The beta-barrel outer membrane protein (OMP) is the major component of the outer membrane of gram-negative bacteria. OMPs come in a variety and play various crucial functions including nutrient exchange, retaining membrane integrity, toxin export, or forming pilus for host-pathogen interaction.
Most of the OMPs are assembled (insertion + protein folding) into the outer membrane by the Beta-barrel Assembly Machinery (BAM) complex. In E. coli, the BAM complex contains BamA, BamB, BamC, BamD, and BamE. The BamA and BamD are essential for cell viability, but other subunits are necessary for full performance. The well-studied central molecular mechanisms of the BAM complex have revealed the function of BamA and BamD. However, the role of the other accessory subunits in the assembly process remains unclear.
To directly assess the function of accessory subunits of the BAM, we employed the in vitro reconstitution assay using E.coli Microsomal Membrane (EMM). Because the EMM contained intact the BAM complex, the EMM assembly assay can directly assess the assembly efficiency, excluding secondary effects such as stress response. We isolated EMM from the WT, bamB, bamC, or bamE deletion strains, respectively. We prepared eight different substrate OMPs classified according to two indexes, the number of beta-strands (8-20 strands)or oligomeric states (monomer, dimer, or trimer). BamE was the most prominently required in all OMPs assembly. Our complex stability analysis revealed that BamE contributed to the stabilization of the BamA-BamD interaction, implying that BamA-D destabilized by lack of BamE impaired all of OMPs assembly. BamB was also required in all OMPs but more effective in the oligomeric OMPs. A previous study showed that BamB stimulated formation gathered BAM complex termed “assembly precinct”, indicating that assebmly precinct was responsible for OMPs origomarization. BamC did not affect the assembly efficiency of all OMPs, but the lack of BamC showed the accumulation of the assembly-intermediate form, indicating that BamC stimulated substrate release from the BAM complex.
The list of model substrates can be adjusted to about more than 80% of the OMPs. This study enable us to know which subunits should be targeted to inhibit the assembly of the protein of interest.