The Escherichia coli β-barrel assembly machinery is sensitized to perturbations under high membrane fluidity.

Integral β-barrel membrane proteins are folded and inserted into the Gram-negative bacterial outer membrane by the β-barrel assembly machine (BAM). This essential complex, comprised of a β-barrel protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE, resides in the outer membrane, a unique asymmetrical lipid bilayer that is difficult to recapitulate in vitro Thus, probing BAM function in living cells is critical to fully understand the mechanism of β-barrel folding. We recently identified an α-BamA monoclonal antibody, MAB1, that is a specific and potent inhibitor of BamA function. Here we show that the inhibitory effect of MAB1 is enhanced when BAM function is perturbed by either lowering the level of BamA or removing non-essential BAM lipoproteins: BamB, BamC, or BamE. Disrupting BAM reduces BamA activity, increases OM fluidity, and activates the σE stress response, suggesting the OM environment and BAM function are interconnected. Consistent with this idea, increasing membrane fluidity through changes in the growth environment or alterations to the lipopolysaccharide in the outer membrane is sufficient to provide resistance to MAB1 and allow BAM to tolerate these perturbations. Substitutions in BamA at positions in the outer membrane spanning region or the periplasmic space that are remote from the extracellular MAB1 binding site also provide resistance to the inhibitory antibody. Our data highlight that the outer membrane environment is a critical determinant in the efficient and productive folding of β-barrel membrane proteins by BamA. IMPORTANCE BamA is an essential component of the β-barrel assembly machine (BAM) in the outer membrane of Gram-negative bacteria. We have used a recently described inhibitory α-BamA antibody, MAB1, to identify molecular requirements for BAM function. Resistance to this antibody can be achieved through changes to the outer membrane or by substitutions in BamA that allosterically affect the response to MAB1. Sensitivity to MAB1 is achieved by perturbing BAM function. By using MAB1 activity and functional assays as proxies for BAM function, we link outer membrane fluidity to BamA activity demonstrating that increasing membrane fluidity sensitizes cells to BAM perturbations. Thus, the search for potential inhibitors of BamA function must consider the membrane environment in which β-barrel folding occurs.