Abstract
The β-barrel assembly machine (BAM) is an essential protein complex that folds and inserts outer membrane β-barrel proteins (OMPs) into the bacterial outer membrane. The BAM complex contains the essential BamA OMP with five soluble polypeptide transport-associated (POTRA) domains, which scaffold the essential BamD lipoprotein and the non-essential lipoproteins BamB/C/E. The importance of each BAM component has been investigated primarily using cell-based phenotypic assays, and structural data have revealed insights into the role of the BamA β-barrel in OMP folding. However, in vitro quantitative analysis for the function of each BAM component has been challenging. We describe the development of a fluorescent reporter of OMP folding, tOmpA-A488, which we use to obtain single-turnover kinetic parameters for wildtype BAM complex in vitro . We observe a k (fold) of 0.78 ± 0.15 min (-1) and approximate substrate affinity of 3.1 ± 1.1 µM consistent with estimates of in vivo requirements. We also find that, contrary to prevailing models, POTRA domain deletions that include POTRA3, which is essential in cells, do not drastically impact activity. This indicates that the first three POTRA domains do not play a major role in binding or folding OMPs under single-turnover conditions, suggesting a different role in cells. Furthermore, we find that BamA alone is inactive in E. coli lipid liposomes, and the gain-of-function mutant BamA E470K does not rescue activity in vitro . The single-turnover kinetics enabled by the fluorescent reporter presented here defines a robust platform for quantitative evaluation of the folding activity of wildtype and mutant BAM complexes. SIGNIFICANCE: The folding of outer membrane proteins (OMPs) in Gram-negative bacteria requires the essential β-barrel assembly machine (BAM). By developing a fluorescent OMP folding reporter, we have unlocked insight into BAM activity in vitro , opening the door for rigorous evaluation of BAM mutants and putative inhibitors. We also discovered that, contrary to current models, the BamA POTRA1-3 domains do not contribute significantly to catalysis, despite being essential for bacterial growth. We propose that, consistent with biochemical, structural, and live cell imaging, the in vivo role of BamA POTRAs1-3 is to support a connection with the Sec translocon to form a transperiplasmic bridge, as well as provide a docking site for the chaperone SurA for substrate delivery.