Extended Data Fig. 8: PbgA interacts with LapB to regulate LpxC stability.

a, Proteins identified by mass spectrometry following co-immunoprecipitation of endogenous PbgA using the anti-PbgA monoclonal antibody 7E7 (n = 3 independent experiments). Hits were classified based on abundance (sum of PSMs) and enrichment in PbgA IPs compared to control purifications (SAINT logOddsScore: anti-PbgA monoclonal antibody 7E7 versus anti-gp120). Identified proteins with a Bayesian FDR <10% are highlighted in red. b, Bacterial two-hybrid system using PbgA-prey and different bait proteins in E. coli cells. Interacting proteins lead to blue colonies on agar plates containing X-gal, whereas non-interacting proteins produce white colonies. A representative agar plate is shown (n = 3) and activity was confirmed in broth cultures. c, Growth of a conditional E. coli K-12 ΔpbgA::pBAD-pbgA after depletion of PbgA in the presence of a IPTG-inducible plasmid expressing wild-type lapB or plsY (Methods) demonstrates that lapB expression does not rescue growth after PbgA depletion. Representative plates are shown and growth assay was repeated three or more times. d, Cell lysates prepared from overnight streaks of E. coli K-12 with pBADpbgA wild-type or mutant plasmids were probed with anti-LpxC, anti-PbgA and anti-GroEL antibodies (Methods), indicating that disturbing the LPS–PbgA interaction interface leads to LpxC stabilization. Representative blots from n = 3 biological replicates are shown. e, Western blot analysis of LpxC after treatment with 1 μM (2× MIC) or 4 μM (8× MIC) of the small molecule MsbA inhibitor G’913, indicating that selective inhibition of MsbA^29,44 and LPS transport impacts LpxC levels; GroEL is the loading control and a representative experiment (n = 3 independent experiments) is shown. f, E. coli K-12 ΔlptD::pBADlptD lysates prepared from cells grown in indicated concentration of arabinose were probed with anti-LpxC, anti-LptD and anti-GroEL antibodies (Methods). Representative blots from n = 3 biological replicates are shown. g, Bacterial two-hybrid assays using LapB-bait (pUT18-lapB) and indicated PbgA-mutant prey constructs (pKT25-pbgA) in E. coli DHM1 cells were performed (Methods). Interacting proteins lead to blue colonies, whereas non-interacting proteins produce white colonies. Note that EptA^TM–PbgA^IFD+PD is a chimeric construct in which the TMD of PbgA has been replaced with the TMD region from EptA²³. Representative plates from n = 3 culture streaks are shown. h, Growth of conditional PbgA strain (E. coli ΔpbgA::pBADpbgA) in the absence of arabinose inducer complemented with, clockwise from the top of plate, wild-type pbgA (PbgA^WT), pbgA encoding only the TMD (PbgA^{TM only}), or a negative control (malE) on plasmids. A representative plate (n = 3) is shown. i. Cell lysates of the conditional pbgA strain (E. coli ΔpbgA::pBADpbgA) in the absence of arabinose inducer complemented with wild-type pbgA or pbgA encoding only the TMD were probed with anti-LpxC antibody (Methods). A representative blot for n = 3 independent experiments is shown. j, Plasmids encoding acpT (right side of plate) or acpS (left side of plate) in conditional-pbgA strain grown in the absence of the pBADpbgA inducer arabinose, with 0.1 mM IPTG at 30 °C. A representative growth plate (n = 3) was imaged. k, Cultures with plasmids expressing pbgA, acpT, acpS, or malE (control) were shifted to no arabinose/plus IPTG if necessary to deplete PbgA (Methods). A representative blot from at least n = 3 biological replicates is shown.