The biogenesis of the outer membrane of bacterial pathogens is a significant pathway in bacterial cell biology. Outer membrane protein assembly is mediated by the beta-barrel assembly machinery, the core component of which is the BAM complex. The translocation and assembly module (TAM) is also capable of assembling outer membrane proteins, but its function has remained enigmatic. To determine the function of the TAM, a pulse chase assay was developed to characterise the specific contributions of the TAM and the BAM complex for a range of outer membrane protein substrates.
Using this technique, eight fimbrial ushers were assigned to the TAM-dependent category of outer membrane proteins. Of these, the usher protein FimD, from which Type I fimbriae emanate, was found to contain an unusual extracellular loop that was readily proteolytically degraded on addition of exogenous proteinase K. Because distinct proteolytic fragmentation "fingerprints" were generated depending on the presence or absence of the TAM, this provided a powerful tool to dissect the contribution of the TAM toward FimD biogenesis. The TAM was thereby demonstrated to initiate β-barrel assembly from the C-terminal end of the substrate. While the BAM complex was also capable of assembling FimD, it was significantly less efficient because it initiated β-barrel assembly from a central region of the protein. The assay has now been adapted to study a diverse range of outer membrane proteins, leading to the conclusion that the TAM is generally important for the assembly of outer membrane proteins with complicated transmembrane topologies.