Fig. 5: Loss of extracellular BAM components results in pleiotropic changes at the cell surface.
From: Structure of a distinct β-barrel assembly machinery complex in the Bacteroidota
a, Volcano plot showing wild-type B. thetaiotaomicron versus ΔbamH quantitative proteomics results (n = 3 biological replicates for each). The vertical dashed lines represent log2(fold change) = ±0.58. Proteins above the horizontal dashed lines have an adjusted P < 0.05. Statistical significance was inferred by two-sided t-test with Benjamini–Hochberg correction for multiple comparisons. Data points of interest are labelled and colour coded according to function or cellular localization. b, B. thetaiotaomicron proteinase K shaving assay for testing protein surface exposure. The top immunoblot was probed with anti-His-HRP conjugate antibodies. The bottom panel shows the same immunoblot re-probed with StrepTactin-HRP. Biotin-carboxyl carrier protein (BCCP, Bt1688) is a cytoplasmic biotinylated protein. SusA is a periplasmic neopullulanase that was used as an OM lysis control. The experiment was done twice with similar results. c, Representative transmission electron microscopy images of thin-sectioned B. thetaiotaomicron cells (wild type n = 17, Bt1927-ON n = 15, Bt1927-ON, ΔbamH n = 26). The white arrowheads point to the capsule that varies in thickness between cells. The black arrowheads point to the S-layer in Bt1927-ON cells. The red arrowheads point to the discontinuous S-layer in the Bt1927-ON, ΔbamH strain. d, Volcano plot showing wild-type P. gingivalis versus ΔbamH quantitative proteomics results (n = 3 biological replicates for each). Statistical significance was inferred by two-sided t-test with Benjamini–Hochberg correction for multiple comparisons. Changes in the T9SS and its substrates are described in Supplementary Discussion. e, Anti-HmuY immunoblot of OM, OMVs and culture medium (Med) fractions of wild type and ΔbamH P. gingivalis strains. The blot shown is representative of two biological repeats. f, SDS-PAGE analysis of P. gingivalis wild type, ΔbamI, ΔbamK and ΔbamH strain whole-cell lysates. g, RagBhis pulldowns from P. gingivalis wild type and ΔbamH strains analysed by size-exclusion chromatography on a Superdex 200 Increase 10/300 GL column. The chromatograms shown are representative of two biological replicates. h, SDS-PAGE analysis of indicated peaks from g. The asterisks denote bands from peaks A and B that correspond to singly cleaved RagA, confirmed by mass spectrometry. SLP, surface-exposed lipoprotein; OMP, outer membrane protein; LP, lipoprotein; CPS, capsular polysaccharide; T9SS, type 9 secretion system; PPAD, peptidylarginine deiminase.
