Figure 1

Subcellular localization of ClyA and ClyA fusions. (a) Electron micrograph of vesicles derived from JC8031 cells expressing ClyA-GFP. Bar is equal to 100 nm. (b) Z-average particle size of 1 ml vesicle suspensions containing ~30 µg/ml total protein obtained from plasmid-free or ClyA-GFP-expressing JC8031 cells. Error bars represent the standard deviation of 3 replicates. (c) Western blot of vesicle fractions isolated from E. coli strain JC8031 expressing GFP, ClyA-GFP and GFP-ClyA. Blot was probed with anti-GFP serum. (d) Western blot and (e) GFP fluorescence of periplasmic (per), cytoplasmic (cyt) and vesicle (OMV) fractions generated from JC8031 or BW25113 nlpI::Kan cells expressing ClyA-His6, ClyA-GFP and GFP-ClyA. ClyA-His6 blot was first probed with anti-polyhistidine. ClyA-GFP and GFP-ClyA blots were probed with anti-GFP. Following stripping of membranes, blots were reprobed with anti-OmpA serum or anti-DsbA serum as indicated. All fractions were generated from an equivalent number of cells. (f) Fluorescence microscopy of vesicles generated from JC8031 cells expressing ClyA-His6, ClyA-GFP and GFP-ClyA.