Extended Data Fig. 6: MST experiments for BAM proteins in the presence of Tetra_n.

(A) Generation of Tetra_n by enzymatic digestion of sacculi from BW25113Δ6LDT. The DD-endopeptidase MepM cleaves the DD-cross-links, generating soluble Tetra_n chains of variable length. (B) HPLC analysis of sacculi from BW25113Δ6LDT (top) and of Tetra_n (bottom), after digestion with the muramidase cellosyl to produce the disaccharide peptide subunits (muropeptides). Schematic representations of the chemical structures of muropeptides are shown on the right side. (C) MST experiment for BamA P1,2 and Tetra_n. Fig. 2f shows that BamA P1,2 does not interact with Tetra_n. Here, the right graph shows the lack of MST response in the control sample (mock PG digest without Tetra_n). (D) MST experiment as in C but with BamA P3,4. BamA P3,4 interacts with Tetra_n (Fig. 2f). (E) MST experiment as in C but with BamA P4,5. BamA P4,5 interacts with Tetra_n (Fig. 2f). Further MST experiments were performed in the presence or absence of Tetra_n or mock PG digests for BamB (F), BamC (G), BamE (H), BamCD (I) and BamCDE (J). (K) Control experiments performed with fluorescent-labelled BSA or BamCD in the presence of Tetra_n, showing that proteins do not generally bind to Tetra_n in MST experiments. (L) Control experiments performed with free fluorescent Red-NHS dye and no protein in the presence of Tetra_n, showing that the fluorescent dye does not bind to Tetra_n. Values shown in panels C-L are mean ± SD of three independent experiments. For source data see Supplementary Table 6.