Bypassing BamD essentiality by mutations in a non-essential substrate

The β-barrel assembly machinery (Bam) is essential for assembling all transmembrane β-barrel outer membrane proteins in gram-negative bacteria. The Bam complex consists of the central β-barrel protein BamA and accessory Bam lipoproteins, including the widely conserved and essential BamD. BamD is assumed to be an essential regulator of OMP assembly, as its absence causes a global defect in OMP biogenesis. Here, we challenge this view by demonstrating that BamD essentiality is both conditional and substrate specific. In Escherichia coli, its function can be bypassed by preventing BamA jamming by a single, non-essential substrate RcsF. Our findings suggest that BamD plays two distinct roles in the Bam complex. It prevents improper RcsF engagement that can jam BamA, and it kinetically enhances BamA-mediated OMP assembly. Contrary to prevailing models, we demonstrate that the second function in general OMP assembly is not essential. We report a genetic background in which each Bam lipoprotein is dispensable for viability, providing a powerful new system for investigating their functions in OMP assembly in the context of unmodified, wild-type BamA.IMPORTANCEThe β-barrel assembly machinery (Bam) complex assembles all outer membrane proteins (OMPs) and is conserved and essential across all gram-negative bacteria. While BamA is critical for the folding and insertion of OMPs into the outer membrane, BamD is also considered essential for OMP assembly because its loss leads to a global reduction in OMP levels and cell death. Our results show that BamD is important but not essential for general OMP assembly. In Escherichia coli, BamD's essentiality arises from its role in preventing a single challenging substrate from jamming BamA and indirectly abolishing OMP assembly. This work challenges long-standing assumptions about BamD's function, underscores the substrate-specific roles of accessory Bam components, and offers important new considerations for interpreting genetic studies of the Bam complex.