The type two secretion system (T2SS) forms a large multi-protein machine spanning both the inner and outer membranes of many Gram negative bacteria.  Bacteria utilize these machines to secrete folded proteins, such as toxins and enzymes, into their extracellular environment. The outer membrane pore of the T2SS is comprised of a protein complex referred to as the secretin ring. The mature ring is made up of 12-15 copies of each secretin monomer but the precise mechanism of secretin assembly and insertion into the outer membrane remains poorly understood.

Secretins often rely on a small lipo-protein, termed pilotin, to catalyze the efficient translocation of secretin monomers from the inner membrane to the outer membrane. In addition to pilotins, other accessory factors may be involved in secretin assembly and stability. We have designed an assay to measure the assembly kinetics of a T2SS secretin from enteropathogenic Escherichia coli E2348/69. The assay provides a means to determine the function of novel accessory proteins, such as candidate pilotins, and to dissect the mechanisms of secretin targeting to the outer membrane for assembly within the outer membrane environment.