Phenotype-Guided Comparative Genomics Identifies the Complete Transport Pathway of the Antimicrobial Lasso Peptide Ubonodin in Burkholderia

New antibiotics are needed as bacterial infections continue to be a leading cause of death, but efforts to develop compounds with promising antibacterial activity are hindered by a poor understanding of-and limited strategies for elucidating-their modes of action. We recently discovered a novel lasso peptide, ubonodin, that is active against opportunistic human lung pathogens from the Burkholderia cepacia complex (Bcc). Ubonodin inhibits RNA polymerase, but only select strains were susceptible, indicating that having a conserved cellular target does not guarantee activity. Given the cytoplasmic target, we hypothesized that cellular uptake of ubonodin determines susceptibility. Although Bcc strains harbor numerous nutrient uptake systems, these organisms lack close homologues of the single known lasso peptide membrane receptor, FhuA. Thus, a straightforward homology-driven approach failed to uncover the identity of the ubonodin transporter(s). Here, we used phenotype-guided comparative genomics to identify genes uniquely associated with ubonodin-susceptible Bcc strains, leading to the identification of PupB as the ubonodin outer membrane (OM) receptor in Burkholderia. The loss of PupB renders B. cepacia resistant to ubonodin, whereas expressing PupB sensitizes a resistant strain. We also examine how a conserved iron-regulated transcriptional pathway controls PupB to further tune ubonodin susceptibility. PupB is only the second lasso peptide OM receptor to be uncovered and the first outside of enterobacteria. Finally, we elucidate the full transport pathway for ubonodin by identifying its inner membrane receptor YddA in Burkholderia. Our work provides a complete picture of the mode of action of ubonodin and establishes a general framework for deciphering the transport pathways of other natural products with cytoplasmic targets.