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.