(a) Model of a defined in vitro assay for TbpB translocation. M. catarrhalis TbpB (folded and urea-unfolded) is translocated inside Slam1-containing proteoliposomes. SM2 biobeads were used to remove …
Quantification of in vitro translocation of purified TbpB.
(a) Sequence identity of N. meningitidis Slam1 and M. catarrhalis Slam1. (b) Overall membrane protein expression and purification pipeline. (c) Sodium dodecyl sulfate–polyacrylamide gel …
(a) Plate reader assay was used to examine the function of Slam homologs. Slams and TbpB were coexpressed in E. coli strain C43(DE3) and probed with α-flag antibodies followed by labeling with the …
Fluorescent signal of TbpB on the surface of E.coli.
(a) Protocol used for insertion of outer membrane proteins (OMPs) into liposomes. OMP-DDM protein–detergent complexes were diluted (below the critical micellar concentration [CMC] of DDM) into …
(a) BamABCDE fractions obtained from a S200 gel filtration column. The BamABCDE complex was obtained using previously described protocols (Hagan et al., 2010). Some non-Bam complex bands (marked in …
(a) Sucrose floatation assay for Slam1 and Bam proteoliposomes. Proteoliposomes were resuspended to a final concentration of 60% sucrose and subsequently layered with 30% sucrose and buffer B (50 mM …
(a) Purified TbpB–DDM complexes obtained from a S200 size chromatography column. The sample was subsequently used for the in vitro translocation assay. (b) Dot blot assay with biotinylated human …
Mixture of urea-unfolded TbpB and urea-unfolded AfuA (1:1 molar ratio) was incubated with empty liposomes, proteoliposomes, or Slam1–DDM complex (1:20 volume ratio), followed by proteinase K …
(a) Model of the proposed in vitro proteoliposomes translocation assay for TbpB secreted directly from E. coli spheroplast. As E. coli cell expressing TbpB, the cells were converted into spheroplast …
Quantification of in vitro translocation of spheroplast-secreted TbpB.
Quantification of time-dependent translocation of spheroplast-secreted TbpB.
(a) Model of the release of SLPs from spheroplasts upon addition of purified E. coli LolA. SLPs are synthesized in the cytoplasm and transported to the periplasm via the Sec translocon. After the …
(a) Western blot accessing the protection of secreted TbpB in the absence of liposomes. Similar background protection was observed in no liposomes and liposome negative control (8.5% and 9.3%, …
(a) Model of pulldown assay using the flag-tag on the C-terminus of TbpB. Samples were analyzed using mass spectrometry (summarized in Table 1) and examined on western blots. (b) Representative …
Mass spectrometry of pulldown samples.
(a) Translocation of TbpB via Slam1 to the surface of E. coli K12 mutants. Depletion of Skp significantly reduces the translocation of TbpB to the surface (by 50% – detecting by using α-flag …
Fluorescence of TbpB on surface of E. coli.
Quantification of in vitro translocatio.
Quantification of in vitro translocation following sucrose floatation assay.
(a) Relative fluorescence intensity accessing the presence of TbpB (65 kDa) on the surface of N. meningitidis mutants using α-TbpB antibody (exposure – top) and biotinylated human transferrin …
Fluorescent signal of TbpB on the surface of N. meningitidis.
(a) The complete knockout process using spot transformation. Gene skp in N. meningitidis B16B6 is replaced by kan cassette. (b) Agarose gel of single colony PCR using external primers (targeting DNA …
Coomassie blue stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of Nme Skp after S75 gel filtration to access its purity prior to antibody production (left panel) and …
(a) OD600 was recorded every 30 min over a 24-hr period. Δskp and Δslam1 mutants were lagging behind but reached OD600 of 0.7 eventually. (b) Representative western blots detecting the expression of …
Growth of N. meningitidis.
Once the surface lipoproteins emerged from the Sec complex, periplasmic chaperone Skp binds to the SLPs to prevent early folding while their N-terminus is modified and lipidated by Lgt, Lsp, and Lnt …
AfuA | TbpB | |||||||
---|---|---|---|---|---|---|---|---|
Proteins | Total peptides | % Coverage | Location | Proteins | Total peptides | % Coverage | Location | |
1 | AfuA | 380 | 78% | P | TbpB | 265 | 31% | – |
2 | TufA | 119 | 70% | IM | LolA | 146 | 73% | P |
3 | LolA | 36 | 72% | P | TufA | 57 | 59% | IM |
4 | OmpF | 12 | 34% | OM | OmpF | 21 | 38% | OM |
5 | DegP | 10 | 30% | P | DegP | 15 | 32% | P |
6 | FlgH | 12 | 34% | P | OmpA | 15 | 34% | OM |
7 | DegQ | 6 | 14% | P | Skp | 9 | 27% | P |
8 | OmpA | 5 | 22% | OM | FlgH | 8 | 26% | P |
9 | DegQ | 5 | 13% | P |
Strains, plasmids, and antibodies used in this study.
Raw images for figures and figure supplements.
Mass spectrometry results.
Original gels and blots used for the figures.