(A) S. flexneri strains 2457T and 2457T icsP^- mutant (icsP^-) were grown in LB and whole cell lysate samples were taken at 0.5, 1, 1.5, 2, 2.5 and 3 h after subculture, followed by electrophoresis on a SDS 15 % polyacrylamide gel and Western immunoblotting with rabbit anti-IcsP antiserum; (B) S. flexneri strains 2457T, icsP^-and icsP^- harbouring pIcsP, pIcsP^HA or pBAD30 (as indicated) were grown in LB for 1.5 h to an OD600 reading of ∼0.4, washed 3 times, and induced with arabinose for 1 h. Pellet and supernatant protein samples were then prepared and electrophoresed on a SDS 15 % polyacrylamide gel, followed by Western immunoblotting with rabbit anti-IcsA antibodies. The size of the full length IcsA protein (120 kDa) and the cleaved form of IcsA (95 kDa) are indicated; (C) S. flexneri strains 2457T and icsP^- harbouring pIcsP^HA or pBAD30 were grown in LB as described in (B), followed by induction with 0%, 0.003%, 0.006%, 0.0125%, 0.025%, 0.05%, 0.1% or 0.2% (w/v) arabinose for 1 h. Whole cell lysates were prepared and electrophoresed on a SDS 15 % polyacrylamide gel, followed by Western immunoblotting with rabbit anti-IcsP antiserum. The size of the full length IcsP protein (36 kDa) is indicated in (A) and (C). Each lane contains 5 x 10⁷bacterial cells of each strain.

https://doi.org/10.1371/journal.pone.0070508.g001

(A) Smooth LPS 2457T icsP^- strains harbouring pIcsP^HA or pBAD30 were grown to an OD₆₀₀ reading of ∼0.8 in LB, washed 3 times, and treated without TP (U), with PMBN only (P) or with TP treatment for 2 h. Strains were then induced with 0.003% (w/v) arabinose for 1 h, washed 3 times, and grown for an additional 3 h for restoration (R) of LPS Oag; (B) Rough LPS 2457T icsP^-/rmlD^- strains harbouring pIcsP^HA and either pRMA727 or pACYC184 (as indicated) were grown to an OD₆₀₀ reading of ∼0.4 in LB, washed 3 times, and induced with arabinose for 1 h. LPS from strains described in (A) and (B) were isolated and detected by silver staining as described in the Methods. The first 15 Oag RUs are indicated on the side of each gel. Each lane contains ∼2 x 10⁸ bacterial cells of each strain; (C) Western blots on whole cell lysates obtained from strains in (B) were probed with rabbit anti-IcsP antiserum. The size of the full length IcsP^HA protein (∼36 kDa) is indicated. Each lane contains 5 x 10⁷ bacterial cells of each strain.

https://doi.org/10.1371/journal.pone.0070508.g004

Smooth LPS 2457T icsP^- strains harbouring pIcsP^HA were subcultured in LB broth to an OD₆₀₀ reading of ∼0.8, washed 3 times in LB, and then further cultured for 2 h; (A) in the absence of TP, (B) in the presence of PMBN only, or (C) in the presence of TP. Arabinose was included in the final hour of treatment at a concentration of 0.03% (w/v). Samples were then fixed and subjected to QD IF using antibodies to HA epitope and IcsA. Non-septating and septating cells (upper and lower rows respectively) are shown for each treatment group. Representative bacteria are shown. Scan  =  Single line-scans measuring the fluorescence intensity of IcsP^HA detected along the surface of the bacterium, Bars  =  1 µm, Arrows  =  direction of line-scan, Arrow heads  =  septa, Control  =  grown in absence of both tunicamycin and PMBN, P  =  PMBN, TP  =  tunicamycin/PMBN, GL  =  Grey level, Phase  =  phase contrast image, IcsP^HA  =  image of fluorescence at 525 nm, IcsA  =  image of fluorescence at 625 nm, Merge  =  overlay of IcsP^HA and IcsA images.

doi:10.1371/journal.pone.0070508.g005