Evolutionary characterization and structure-guided discovery of the β-barrel assembly machinery in Treponema pallidum subsp. pallidum reveals a putative BamD-like protein.

Abstract

Gram-negative bacteria rely on the β-barrel assembly machinery (BAM) for the biogenesis of their outer membrane proteins (OMPs). In most bacterial species, the BAM complex consists of BamA together with several accessory proteins, including BamD, a highly conserved and indispensable component. However, no BAM complex components have previously been recognized in Treponema pallidum subsp. pallidum. By integrating phylogenetic analysis, AlphaFold 3-based structural prediction, and biochemical validation, we identified Tp0625 as a previously unrecognized BamD-like protein of the T. pallidum BAM system. Tp0625 binds directly to the POTRA domain of BamA (Tp0326), with preferential interaction involving the POTRA5-containing region, and forms a stable complex stabilized by extensive electrostatic interactions. Biophysical analysis reveals that Tp0625 binds BamA with moderate affinity (KD = 3.28 μM), and structural comparison shows that Tp0625 retains a conserved tetratricopeptide repeat (TPR) fold despite low sequence identity and a reduced number of TPR domains. These results offer evidence for a putative BamD-like protein in T. pallidum and support the existence of a conserved but simplified BAM complex in this pathogen. Our study establishes a structure-guided strategy for discovering divergent homologs of essential macromolecules, and expands our understanding of how spirochaetes maintain outer membrane integrity without a full complement of accessory proteins.