Structural architecture of a hybrid β-barrel assembly machinery BAM-TAM complex in Borrelia burgdorferi.

The β-barrel assembly machinery (BAM) inserts outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria. In well-studied organisms such as Escherichia coli, BAM consists of BamA and four accessory lipoproteins (BamB-E) that coordinate substrate recognition, membrane remodeling, and β-barrel insertion. In the Lyme disease spirochete Borrelia burgdorferi, however, the BAM system is simplified, comprising only BamA (BB0795), BamD (BB0324), and BamB (BB0028), while lacking BamC and BamE. In addition, B. burgdorferi encodes a TamB homolog (BB0794), suggesting a hybrid BAM-TAM organization. Here, we define the structural architecture of the spirochetal BAM-TAM system. AlphaFold3 modeling predicts that BamD binds to BamA periplasmic domains POTRA4-5, while BamB interacts with POTRA1, POTRA3, POTRA4, and POTRA5, forming a closed periplasmic ring distinct from the enterobacterial arrangement. Microscale thermophoresis confirms micromolar-affinity binding of BamD to BamA POTRA3-5, no detectable interaction with POTRA1-3, and loss of POTRA3-5 interaction upon mutation of key interface residues. Crystal structures of BamA POTRA1-2 and POTRA2-3 further define the architecture and flexibility of the periplasmic region. Strikingly, structural modeling of the full complex suggests that the C-terminal β-strands of TamB complete the BamA β-barrel through β-strand augmentation, forming a continuous hybrid barrel with a hydrophobic exterior and hydrophilic interior. This arrangement may provide a direct structural link between periplasmic protein handling and membrane insertion. Together, these findings support a reorganized BAM architecture in B. burgdorferi and suggest that the hybrid BAM-TAM organization is conserved across Lyme disease-associated Borrelia species, providing a framework for understanding outer membrane assembly in spirochetes.