# A fluorescent reporter and single-turnover kinetics reveal insight into BAM complex function

## TL;DR

A fluorescent reporter and kinetic analysis reveal new insights into how the BAM complex folds outer membrane proteins in bacteria.

## Contribution

A new fluorescent reporter and single-turnover kinetics provide quantitative insights into BAM complex function and subunit roles.

## Key findings

- BamA alone is inactive, but its activity is rescued by BamD or BamB.
- POTRA1-3 domains of BamA do not significantly contribute to OMP folding under single-turnover conditions.
- BamAB and BamAD subcomplexes show activity similar to the full BAM complex.

## Abstract

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. Furthermore, we found that BamA alone is inactive, but its activity can be rescued by either the essential BamD or the nonessential BamB, suggesting partially redundant roles in stabilizing active BamA conformations. We also found that, contrary to current models, the BamA POTRA1-3 domains do not contribute significantly to catalysis, despite being essential for bacterial growth. Thus, our quantitative approach allows for robust testing of mechanistic BAM models.

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 nonessential 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, bOmpA-A488, which we use to obtain single-turnover kinetic parameters for wild-type BAM complex in vitro. We observe a kfold of 0.78 ± 0.15 min−1 and approximate substrate affinity of 3.1 ± 1.1 µM consistent with estimates of in vivo requirements. Furthermore, we find that while BamA alone is inactive in Escherichia coli lipid liposomes, BamAB and BamAD subcomplexes have activities similar to that of the holoBAM complex. This suggests that OMP folding and insertion is catalyzed by BamA while the accessory lipoproteins maintain BamA in a catalytically competent conformation. 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.