The importance of the Gram-negative outer membrane is underscored by its physical location: because it forms the bacterial cell surface, it is the first point of contact for antimicrobial agents and host immune cells. From the signals that regulate gene expression, to the insertion of new cell surface proteins, rapid and precise remodeling of the proteome is essential for the bacterium to become a successful pathogen. As the final step in this cellular process, the β-barrel assembly machinery (BAM) mediates outer membrane protein biogenesis and central to its operation is the BAM complex and the translocation and assembly module (TAM). While the BAM complex is embedded within the outer membrane only, the TAM is an intermembrane-spanning nanomachine that is particularly important for the assembly of complex substrates, especially those involved in type 5 secretion systems and chaperone-usher pathways [1-3].
Fundamental to the function of the BAM are two proteins belonging to the TpsB/Omp85 superfamily: BamA in the BAM complex and TamA in the TAM. Some members of this superfamily contain a lateral gate between the first and last transmembrane β-strands that, upon separating, would allow direct insertion of folded protein into a lipid environment. Therefore, due to the presence of a lateral gate in BamA [4], BamA was initially thought to play the major enzymatic role in membrane protein biogenesis, with TamA playing a supporting role through destabilisation of the protein-lipid interface. However, structural analysis of TamA has revealed the likely presence of its own lateral gate and preliminary studies have demonstrated that the β-barrel domain of TamA directly engages its substrate [3,5]. Using a series of site-directed cysteine mutants to lock the putative lateral gate and prevent substrate folding and insertion, TamA was found to contain a lateral gate that was necessary for protein insertion into the membrane.