Fig. 4: Depletion analysis of the essential BAMFj subunits. | Nature

Fig. 4: Depletion analysis of the essential BAMFj subunits.

From: A new paradigm for outer membrane protein biogenesis in the Bacteroidota

Fig. 4

Strains are the wild-type and depletion strains for BamA (bamAdep), BamG (bamGdep) and BamH with either a strong (bamHdep) or weak (bamHlow) inducible promoter. SkpA is a periplasmic protein to control for OM integrity; GroEL is a loading control. ad, Strains were cultured in rich (Casitone yeast extract, CYE) medium. The aTC inducer of the target gene was removed at 0 h where indicated (−aTC) to initiate subunit depletion. Samples in bd were taken at the indicated time points in a. a, Growth curves. Data are mean ± s.d. b, Immunoblots of whole-cell lysates. c, Representative transmission electron microscopy images showing OM defects in the depletion strains. Yellow arrows highlight budding OM vesicles; black arrows highlight OM blebbing and rupture. Scale bar, 500 nm. d, Depletion of BAMFj subunits for 6 h does not change the surface exposure of the SLP SusE as assessed by proteinase K accessibility. Triton X-100 permeabilizes the OM. Reactions were stopped immediately (t0) or after 20 min (t20) and analysed by immunoblotting. e, Comparative whole-membrane proteome analysis of depleted (−aTC) versus induced (+aTC) strains collected at the 6 h time point in a. Data points for OMPs and SLPs are coloured as indicated and the most highly expressed OM proteins are labelled. A significance threshold is drawn according to a two-tailed t-test with a false discovery rate (FDR) of 0.1 and a variance correction constant S0 of 0.1. Data are averaged over three biological repeats. FC, fold change. f,g, Analysis of chronic BamH depletion in an induced strain (bamHlow + aTC) in which a weak promoter results in the incomplete restoration of wild-type BamH levels. f, Whole-cell immunoblots. Arrow indicates BamH; asterisk indicates a non-specific band. g, As e but comparing chronic BamH depletion (bamHlow strain + aTC) relative to the wild type. ad,f, Similar data were obtained for three biological repeats.

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