Extended Data Fig. 4: SlyB expression and interactome under stressed and non-stressed conditions.
From: SlyB encapsulates outer membrane proteins in stress-induced lipid nanodomains
(a) WB of whole cell SDS PAGE of BW25113 (WT), the complemented knockout strain BW25113 ΔslyB attTn7::slyB (‘C’) and BW25113 ΔphoP (right) grown on LB supplemented with 200 mM Mg2+ (1, LB+), on LB (2), or on LB supplemented with 1mM EDTA (3) or 100 μg.mL−1 Bac2A (4). Experiment shows the proportion of PhoP-independent SlyB expression and the PhoP-dependent induction of SlyB during LPS destabilizing conditions. (b) α-SlyB Western analysis of snPAGE of BW25113 ΔphoP whole cell lysates. In absence of phoP, little to no SlyB OMDs (SlyBC) are formed. (c) α-SlyB stained snPAGE of BW25113 cells grown up to OD600 of 0.1 on N minimal medium with indicated Mg2+ concentrations. (d, e) SEC profile and fractionated coomassie-stained (d) snPAGE and its α-SlyB Western blot (e) of Ni-NTA pulldowns from BW25113 ΔslyB::slyBTEV_His grown on LB-EDTA (5mM) medium. Elution volumes of MW standards indicated by vertical lines. SEC elution fractions are indicated above lanes. Bands corresponding to monomeric SlyB or high molecular weight SlyB complexes are labelled SlyBM and SlyBC, resp. (f, g) Western analysis of the snPAGE (f) and denaturing SDS-PAGE (g) of the SEC fractionated Ni-NTA pulldowns as in panel a. Western blot analysis use α-BamA, α-LptD, α-OmpA, α-OmpC or α-SlyB as primary immune serum; as indicated. MW: standards with molecular weight indicated in KDa. (h, i) Representative 2D cryoEM classes from Ni-NTA pulldowns of BW25113 ΔslyB::slyBTEV_His grown in LB (h) or LB + 5mM EDTA medium (i), generated by cryoSPARC52. Both datasets contain a series of homogeneous classes corresponding to a SlyB:YnfB complex (see Extended Data Fig. 1g, h, j) corresponding to 6 and 14 % of the aligned particles in the LB and LB + EDTA datasets, resp. (top row in h and i). The YnfB protein was identified as the dominant tryptic peptide in the MS peptide fingerprint and the sole significant periplasmic SlyB binding partner in the LB dataset (see SI Table 3). The remaining particles in the LB correspond to SlyB oligomers (SlyBO; middle five rows) of variable diameter and protomer number (as judged by top views), as well as a smaller fraction of particles corresponding to micelles with a low molecular weight SlyB complex (a single to a few SlyB protomers only; bottom row). In the LB + EDTA dataset, the majority of particles corresponds to SlyB oligomers as well as different SlyB:OMP complexes (bottom five rows, and Fig. 3f). For some classes, the molecular identity of the encapsulated OMP can be decerned by its structural properties (See Fig. 3f). Box size = 275 Å.
