Article Figures & Data
Figures
- FIG 1
Synthetic TAMPs and type II IMPs. (a) Schematic representation of the topology of the NG-WALP and NG-WALP-TolR constructs. (b) The constructs consist of N-terminal His-tagged mNeonGreen, followed by a flexible linker and a TMD with various degrees of hydrophobicity, as indicated by GRAVY scores. NG-WALP constructs contain the two residues GS and a C-terminal tail, whereas NG-WALKP-TolR constructs are extended with the periplasmic domain of TolR to create a (non-tail-anchored) type II IMP.
- FIG 2
Subcellular localization of NG-WALP and NG-WALP-TolR constructs. E. coli MC4100 cells expressing His-tagged NG-WALP and NG-WALP-TolR constructs were lysed and subjected to ultracentrifugation or fixed for fluorescence microscopy. (a and c) Whole-cell (WC), high-speed supernatant (HSS), and high-speed pellet (HSP) fractions were analyzed by Western blotting using anti-His serum. Lep was detected by specific antibodies as an inner membrane protein control. (b and d) A portion of the cells was fixed with formaldehyde and analyzed by fluorescence microscopy. Bars, 3 μm.
- FIG 3
Role of DnaJ/K in the targeting of NG-WALP and NG-WALP-TolR constructs. (a to o) NG-WALP and NG-WALP-TolR constructs were expressed in a DnaJ/K-knockout strain and its isogenic wild-type MC4100. Cells were fixed with formaldehyde and analyzed by fluorescence microscopy. mNeonGreen (NG) was used as a cytoplasmic control protein. Bars, 3 μm.
- FIG 4
Role of SRP in the targeting of NG-WALP and NG-WALP-TolR constructs. (a to o) NG-WALP and NG-WALP-TolR constructs were expressed under depleting and nondepleting conditions in the conditional Ffh strain HDB51. Cells were fixed with formaldehyde and analyzed by fluorescence microscopy. mNeonGreen (NG) was used as a cytoplasmic control protein. Bars, 3 μm.
- FIG 5
Role of YidC in the membrane insertion of NG-WALP and NG-WALP-TolR constructs. (a to o) NG-WALP and NG-WALP-TolR constructs were expressed under depleting and nondepleting conditions in the conditional YidC strain MK6s. Cells were fixed with formaldehyde and analyzed by fluorescence microscopy. mNeonGreen (NG) was used as a cytoplasmic control protein. Bars, 3 μm.
- FIG 6
Role of SecYEG in the membrane insertion of NG-WALP and NG-WALP-TolR constructs. (a to o) NG-WALP and NG-WALP-TolR constructs were expressed under depleting and nondepleting conditions in a strain conditional for the essential SecYEG component SecE. Cells were fixed with formaldehyde and analyzed by fluorescence microscopy. mNeonGreen (NG) was used as a cytoplasmic control protein. Bars, 3 μm.
- FIG 7
NG-WALPs with low-hydrophobicity TMDs can insert spontaneously into protein-free liposomes. Purified NG and NG-WALPs were incubated with protein-free liposomes consisting of 75% PE and 25% PG. Subsequently, samples were subjected to sucrose density gradient centrifugation, and three fractions were taken from top to bottom (1 to 3). Fractions were analyzed by Western blotting using anti-His serum.
Supplemental Material
FIG S1
Membrane insertion analyses of NG-WALP and NG-WALP-TolR constructs. (a and c) Crude membranes from E. coli MC4100 expressing NG-WALP and NG-WALP-TolR constructs acquired as described in the legend to Fig. 2 were extracted with PBS or 2% DDM. Subsequently, samples were centrifuged and supernatant (S) and pellet (P) fractions were analyzed by Western blotting using anti-His and anti-Lep sera. Lep served as inner membrane protein control. (b and d) E. coli MC4100 cells expressing NG-WALP and NG-WALP-TolR constructs were converted to spheroplasts and treated with PBS, ProtK alone, or ProtK and Triton. Samples were analyzed by Western blotting using anti-His, antiopsin, and anti-TF sera. TF was used as a cytoplasmic protein control. Truncated forms of NG-WALP-TolR-E/F of ∼30 kDa that also appear in PBS-treated samples might indicate intrinsic cleavage of these constructs. Download FIG S1, TIF file, 1.0 MB.
