Journal: Molecular microbiology

Article Title: Secretion and functional display of fusion proteins through the curli biogenesis pathway.

doi: 10.1111/mmi.12515

Figure Lengend Snippet: Fig. 1. ERD10 fused to CsgA is expressed on the surface of the E. coli bacteria. A. Representation of pNA1 and pNA36 vectors. pNA1 harbours 6xHis-tagged (H6) csgA under the control of the arabinose inducible PBAD promoter. pNA36 is derived from pNA1 by introducing ERD10 and a flexible linker with sequence SGSGSG (L) in the SmaI site in between csgA and H6. B–D. Immunofluorescence microscopy using a primary mouse anti-6xHis and a secondary anti-mouse AlexaFluor488 labelled antibody, of induced DH5α(pNA36) cells (B), DH5α(pBAD33) (C) or DH5α(pNA48), producing ERD10-6xHis in the periplasm (D). E. Dot blot analysis on whole cells using a primary mouse anti-6xHis antibody. LSR10 (i.e. MC4100ΔcsgA) or NVG1 (i.e. LSR10ΔcsgG) were tested, expressing either the empty vector (pBAD33), periplasmic ERD1O-6xHis (pNA48) or the csgA–ERD10-6xHis fusion (pNA36). Cells were left untreated (−) or treated with lysozyme and EDTA (+) in 20% sucrose prior to blotting. F. Anti-6xHis immunogold TEM of LSR10 (pNA36), scale bar is 100 nm. G. TEM micrographs of the negative control LSR10 (pBAD33), scale bar represents 200 nm.

Article Snippet: Samples were subjected to formic acid treatment and evaluated in Western blotting using an anti-6xHis mAb (MCA1396, AbD Serotec) or a rabbit anti-CsgA antiserum (kindly provided by M. R. Chapman).

Techniques: Bacteria, Control, Derivative Assay, Sequencing, Microscopy, Dot Blot, Expressing, Plasmid Preparation, Negative Control

Journal: Molecular microbiology

Article Title: Secretion and functional display of fusion proteins through the curli biogenesis pathway.

doi: 10.1111/mmi.12515

Figure Lengend Snippet: Fig. 2. Expression of different CsgA fusion proteins on the surface of bacteria. Immunofluorescence microscopy, using a primary mouse anti-6xHis and a secondary anti-mouse AlexaFluor488 labelled antibody of E. coli LSR10 expressing the different CsgA fusion proteins. LSR10 (pBAD33), harbouring the empty vector (pBAD33 in figure), LSR10 (pNA15) (A-Nb208), LSR10 (pNA32) (A-FedF), LSR10 (pNA30) (A-FimC), LSR10 (pNA34) (A-mCherry), LSR10 (pNA29) (A-RNase1), LSR10 (pNA31) (A-Bla), and LSR10 (pNA33) (A-PhoA).

Article Snippet: Samples were subjected to formic acid treatment and evaluated in Western blotting using an anti-6xHis mAb (MCA1396, AbD Serotec) or a rabbit anti-CsgA antiserum (kindly provided by M. R. Chapman).

Techniques: Expressing, Bacteria, Microscopy, Plasmid Preparation

Journal: Molecular microbiology

Article Title: Secretion and functional display of fusion proteins through the curli biogenesis pathway.

doi: 10.1111/mmi.12515

Figure Lengend Snippet: Fig. 3. Display of heterologous proteins fused to CsgA. A. Whole cell ELISA of MC4100 (CsgA) or E. coli LSR10 producing the different CsgA fusion proteins. Anti-6xHis (His) and anti-peptidoglycan (pep) were used as primary antibodies: results are normalized to anti-E. coli antibodies and shown in arbitrary units (A.U.). SD are shown for 3 independent experiments, done in triplicate. Statistics were done with the Mann–Whitney test, using pBAD33 as reference (for anti-pep response: *P < 0.05, **P < 0.001). B and C. Extracellular protease accessibility of proteins fused to CsgA. LSR10 cells harbouring different proteins fused to CsgA were treated with formic acid and cell lysates were subjected to SDS-PAGE and subsequent Western blotting using an anti-6xHis mAb (B) or an anti-DsbA antiserum (C). Prior to formic acid treatment, cells were incubated with proteinase K (Prot K) (+), or PBS buffer (−). As a control, LSR10 (pNA15) cells were subjected to sonication prior to Prot K treatment (A-Nb208 sonic). The symbol ‘§’ indicates the bands corresponding to the respective fusion proteins; ‘°’ indicates the band corresponding to the passenger proteins only.

