Extended Data Fig. 7: Controls for PG pull-down experiments and BAM activity assays performed with tetrapeptide-rich and pentapeptide-rich PG.

(A) HPLC muropeptide analysis of tetrapeptide-rich PG from MC1061 and pentapeptide-rich PG from CS703-1. Schematic representation of the main muropeptides are shown. (B) PG pull-down assays for BAM proteins performed in the presence of tetrapeptide-rich PG (MC1061) or pentapeptide-rich PG (CS703-1), analysed by SDS-PAGE and Coomassie Blue staining (for gel source data, see SI Fig. 1). S, supernatant fraction; W, wash fraction; P, pellet fraction. Marker lanes are indicated by asterisks. Representative results from two independent replicates are shown. (C) BAM-mediated OmpT assembly in vitro. Control experiments without BAM (empty liposomes), OmpT or SurA are included. Values are mean ± SD of three independent experiments. (D) Effect of tetrapeptide-rich PG on OmpT activity. Sacculi did not reduce the cleavage of the fluorogenic peptide when added to reactions containing already folded OmpT (n = 3, P = 0.69). (E) Interaction of SurA with tetrapeptide-rich (MC1061) and pentapeptide-rich PG (CS703-1) in vitro, analysed by SDS-PAGE and Coomassie Blue staining (for gel source data, see SI Fig. 1). S, supernatant fraction; W, wash fraction; P, pellet fraction. (F) BAM activity measured in the presence of a 15 µM excess of SurA and tetrapeptide-rich PG, showing that SurA binding to PG is not a limiting factor for BAM activity in vitro (n = 3; control vs sample containing excess SurA: P = 0.21; control vs sample containing PG: P = 0.001; control vs sample containing PG + excess SurA: P = 0.0002; sample containing PG vs sample containing PG + excess SurA: P = 0.89. For gel source data, see SI Fig. 1). (G) Effect of Tetra_n on BAM-mediated OmpT assembly in vitro (n = 3, P = 0.02). Statistical significance of data shown in panels D, F and G was calculated using two-tailed Student’s unpaired t-test. Values are mean ± SD.