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. 2021 Apr 13;10:e67817. doi: 10.7554/eLife.67817

Figure 3. DolP opposes an envelope detrimental effect caused by BamA overaccumulation in the OM.

(A) BW25113 cells harbouring pBAMHis where indicated were cultured and supplemented with no IPTG or 400 μM IPTG for 1 hr prior to collecting cells. The protein contents of the envelope fractions were analysed by SDS-PAGE and coomassie staining. Prior to loading, samples were heated for 5 min at 90°C, a temperature which is not sufficient to fully denature OmpA (folded OmpA, fOmpA). The band of BamB overlaps with the band of the major porin unfolded OmpC (uOmpC). (B) The BW25113 and the derivative ΔdolP, Δskp, or ΔompA strains carrying an empty control vector (pCtrl) or pBAMHis were serially diluted and spotted onto LB agar supplemented with IPTG as indicated. (C) BW25113 cells carrying a control empty vector (pCtrl), or the indicated plasmids for ectopic overproduction of BAM, or subsets of BAM subunits, or OmpAHis were serially diluted and spotted onto LB agar containing 400 μM IPTG. The diagrams depict the overproduced proteins. (D) BW25113 and derivative ΔdolP cells carrying the indicated plasmids for ectopic overproduction of either BamA alone or both DolPHis and BamA were serially diluted and spotted onto LB agar supplemented with IPTG as indicated. (E) BW25113 cells carrying the indicated plasmids were cultured overnight and streaked onto LB agar containing IPTG and vancomycin as indicated. (F) Heat-modifiability of BamA in wild-type and ΔdolP cells carrying the indicated plasmids. When the cultures reached the mid-exponential phase, the expression of BamA was induced for 2 hr with 200 μM IPTG. Total cell proteins were incubated at 25°C (Boiling −) or at 99°C (Boiling +), separated by SDS-PAGE and analysed by immunoblotting using the indicated antisera. u, unfolded; f, folded. (G) Heat modifiability of the protein contents of the envelope fraction of BW25113 (dolP+) or ΔdolP cells carrying no vector or transformed with pBamA or pDolPHis-BamA. Plasmid-borne genes were induced with 200 μM IPTG for 2 hr prior to collecting cells. The envelope fractions were mixed with SDS-PAGE loading buffer, incubated at 25°C (Boiling −) or 99°C (Boiling +) for 10 min, and analysed by SDS-PAGE and coomassie staining. u, unfolded.

Figure 3.

Figure 3—figure supplement 1. The detrimental effect of BAM overproduction is caused by the overaccumulation of BamA in the OM.

Figure 3—figure supplement 1.

(A) Spot dilution test of BW25113 cells transformed with the indicated plasmids. In the presence of IPTG, overproduction of BAMΔP1/ProtA impaired growth to a lower extent compared to overproduction of BAMProtA or BAMΔP2/ProtA. (B) The crude envelope fractions of cells overproducing BAMProtA, BAMΔP1/ProtA, or BAMΔP2/ProtA were solubilized with 1% (w/v) digitonin and 0.1% (w/v) DDM, and subjected to native IgG-affinity chromatography. Elutions were analysed by blue native-PAGE and coomassie staining (lanes 1–3) or SDS-PAGE and immunoblotting (lanes 4–6). Blue native-PAGE of the elution fraction containing wild-type BamA resolved a coomassie stainable complex migrating with an apparent mass of 250 kDa, which corresponds to the BAM complex (lane 1). A roughly similar amount of the BAM complex was detected in the elution obtained with BamAΔP2/ProtA samples, although, in this case, the BAMΔP2 complex migrated slightly faster accounting for the BamAΔP2 mass difference (lane 3). In contrast, the amount of BAMΔP1/ProtA variant was considerably lower (lane 2). The asterisk indicates that BamE is obtained from TEV digestion of BamEProtA. (C) Cells overproducing the indicated variants of the BAM complex were fractionated. The membrane and soluble fractions obtained upon treatment of collected cells with lysozyme and EDTA were analysed by SDS-PAGE and immunoblotting. BamAΔP1 was depleted from the total membrane fraction (lanes 1–3) and accumulated in the soluble fraction containing the periplasmic protein DsbA (lanes 4–6). (D) The periplasm and spheroplast fractions of cells overproducing BamA or both DolPHis and BamA (as in Figure 3F) were analysed by SDS-PAGE and immunoblotting using the indicated antisera. (E) Heat-modifiability of BamA and overproduced OmpAHis in BW25113 and ΔdolP cells carrying pOmpAHis. Upon reaching the mid-exponential phase, cultures were supplemented with 200 μM IPTG for 2 hr. The whole-protein content of collected cells were incubated at 25°C (Boiling −) or at 99°C (Boiling +), separated by SDS-PAGE, and analysed by immunoblotting using the indicated antisera. u, unfolded; f, folded. * indicates a non-specific cross-reaction.