Extended Data Fig. 9: Isolation of BamA complexes from subunit depleted backgrounds and phenotypic characterisation of the bamHsup strain.
From: A new paradigm for outer membrane protein biogenesis in the Bacteroidota
a,b, Isolation of BamA complexes either (a) in the absence of BamM or (b) after 6 h of depletion of the essential BamG or BamH subunits. Size exclusion chromatography profile of Twin-Strep-tagged BamA complexes purified by Streptactin affinity chromatography (Left) and a Coomassie-stained SDS–PAGE gel of the indicated peak fractions (Right). BamA* indicates a proteolysis product of BamA. The identities of the BamA* and BamD + BamP bands were assigned by peptide fingerprinting. Similar results were obtained from 2 biological repeats. c-f, Characterization of the recreated bamHsup mutant (bamAQ801K ΔbamH ΔbamH2). wt, wild type. Similar results were obtained for three biological repeats. c, OM integrity assays. Cells were grown on CYE agar with the indicated additions. d, Surface exposure of the SLP SusE. Strains expressing a protease-sensitive His-tagged variant of SusE (SusEHis) were treated as indicated with Proteinase K and the detergent Triton X-100 (to permeabilise the OM). Reactions were stopped immediately (t0) or after 20 min (t20) and analysed by immunoblotting with His tag antibodies. The periplasmic protein SkpA serves as an OM integrity control. e, Spreading (gliding) morphology of colonies on agar. Scale bar, 5 mm. f, Purification of the native SusCDE complex via a Twin-Strep tag on the N-terminus of SusC followed by size exclusion chromatography. Analysed on a Coomassie-stained SDS–PAGE gel.
