Bacteria have outer membrane proteins of at least three distinct architectures: β-barrel proteins, lipoproteins and secretins. The proteins of β-barrel architecture are of diverse function including autotransporters, ushers, intimins and pores, and are assembled by the β-barrel assembly machinery (the BAM) with assistance from the translocation assembly module (the TAM). Lipoproteins carry covalently attached lipids and are assembled into the outer membrane by the Lol machinery. Secretins assemble to form channels in the outer membrane, with examples including the Type II Secretion Systems (T2SS), Type III Secretion Systems (T3SS) and Type IV fimbrae. These sophisticated molecular machines drive the translocation of large cargoes across the bacterial outer membrane. What currently remains unclear is the identity of the assembly machinery for the T2SS, T3SS and other related molecular machines.

We have used comparative sequence analysis to find that distinct forms of T2SSs can be distinguished based on the characteristics of their secretin subunits. Detailed analysis of these secretins, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the ‘pilotin’ chaperone that mediates secretin transport and assembly into the outer membrane. We determined the crystal structure of the AspS pilotin found in Vibrio cholerae, Escherichia coli and Shigella, showing it to be functionally equivalent and yet structurally unrelated to the PulS pilotins found in Klebsiella and related bacteria. We developed new FlAsH-based assembly assays to find that AspS binds to a specific targeting sequence in the Vibrio-type secretins, enhancing the kinetics of secretin transport and assembly. The new assay system provides a reliable biochemical approach to identify and characterize the components of the assembly machinery, thereby defining the pathway through which T2SS and other related molecular machines are built in bacterial outer membranes.