Figure 1. Slam1 is necessary for translocation of unfolded TbpB.

(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 DDM detergent from TbpB before adding proteoliposomes for translocation. Efficiency of TbpB translocation/insertion was calculated based on percentage of TbpB that was protected from proteinase K. (b) Representative proteinase K protection assay results obtained for Slam1 or Slam1 + Bam incubated with purified TbpB (folded or kept unfolded by 8 M urea). Proteoliposomes containing Empty or Bam were used as controls. Each sample was treated with PK or PK + Triton X-100 and examined by western blot. α-flag antibody western blots were used to quantify the amount of TbpB. Asterisk (*) The lower band in the (+PK and −Triton X) samples in Slam1 and Bam + Slam1 proteoliposomes treatment likely represents incompletely translocated TbpB that has been partially degraded by proteinase K. (c) Quantification of TbpB protection in proteoliposomes through densitometry analysis. The % TbpB insertion was calculated by dividing the protected TbpB of + PK sample by TbpB of the input sample. The plot contains results obtained from three biological replicates. Individual data points were included on the graph. Two-way analysis of variance (ANOVA) test was performed to determine the statistical significance for the translocation of unfolded TbpB by Slam1 proteoliposomes and Slam1 + Bam proteoliposomes treatment versus by the negative controls (empty liposomes and Bam proteoliposomes). Only statistical significance of the unfolded TbpB translocation by Slam1 proteoliposomes against the two negative controls are included on the blot for simplification. (***) represents p-value < 0.001.

Figure 1—figure supplement 1. Purification of M. catarrhalis Slam1.

(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 electrophoresis (SDS–PAGE) gels of pure MonoQ fractions from Mcat Slam1 purification. Pure Slam–DDM detergent complex eluted at 50 mM Tris–HCl pH 8, 40 mM NaCl from a MonoQ column. The proteins were used for the downstream functional assays. Proteins that were used for structural studies were passed through S200 gel filtration.

Figure 1—figure supplement 2. Translocation of Mcat TbpB to the surface of E. coli cells.

(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 secondary antibody conjugated with fluorescent probe phycoerythrin (PE). The fluorescence was quantified using a plate reader. (b) Quantification of surface display of Mcat TbpB by Slam1 and Slam2 homologs. Slam2 are negative controls as they have different substrate specificity (HpuA) for translocation. Normalized fluorescence values obtained for each of the Slam homologs is shown. The results represent at least three biological replicates and demonstrate that Mcat Slam1 is functional in translocating TbpB in the E. coli model. (***) represents p-value < 0.001 and (ns) represents p-value > 0.05.

Figure 1—figure supplement 3. Generation of Slam1 and Bam proteoliposomes.

(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 preformed liposomes. Detergent was further removed using SM2 biobeads. (b) Quantification of Slam1 and BamABCDE insertion using Coomassie staining. Bovine Serum Albumin (BSA) was used as a control for estimating absolute protein quantity. Insertion percentages were calculated by dividing the band intensity of protein inserted in liposomes (l) by total protein incubated with liposomes (T). For the Bam complex, BamA intensity was used to calculate insertion efficiency. (c) Confirmation of Slam1 and BamABCDE insertion using western blots with α-His antibodies.

Figure 1—figure supplement 4. Purification and characterization of E. coli Bam complex.

(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 asterisk) were observed, and they most likely correspond to common E. coli proteins that have been reported in previous His-tag purified proteins. (b) Design of an in vitro translocation assay for testing the function of the Bam complex. E. coli spheroplasts secrete porins such as OmpA into the supernatant. When incubated with Bam complex proteoliposomes, secreted OmpA is successfully inserted into Bam proteoliposomes. (c) An α-OmpA western blot to access the folding states of secreted OmpA over time in Tris pH 8 buffer, empty liposome, and Bam proteoliposomes. Approximately 95% of OmpA achieved folded form in the presence of Bam proteoliposomes within the first 10 min of incubation while self-folding in the empty liposome remained at 50%.

Figure 1—figure supplement 5. Characterization of Mcat Slam1 and BamABCDE containing proteoliposomes.

(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 Tris pH 7, 200 mM NaCl). Ten fractions were collected from the top and alternate fractions were run on an sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel. Western blots using α-His antibodies are shown indicating the amount of protein present in each fraction. (b) Proteinase K protection assay on Slam1 and Bam proteoliposomes. Proteoliposomes were incubated with 0.1 mg/ml proteinase K for 15 min at room temperature. Coomassie blue stained gel and α-His western blot were used to assess orientation of the proteins in liposomes. Approximately 18% of Slam1 inserted with N-terminal his-tag residing in the lumen of liposomes and was protected from PK digestion. Percentage protection was calculated using densitometry. Asterisk (*) indicates fragments of Slam1 (potentially C-terminal barrel) remaining after PK shaving.

Figure 1—figure supplement 6. Purification and functional characterization of M. catarrhalis TbpB.

(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 transferrin (bio-hTf) for detecting the function of TbpB. 0.5 μl TbpB and respective controls (TbpA, BamABCDE, and BSA) were spotted on nitrocellulose membrane and blotted with bio-hTf (50 μg/ml) followed by streptavidin–horseradish peroxidase (HRP). TbpB is bound tightly with bio-hTf indicating it is functional.

Figure 1—figure supplement 7. Protection of urea-unfolded TbpB (flag-tag) and negative control urea-unfolded AfuA (his-tag) by liposomes, proteoliposomes, and purified Slam1–DDM complex.

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 treatment (0.1 mg/ml). Reaction was inhibited by 1 mM phenylmethylsulfonyl fluoride (PMSF) before loading on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel and western transferred to PVDF blot. Blot was cut and the two halves were incubated with α-flag (for 75 kDa TbpB) or α-his antibodies (for 38 kDa AfuA) accordingly.