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

Area of Science:

• Biochemistry and Molecular Biology
• Bacterial Outer Membrane Protein Biogenesis
• Enzyme Kinetics

Background:

• The β-barrel assembly machine (BAM) complex is essential for folding and inserting outer membrane proteins (OMPs) into the bacterial outer membrane.
• Previous studies relied on cell-based assays, limiting in vitro quantitative analysis of BAM component function.
• The structure of BAM reveals BamA with five POTRA domains, scaffolding other lipoproteins.

Purpose of the Study:

• To develop an in vitro method for quantitative analysis of BAM complex activity.
• To determine single-turnover kinetic parameters for wildtype BAM complex.
• To investigate the role of specific BAM components, particularly POTRA domains, in OMP folding.

Main Methods:

• Development of a fluorescent reporter, tOmpA-A488, for OMP folding.
• Single-turnover kinetic assays using the fluorescent reporter to measure folding rates and substrate affinity.
• Analysis of wildtype BAM complex and mutant variants (POTRA domain deletions, BamA E470K) in E. coli lipid liposomes.

Main Results:

• Wildtype BAM complex exhibited a folding rate (kfold) of 0.78 ± 0.15 min⁻¹ and substrate affinity of 3.1 ± 1.1 µM in vitro.
• Contrary to prevailing models, POTRA domain deletions, including the essential POTRA3, did not significantly impact in vitro activity.
• BamA alone was inactive, and the gain-of-function mutant BamA E470K did not restore activity in vitro, suggesting essential roles for other BAM components.

Conclusions:

• The developed fluorescent reporter provides a robust platform for quantitative evaluation of BAM complex activity in vitro.
• The first three POTRA domains of BamA may not be primarily involved in OMP binding or folding under single-turnover conditions.
• The in vivo roles of BamA POTRA1-3 likely involve interactions with the Sec translocon and the SurA chaperone for substrate delivery.