Article Figures & Data
Figures
- FIG 1
The passenger domains of UpaGΔ2 are secreted rapidly. (A) Homology-based model of the structure of UpaGΔ2 generated using PHYRE2 and GalaxyHOMOMER software. The approximate locations of proteinase K (PK) cleavage sites and a previously identified Him motif (55) are shown. The larger C-terminal fragment was produced by the treatment of native UpaGΔ2 trimer with PK (PK fragment), and the slightly smaller fragment (PK fragment′) was produced by PK treatment of incompletely folded derivatives. The numbers shown here and throughout refer to positions in the full-length UpaG sequence. (B) Illustration of the ST-UpaGΔ2 protein. HA, HA tag; ST, SpyTag. (C) AD202 cells transformed with a plasmid encoding ST-UpaGΔ2 (pRS31) were subjected to pulse-chase labeling. After cells were either incubated with SpyCatcher or mock treated, immunoprecipitations were conducted using an anti-UpaG antiserum and proteins were resolved by SDS-PAGE.
- FIG 2
The length of the passenger domain does not affect the kinetics of UpaG assembly. (A) Illustration of the ST-UpaGΔ1 protein. HA, HA tag; ST, SpyTag. (B) AD202 cells transformed with a plasmid encoding ST-UpaGΔ1 (pRS37) were subjected to pulse-chase labeling. After cells were either incubated with PK or mock treated, immunoprecipitations were conducted using an anti-UpaG antiserum and proteins were resolved by SDS-PAGE. (C) The experiment represented in panel part B was repeated, except that cells were incubated with SpyCatcher instead of PK. In panels B and C, unidentified bands that appear to have been derived from ST-UpaGΔ1 are labeled with an asterisk.
- FIG 3
The presence of an intrinsically disordered segment delays the completion of passenger domain secretion. (A) Illustration of the HA–RTX-UpaGΔ2 protein. HA, HA tag. (B) AD202 cells transformed with a plasmid encoding HA–RTX-UpaGΔ2 (pRS42) were subjected to pulse-chase labeling. After cells were either incubated with PK or mock treated, immunoprecipitations were conducted using an anti-UpaG antiserum and proteins were resolved by SDS-PAGE. (C) AD202 cells were transformed with a plasmid encoding ST–RTX-UpaGΔ2 (pRS43). The experiment represented in panel B was repeated, except that cells were incubated with SpyCatcher instead of PK. The OM of half of the cells was permeabilized prior to the addition of SpyCatcher. (D) Model for the secretion of the ST–RTX-UpaGΔ2 passenger domain. The secretion of the segment of the chimeric passenger domain derived from UpaG is fast and is potentially driven by the formation of a coiled-coil structure. Because the RTX segment cannot fold, its rate of secretion and the level of concomitant surface exposure of the N-terminal SpyTag are considerably lower.
- FIG 4
The secretion of disordered passenger domains is completed in a stepwise fashion. (A) Illustration of the HA-(GGS)33-UpaGΔ4 protein. HA, HA tag. (B) AD202 cells transformed with a plasmid encoding HA-(GGS)33-UpaGΔ4 (pRS22) were subjected to pulse-chase labeling. After cells were either incubated with PK or mock treated, immunoprecipitations were conducted using an anti-UpaG antiserum and proteins were resolved by SDS-PAGE. (C) AD202 cells were transformed with a plasmid encoding ST–(GGS)33-UpaGΔ4 (pRS36). The experiment represented in panel B was repeated, except that cells were incubated with SpyCatcher instead of PK. The OM of an equal number of cells was permeabilized prior to the addition of SpyCatcher.
- FIG 5
The secretion of passenger domains that contain a nonnative disulfide bond is completed in a stepwise fashion. (A) AD202 cells transformed with a plasmid encoding HA-UpaGΔ2 (S1624C/S1652C) or HA-UpaGΔ2 (S1620C/S1678C) (pRS29 or pRS30) were subjected to pulse-chase labeling. After cells were either incubated with PK or mock treated, immunoprecipitations were conducted using an anti-UpaG antiserum and proteins were resolved by SDS-PAGE. (B) AD202 cells were transformed with a plasmid encoding ST-UpaGΔ2 (S1624C/S1652C) or ST-UpaGΔ2 (S1620C/S1678C) (pRS33 or pRS34). The experiment represented in panel A was repeated, except that cells were incubated with SpyCatcher instead of PK. The OM of an equal number of cells that produced ST-UpaGΔ2 (S1624C/S1652C) was permeabilized prior to the addition of SpyCatcher. Unidentified bands that might have been breakdown products are labeled with an asterisk. (C) Model for the stepwise secretion of UpaGΔ2 passenger domain derivatives that contain a disulfide bond. Passenger domain translocation stalls because the disulfide-bonded segments cannot pass readily through the linked β barrel domain. We propose that as a result of a series of conformational changes, the β barrel expands transiently and enables each passenger domain to be fully secreted.
