Immunoblot analyses of PrtV expression and secretion and ultrastructural analysis of <i>V</i>. <i>cholerae</i> surface structures.

<p>(A) Immunoblot analysis of expression and secretion of PrtV in different <i>V</i>. <i>cholerae</i> O1 isolates. Bacterial strains were grown at 30°C and samples were collected at OD<sub>600</sub> 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 <i>V</i>. <i>cholerae</i> El Tor O1 strains A1552, C6706, and P27459 respectively. (B) Ultrastructural analysis of <i>V</i>. <i>cholerae</i> 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 <i>V</i>. <i>cholerae</i> isolates. Immunoblot analysis of OMVs from different <i>V</i>. <i>cholerae</i> 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 <i>V</i>. <i>cholerae</i> non-O1/non-O139 serogroup: V:52, V:5/04, V:6/04, KI17036, 93Ag19, and NAGV6; lanes 7–9: OMVs from <i>V</i>. <i>cholerae</i> O1 El Tor clinical isolates: P27459, C6706, and A1552; lane 10: OMVs from <i>V</i>. <i>cholerae</i> classical O1 strain 569B; lanes 11–13: OMVs from <i>V</i>. <i>cholerae</i> O1 environmental isolates: AJ4, AJ3, and AJ2. Lane 14, C6706 Δ<i>prtV</i> mutant.</p>