Article Snippet: Samples were subjected to formic acid treatment and evaluated in Western blotting using an anti-6xHis mAb (MCA1396, AbD Serotec) or a rabbit anti-CsgA antiserum (kindly provided by M. R. Chapman).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, SDS Page, Western Blot, Incubation, Control, Sonication

Journal: Molecular microbiology

Article Title: Secretion and functional display of fusion proteins through the curli biogenesis pathway.

doi: 10.1111/mmi.12515

Figure Lengend Snippet: Fig. 5. CsgG-mediated secretion is compatible with small CsgA-fused passengers in non-extended conformation. A–D. Disulphide formation in Nb208 is necessary for GFP binding. (A and C) Anti-6xHis and anti-mouse AlexaFluor594 IF of induced LSR10 (pNA35) expressing CsgA–Nb208C22S (A) or MC1000 ΔdsbA (pNA15) expressing CsgA–Nb208 (C). (B and D) Exogenously added GFP fails to bind to induced LSR10 (pNA35) (B) or MC1000 ΔdsbA (pNA15) (D). E–H. The conformationally selective anti-FedF nanobody Nb231 recognizes folded FedF on the surface of bacteria. (E insert) Dot blot of boiled (B) and native FedF (NB), using Nb231. (E and F) IF using a FITC-labelled Nb231 of induced LSR10 (pNA32), expressing the CsgA–FedF fusion protein and untreated (E) or treated (F) with DTT and 2-ME prior to IF. (G and H) IF of induced MC1000 ΔdsbA (pNA32), stained with an anti-6xHis mAb and an anti-mouse AlexaFluor594 labelled secondary antibody (G) or with the FITC-labelled Nb231 (H).

Article Snippet: Samples were subjected to formic acid treatment and evaluated in Western blotting using an anti-6xHis mAb (MCA1396, AbD Serotec) or a rabbit anti-CsgA antiserum (kindly provided by M. R. Chapman).

Techniques: Binding Assay, Expressing, Bacteria, Dot Blot, Staining

Journal: Molecular microbiology

Article Title: Secretion and functional display of fusion proteins through the curli biogenesis pathway.

doi: 10.1111/mmi.12515

Figure Lengend Snippet: Fig. 6. TEM analysis of secreted CsgA–Nb208 deposits. A. Negative TEM image of LSR10 (pNA15) shows the predominant formation of a dense matrix of positively staining aggregates. B. MC4100 showing native curli fibres as revealed by negative staining TEM. C. Besides aggregates, TEM and Ni-NTA-gold (5 nm) staining shows LSR10 (pNA15) displays negatively staining filamentous threads that contain CsgA–Nb208-6xHis. D. Ni-NTA-gold labelled CsgA–6xHis fibrils as found on the surface of LSR10 (pNA1). Black bars indicate a 100 nm scale.

Article Snippet: Samples were subjected to formic acid treatment and evaluated in Western blotting using an anti-6xHis mAb (MCA1396, AbD Serotec) or a rabbit anti-CsgA antiserum (kindly provided by M. R. Chapman).

Techniques: Staining, Negative Staining

Journal: Molecular microbiology

Article Title: Secretion and functional display of fusion proteins through the curli biogenesis pathway.

doi: 10.1111/mmi.12515

Figure Lengend Snippet: Fig. 7. Western blotting and TEM analysis of secreted CsgA fusions and SDS-insoluble surface-bound filaments. A. Anti-6xHis Western blot analysis of cell lysates of LSR10 cells expressing CsgA–Nb208 (pNA15), CsgA–FedF (pNA32), CsgA–RNase1 (pNA29) or CsgA–ERD10 (pNA36), treated with (FA +) or without (FA −) formic acid. B–D. SDS-insoluble material was isolated from LSR10 cells expressing different fusion proteins, visualized by negative staining TEM in case of the CsgA–Nb208 fusion (B), or after formic acid treatment subjected to SDS-PAGE, followed by anti-6xHis (C) or anti-CsgA (D) Western blotting. Arrow, ‘°’ and ‘§’ indicate the band corresponding to SDS-insoluble CsgA fusions, the fused proteins and the various intact fusion proteins respectively. For unknown reasons, the CsgA–ERD10 fusion is poorly recognized by the anti-CsgA antibody. Black bar indicates a 100 nm scale.

Article Snippet: Samples were subjected to formic acid treatment and evaluated in Western blotting using an anti-6xHis mAb (MCA1396, AbD Serotec) or a rabbit anti-CsgA antiserum (kindly provided by M. R. Chapman).

Techniques: Western Blot, Expressing, Isolation, Negative Staining, SDS Page