BPS2026 – Electrostatics within the BamA lumen influence OMP folding

Proper assembly of the outer membrane (OM) is essential to the survival and pathogenicity of diderm bacteria. The β-barrel assembly machinery (BAM) complex facilitates the accurate and precise insertion of outer membrane proteins (OMPs) of various size, function, and complexity into the OM. Electrostatics have long been associated with this process, due to the asymmetrically charged nature of the OM, alongside the “positive outside” rule for OMP topology. Although the exact mechanism for BAM-mediated OMP assembly remains ambiguous, the BAM complex must be adeptly equipped to manage those charged residues, particularly the BamA subunit which physically interacts with the folding OMP. Inside the BamA lumen the spatial organization of charged residues can be categorized into charge site 1 (CS1) toward the front of the barrel with a net negative charge, and CS2 at the back of the barrel with a net positive charge. The necessity for such charged residues has been screened via a vancomycin sensitivity assay in vivo, revealing an incremental loss of membrane integrity as the net charge of CS1 was increased. The double mutant BamA E470KD512K was found to contain the most significant defects in membrane permeability despite being able to maintain cell viability, suggesting the BAM complex remained productive. The rate of OMP folding in vitro was also significantly perturbed in BamA E470KD512K compared to that of Wt. Moreover, a clear relationship was observable between the net charge at Site1 in BamA and the positive external patch (PEP) residues in the folding OMP. In summary, the negative charge at CS1 inside the BamA lumen appears to play a specific role within OMP folding mechanism.