Phage, plasmid and bacterial strains
M13 phage was from our lab collection . M13am9 with an amber mutation in the second codon of gIX was constructed by site-directed mutagenesis . For the construction of gp9-T7, gp9-DT7, gp9-HA and gp9-DHA RF-DNA of M13mp19 served as template for PCR amplification. The PCR amplified gIX was subcloned into pMS119  and an unique MunI restriction site was introduced by QuikChangeTMin vitro mutagenesis between the codons 2 and 3. Into this site RF-DNA of M13mp19 served as template for the amplification of gIX by PCR. The gIX fragment was subcloned into pMS119, DNA fragments encoding the T7 and HA tag sequences were introduced by ligation, resulting in pMS-g9-T7 and pMS-g9-HA. Also, longer epitopes were introduced to construct pMS-g9-DT7 and pMS-g9-DHA, respectively. For protein expression and complementation experiments E. coli K38 (HfrC T2R relA1 pit-10 spoT1 tonA22 ompF627 phoA4 λ-)  was transformed as a non-suppressor strain. E. coli K37 (HfrC supD32 relA1 pit-10 spoT1 tonA22 ompF627 phoA4 T2R λ-) [19, 20] was used as a suppressor strain and E. coli JS7131 (MC1060 ΔyidC attB::R6Kori ParaBADyidC+ Specr) as a depletion strain of the membrane insertase YidC .
Complementation test of phage expressing modified gp9 proteins
On agar plates 4 mL melted LB top agar (47°C) containing 1 mM IPTG was mixed with 500 μL of a fresh E. coli K38 overnight culture bearing either pMS-g9/7 pMS-g9-T7, pMS-g9-DT7, pMS-g9-HA or pMS-g9-DHA. After solidification of the top agar, 10 μL of a phage suspension was applied on top of the agar from serial dilutions of a phage stock. Plaque formation was observed after incubation at 37°C overnight.
Expression of the modified gp9 proteins
2 mL cultures of E. coli K38 bearing plasmids encoding a respective gp9 variant were grown at 37°C to the early exponential phase in M9 minimal medium. Protein expression was induced by adding 1 mM IPTG and 10 min later the newly synthesised proteins were pulse-labelled for 10 min with 20 μCi 35S-methionine. To remove the non-incorporated 35S-methionine the total bacterial proteins were precipitated with 12% TCA on ice overnight, washed with cold acetone and resuspended in 10 mM Tris/HCl 2% SDS, pH 8.0. The samples were immunoprecipitated with antiserum to the T7 tag (Novagen) or to the HA tag (Roche), respectively, and analysed by SDS tricine PAGE and phosphorimaging.
Membrane insertion of gp9
To test the membrane insertion of gp9, E. coli K38 bearing pMS-g9-T7 was grown to the early exponential phase in M9 minimal medium. Cells were induced for 10 min with 1 mM IPTG and labelled with 35S-methionine for 10 min. To generate spheroplasts, the cells were centrifuged at 12 000 g for 3 min and resuspended in 500 μL of ice-cold spheroplast buffer (40% w/v sucrose, 33 mM Tris/HCl, pH 8.0). Lysozyme (5 μg/mL, final concentration) and 1 mM EDTA were added for 15 min. Aliquots of the spheroplast suspension were incubated on ice for 1 h either in the presence or absence of 0.5 mg/mL proteinase K. The samples were precipitated with 12% TCA, washed with cold acetone and resuspended in 10 mM Tris/HCl, 2% SDS, pH 8.0 and immunoprecipitated with antibodies against T7, OmpA (a periplasmic control), or GroEL (a cytoplasmic control). Samples were analysed by SDS tricine PAGE and phosphorimaging.
In vivo assay of YidC dependent membrane insertion
To test the requirement of YidC for the membrane insertion of gp9-T7, the YidC depletion strain E. coli JS7131 bearing pMS-g9-T7 was grown to the early exponential phase in LB with 0.2% arabinose. After back-dilution, the cells were grown in M9 minimal medium with either 0.2% arabinose (YidC+) or 0.2% glucose (YidC-) for 2 h. To induce expression of gp9-T7, 1 mM IPTG was added and after 10 min the cells were pulse-labelled with 35S-methionine for 10 min and then converted to spheroplasts by lysozyme treatment as described above. Samples were immunoprecipitated with antibodies to T7, OmpA (a periplasmic control), or GroEL (a cytoplasmic control). For testing the YidC depletion, samples of the cultures were drawn and precipitated with TCA (12%, final concentration), washed with cold acetone, resuspended in 10 mM Tris/HCl, 2% SDS, pH 8.0 and analysed by SDS/PAGE and Western blot using YidC antiserum.
M13am9 phage presenting gp9 variant proteins
50 mL cultures of E. coli K38 cells harbouring either pMSg9-T7, pMSg9-DT7, pMSg9-HA or pMSg9-DHA were grown at 37°C in LB-medium to a density of 2 × 108 cells/mL. The expression of the gp9 variant proteins was induced by adding 1 mM IPTG and the cells were infected with M13am9 at m.o.i 10. Adsorption of the phage was allowed for 5 min at room temperature without shaking. Subsequently, the infected cells were shaken overnight at 37°C. The phage was harvested from the supernatant after removing the cells by centrifugation. Then, the phage titer was determined by serial dilutions on E. coli K37. Every dilution was plated three times on LB agar plates to control variations in plating and pipetting. The agar plates were incubated at 37°C overnight and the plaques were counted and averaged for each dilution step.
For detection of the plasmid-encoded variants on the phage via dot-blot, serial dilutions of the above described phage stocks were prepared resulting in equal amounts of phage particles/400 μL for every variant. 400 μL of each suspension was adsorbed on a nitrocellulose membrane (Hybond ECL Nitrocellulose, Amersham) via dot-blot equipment (MiniFold®, Schleicher & Schuell) and treated overnight with blocking solution (1x Tris-buffered saline (TBS) pH 8, 5% non-fat dry milk w/v). The blot was washed three times with 1x TBS and incubated with antiserum to M13 gp8, to T7 or to HA tag, respectively. The presence of gp9 variants was analysed with a secondary peroxidase-coupled antibody by chemoluminescence.
Immunogold labelling of M13gp9 variant phage for TEM
For testing the exposure of an antigenic epitope 50 μL of each phage stock solution (about 1011 phage/mL) of M13gp9-DT7 and M13gp9-DHA was incubated with 1 × TBS containing 0.1% BSA for 30 min to avoid unspecific binding of the primary antibody to the sample. Each sample was then incubated with the respective serum (diluted 1:20 in 1x TBS) for 1 h. Then, protein A coupled immunogold particles (Protein A - 20 nm colloidal gold, Sigma-Aldrich) was added 1:20 in 1x TBS for 1 h. After immunogold labelling, 10 μL of the phage stock solution was adsorbed on carbon-coated copper grids (Athene 200, Plano, Wetzlar/Germany) that had been glow discharged shortly before use . The suspensions were allowed to adsorb for 5 min, unbound material was removed by touching the grid to filter paper. The grid was then washed by touching the surface of a drop of distilled water for 2 sec. The excess water was removed by touching the grid to filter paper. A drop (5 μL) of 5% phosphotungstic acid (pH 7) was then applied to the grid and after 30 sec the excess stain was removed by touching the grid to a drop (50 μL) of ddH20 for 2 sec. The excess liquid was drawn off with filter paper. The grid was dried at room temperature and examined by electron microscopy.