding a TssJ:TssL:TssM stoichiometry of 3:2:2, a 11-residue loop in TssM, protruding inside the lumen of the MC and constituting a functionally important periplasmic gate, and hinge regions. Based on these data, we revisit the model on the mechanism of action of the MC during T6SS assembly and function.
