Immunoblot analyses of PrtV expression and secretion and ultrastructural analysis of V. cholerae surface structures.

(A) Immunoblot analysis of expression and secretion of PrtV in different V. cholerae O1 isolates. Bacterial strains were grown at 30°C and samples were collected at OD600 2.0. Samples of whole cell extracts from overnight cultures (lanes 1–3, 5 μl) and culture supernatants (lanes 4–6, 10 μl, corresponding to tenfold concentration compared with the whole cell samples) were loaded in the gel. Immunoblotting was done using anti-PrtV polyclonal antiserum. Lanes 1–3; whole cell lysates; lanes 4–6: culture supernatants from wild type V. cholerae El Tor O1 strains A1552, C6706, and P27459 respectively. (B) Ultrastructural analysis of V. cholerae by electron microscopy. An electron micrograph showing the flagella (open arrows) and OMVs (closed arrows). Bar, 500 nm. (C) PrtV association with OMVs from different V. cholerae isolates. Immunoblot analysis of OMVs from different V. cholerae isolates using PrtV polyclonal antiserum. Bacterial strains were grown at 30°C for 16 h and OMVs were isolated using the procedure described in Materials and Methods. 10 μl of OMV samples were loaded for immunoblot analyses and SDS-PAGE analyses by Coomassie blue staining. Lanes 1–6; OMVs from V. cholerae non-O1/non-O139 serogroup: V:52, V:5/04, V:6/04, KI17036, 93Ag19, and NAGV6; lanes 7–9: OMVs from V. cholerae O1 El Tor clinical isolates: P27459, C6706, and A1552; lane 10: OMVs from V. cholerae classical O1 strain 569B; lanes 11–13: OMVs from V. cholerae O1 environmental isolates: AJ4, AJ3, and AJ2. Lane 14, C6706 ΔprtV mutant.