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FIG S3
Fluorescence profiles MC4100 and MC4100ΔdnaJ/K cells (a to n). For Fig. S3 to S6, software ImageJ, plugin ObjectJ, Coli-Inspector, and CrossProfilesMacro1.0 were used. Cross profiles of cells (n) were used to calculate the average fluorescence profile perpendicular to the cell axis of MC4100 (+ DnaJ/K) and MC4100ΔdnaJ/K (-DnaJ/K) cells expressing NG-WALP and NG-WALP-TolR constructs. Cell count was determined automatically by the software, and variation stems from clumping of cells leading to different cell densities on the agarose pad. Download FIG S3, TIF file, 2.7 MB.
Copyright © 2019 Peschke et al.
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FIG S7
Center/border ratio of the fluorescence signal (a to h). Center/border ratios of the fluorescence signal were calculated from cross profiles (see Fig. S3 to S6). Black lines indicate the median of the center/border ratios automatically calculated by software GraphPad Prism8. NG was used as a cytoplasmic control protein. Download FIG S7, TIF file, 1.9 MB.
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FIG S2
Control blotting assays for depletion and deletion strains. NG-WALP and NG-WALP-TolR constructs were expressed in a DnaJ/K-knockout strain, its isogenic wild-type strain, and strains conditional for the expression of Ffh, YidC, and SecE (Fig. 3 and 6). (a) Whole-cell samples of E. coli MC4100ΔdnaJK (−) and MC4100 (+) were analyzed by Western blotting using anti-DnaK serum. (b to d) Levels of Ffh and YidC in strains HDB51, MK6s, and CM124 were checked under depleted (−) and nondepleted (+) conditions using anti-Ffh and anti-YidC sera. For strains HDB51 and MK6s, successful processing of SurA was used as an overdepletion control. Control samples (Ctrl) showing accumulation of unprocessed SurA were loaded for HDB51 and MK6s samples. The pre-SurA control sample was obtained from whole cells of the temperature-sensitive SecA strain MM52 grown under nonpermissive conditions. For CM124, accumulation of pre-SurA served as a control for successful depletion of SecE. Download FIG S2, TIF file, 1.6 MB.
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FIG S4
Fluorescence profiles of HDB51 cells (a to n). Cross profiles of cells (n) were used to calculate the average fluorescence profile perpendicular to the cell axis of HDB51 cells expressing NG-WALP and NG-WALP-TolR constructs under nondepleted (+SRP) and depleted (-SRP) conditions. Cell count was determined automatically by the software, and variation stems from clumping of cells leading to different cell densities on the agarose pad. The filamentation of cells in the absence of Ffh additionally creates a big variation in cell count compared to cells that properly separate. Download FIG S4, TIF file, 2.7 MB.
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FIG S5
Fluorescence profiles of MK6s cells (a to n). Cross profiles of cells (n) were used to calculate the average fluorescence profile perpendicular to the cell axis of Mk6s cells expressing NG-WALP and NG-WALP-TolR constructs under nondepleted (+YidC) and depleted (-YidC) conditions. Cell count was determined automatically by the software, and variation stems from clumping of cells leading to different cell densities on the agarose pad. Download FIG S5, TIF file, 2.7 MB.
Copyright © 2019 Peschke et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S6
Fluorescence profiles of CM124 cells (a to n). Cross profiles of cells (n) were used to calculate the average fluorescence profile perpendicular to the cell axis of CM124 cells expressing NG-WALP and NG-WALP-TolR constructs under nondepleted (+SecE) and depleted (-SecE) conditions. Cell count was determined automatically by the software, and variation stems from clumping of cells leading to different cell densities on the agarose pad. Download FIG S6, TIF file, 2.7 MB.
Copyright © 2019 Peschke et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S1
List of PCR primers used to create the constructs in this study. Download Table S1, DOCX file, 0.01 MB.
Copyright © 2019 Peschke et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S2
List of plasmid constructs used in this study. Download Table S2, DOCX file, 0.01 MB.
Copyright © 2019 Peschke et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