- FIG 6
UpaGΔ2 remains associated with the Bam complex until passenger domain secretion is complete. AD202 cells transformed with plasmids encoding pDULE-Bpa and HA-UpaGΔ2 (W1778am) or HA-UpaGΔ2 (S1624C/S1652C/1778am) (pRS45 or pRS46) were subjected to pulse-chase labeling. Half of the cells were irradiated with UV light, and the other half were left untreated. Immunoprecipitations were then conducted with a monoclonal anti-HA antibody or a polyclonal antiserum raised against a BamA C-terminal peptide, and proteins were resolved by SDS-PAGE. An unidentified cross-linking product that served as an internal gel loading control is labeled with an asterisk.
- FIG 7
Model for the assembly of TAAs. The three subunits of a TAA form an asymmetric trimer in the periplasm (step i). The partially folded trimer is then targeted to the OM, where the Bam complex catalyzes the membrane integration of the β barrel domain (step ii). A conformational change in the β barrel domain may be required to expose the passenger domains on the cell surface and to initiate translocation. Translocation proceeds through a hybrid channel comprised of open forms of the linked β barrel domain and the BamA β barrel (step iii). Translocation is driven at least in part by the folding of the passenger domains into a coiled-coil structure. Following the completion of the translocation reaction, the β barrel domain folds completely and dissociates from the Bam complex.
Supplemental Material
FIG S1
UpaG derivatives that contain only the β barrel and the embedded linker segment are assembled extremely rapidly. (A) Illustration of HA-UpaGΔ3, UpaGΔ4, and UpaGΔ5 constructs. HA, HA tag. The site of each deletion is shown on a homology-based model of the C terminus of an UpaG monomer. (B) Strain AD202 cells transformed with a plasmid encoding HA-UpaGΔ3 (pRS18), UpaGΔ4 (pRS19), or UpaGΔ5 (pRS20) were subjected to pulse-chase labeling. After the cells were either incubated with PK or subjected to mock treatment, immunoprecipitations were conducted using a polyclonal anti-HA antiserum (for HA-UpaGΔ3) or an anti-UpaG antiserum (for all constructs). Proteins were resolved by SDS-PAGE on either 8% to 16% Tris-glycine minigels (for HA-UpaGΔ3) or 12% Bis-Tris minigels using a morpholineethanesulfonic acid (MES)-based buffer system (Thermo Fisher Scientific) (for UpaGΔ4 and UpaGΔ5). (C) The experiment performed with UpaGΔ4 was repeated except that the cells were transferred to a prewarmed 25°C water bath for 15 min prior to pulse-chase labeling at the same temperature. Immunoprecipitations were conducted using an anti-UpaG antiserum, and proteins were resolved by SDS-PAGE on an 8% to 16% Tris-glycine minigel. Unidentified background bands are labeled with an asterisk in panels B and C. Download FIG S1, PDF file, 0.9 MB.
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FIG S2
The faster-migrating form of an RTX-UpaGΔ2 trimer contains a disordered RTX domain. AD202 cells transformed with a plasmid encoding HA–RTX-UpaGΔ2 (pRS42) were grown in MOPS minimal media to mid-log phase and induced with 0.2% l-rhamnose. Cultures were divided in half, and 2 mM CaCl2 was added to one half 5 min prior to pulse-chase labeling. After the cells were either incubated with PK or mock treated, immunoprecipitations were conducted using an anti-UpaG antiserum and proteins were resolved by SDS-PAGE. The observation that the level of the faster-migrating (∼110-kDa) form of the RTX-UpaGΔ2 trimer was greatly reduced when calcium was added to induce folding indicates that it contained an unfolded RTX moiety. Download FIG S2, PDF file, 0.4 MB.
