Bacterial conjugation mediates contact-dependent transfer of DNA from donor to recipient bacteria, thus facilitating the spread of virulence and resistance plasmids. Here we describe how variants of the plasmid-encoded donor outer membrane (OM) protein TraN cooperate with distinct OM receptors in recipients to mediate mating pair stabilization and efficient DNA transfer. We show that TraN from the plasmid pKpQIL (Klebsiella pneumoniae) interacts with OmpK36, plasmids from R100-1 (Shigella flexneri) and pSLT (Salmonella Typhimurium) interact with OmpW, and the prototypical F plasmid (Escherichia coli) interacts with OmpA. Cryo-EM analysis revealed that TraNpKpQIL interacts with OmpK36 through the insertion of a β-hairpin in the tip of TraN into a monomer of the OmpK36 porin trimer. Combining bioinformatic analysis with AlphaFold structural predictions, we identified a fourth TraN structural variant that mediates mating pair stabilization by binding OmpF. Accordingly, we devised a classification scheme for TraN homologues on the basis of structural similarity and their associated receptors: TraNα (OmpW), TraNβ (OmpK36), TraNγ (OmpA), TraNδ (OmpF). These TraN-OM receptor pairings have real-world implications as they reflect the distribution of resistance plasmids within clinical Enterobacteriaceae isolates, demonstrating the importance of mating pair stabilization in mediating conjugation species specificity. These findings will allow us to predict the distribution of emerging resistance plasmids in high-risk bacterial pathogens.

The Mycobacterium tuberculosis ( Mtb ) LpqY-SugABC ATP-binding cassette transporter is a recycling system that imports trehalose released during remodeling of the Mtb cell-envelope. As this process is essential for the virulence of the Mtb pathogen, it may represent an important target for tuberculosis drug and diagnostic development, but the transporter specificity and molecular determinants of substrate recognition are unknown. To address this, we have determined the structural and biochemical basis of how mycobacteria transport trehalose using a combination of crystallography, saturation transfer difference NMR, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the synthesis of trehalose analogs. This analysis pinpoints key residues of the LpqY substrate binding lipoprotein that dictate substrate-specific recognition and has revealed which disaccharide modifications are tolerated. These findings provide critical insights into how the essential Mtb LpqY-SugABC transporter reuses trehalose and modified analogs and specifies a framework that can be exploited for the design of new antitubercular agents and/or diagnostic tools.