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FIG S3
Properties of monomeric and slower-migrating trimeric forms of ST–RTX-UpaGΔ2. (A) Regions of the gels presented in Fig. 3C that contained the RTX-UpaGΔ2 monomer (which is located entirely in the periplasm) are shown. The results indicate that the periplasm of cells that produce ST–RTX-UpaGΔ2 remained inaccessible to the SpyCatcher protein unless the OM was permeabilized. Perhaps due to the use of a cell permeabilization buffer, a greater amount of a polypeptide that likely corresponded to the precursor form of the ST–RTX-UpaGΔ2 monomer (*) was reproducibly immunoprecipitated from samples generated from permeabilized cells than from intact cells. (B) Regions of the gels presented in Fig. 3C that contained the slower-migrating (alternative) form of the ST–RTX-UpaGΔ2 trimer are shown. The results indicate that the secretion of the passenger domains of the slower-migrating form of the ST–RTX-UpaGΔ2 trimer was completed in a stepwise fashion. Download FIG S3, PDF file, 0.2 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S4
The secretion of chimeric passenger domains that contain an unfolded segment is completed in a stepwise fashion. (A) Illustration of the HA-CtxB*-UpaGΔ2 protein. HA, HA tag. (B) AD202 cells transformed with a plasmid encoding HA-CtxB*-UpaGΔ2 (pRS39) were subjected to pulse-chase labeling. After cells were either incubated with PK or mock treated, immunoprecipitations were conducted using an anti-UpaG antiserum and proteins were resolved by SDS-PAGE. (C) AD202 cells were transformed with a plasmid encoding ST-CtxB*-UpaGΔ2 (pRS41). The experiment represented in panel B was repeated, except that cells were incubated with SpyCatcher instead of PK. Download FIG S4, PDF file, 0.3 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S5
Rapid secretion of chimeric passenger domains that contain a disordered segment. (A) AD202 cells transformed with a plasmid encoding HA-(GGS)33-UpaGΔ2 (pRS21) were subjected to pulse-chase labeling. After cells were either incubated with PK or mock treated, immunoprecipitations were conducted using an anti-UpaG antiserum and proteins were resolved by SDS-PAGE. (B) AD202 cells were transformed with a plasmid encoding ST–(GGS)33-UpaGΔ2 (pRS35). The experiment represented in panel A was repeated, except that cells were incubated with SpyCatcher instead of PK. Download FIG S5, PDF file, 0.3 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S6
Nonnative cysteine residues introduced into the passenger domain of UpaGΔ2 form disulfide bonds. AD202 and RI2 (AD202 dsbA::cm) cells transformed with a plasmid encoding HA-UpaGΔ2 (S1624C/S1652C) or HA-UpaGΔ2 (S1620C/S1678C) (pRS29 or pRS30) were subjected to pulse-chase labeling. Samples were divided in half, and one aliquot was incubated with EZ-Link maleimide-PEG2-biotin to biotinylate free cysteine residues. After immunoprecipitations were conducted using a monoclonal anti-HA antibody, biotinylated proteins were isolated from the treated aliquots using NeutrAvidin-agarose beads. Proteins were subsequently resolved by SDS-PAGE. Download FIG S6, PDF file, 0.5 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S7
The efficiency of the SpyTag/SpyCatcher reaction is not increased by the use of a Tris-based buffer. AD202 cells transformed with a plasmid encoding HA-UpaGΔ2 (S1624C/S1652C) (pRS29) were subjected to pulse-chase labeling. Cells were resuspended in spheroplast buffer (33 mM Tris [pH 7.0], 40% sucrose) and divided in half (intact cells) or incubated with lysozyme to permeabilize the OM before they were divided in half (permeabilized cells). SpyCatcher was then added to one of the two halves of each sample. Immunoprecipitations were conducted using an anti-UpaG antiserum, and proteins were resolved by SDS-PAGE. Unidentified bands that might represent breakdown products are labeled with an asterisk. Download FIG S7, PDF file, 0.3 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S8
Isolation of a high-molecular-weight crosslinking product by sequential immunoprecipitation. AD202 cells transformed with plasmids encoding pDULE-Bpa and HA-UpaGΔ2 (W1778am) or HA-UpaGΔ2 (S1624C/S1652C/1778am) (pRS45 or pRS46) were subjected to pulse-chase labeling and UV irradiation, and immunoprecipitations were conducted with a monoclonal anti-HA antibody. Immunoprecipitated proteins were then released by resuspending protein A beads in 50 μl 100 mM Tris base–8 mM EDTA–2% SDS–7% glycerol and heating them at 99°C for 5 min. After the beads were pelleted, 10 μl of each sample was placed on ice and the remaining 40 μl was mixed with 800 μl RIPA buffer and used for a second round of immunoprecipitations with a polyclonal antiserum raised against a BamA C-terminal peptide. Proteins isolated after both single and sequential immunoprecipitations were then resolved by SDS-PAGE. A larger portion of each sample was used for the sequential immunoprecipitations because the anti-BamA antiserum binds to BamA relatively weakly. Download FIG S8, PDF file, 1.1 MB.
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FIG S9
Expression of ST-UpaGΔ2 (S1624C/S1652C) in cells that produce a low level of BamA creates a synthetic lethal phenotype. AD202 and RS959 (bamA101) cells were transformed with a plasmid encoding either ST-UpaGΔ2 or ST-UpaGΔ2 (S1624C/S1652C) under the control of a rhamnose-inducible promoter (pRS47 or pRS48). Cells were then streaked on LB agar plates with or without 0.2% l-rhamnose and incubated overnight at 37°C. Download FIG S9, PDF file, 1.9 MB.
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TABLE S1
Plasmids and oligonucleotides used in this study. Download Table S1, DOCX file, 0.03 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
