Assembly of the Type II Secretion System such as Found in Vibrio cholerae Depends on the Novel Pilotin AspS
(A) The expression cassette in pETDuet plasmids GspD-C4, GspD-C4+AspS and GspD-C4+YacC are represented diagrammatically. The pETDuet-1 vector (Novagen) has two multi-cloning sites (MCS) represented as black squares, and cloning into the NcoI and NdeI sites is optimal with respect to the ribosome-binding sites: NcoI and HindIII sites were used to clone the open-reading frame corresponding to GspD-C4; NdeI and XhoI sites were used to clone the open-reading frame corresponding to AspS and YacC. The T7 terminator sequence (T) in the plasmid is represented by a black triangle. (B) E. coli BL21(DE3)(ΔgspD,ΔaspS) complemented with pETDuet (GspD-C4) or pETDuet (GspD-C4+AspS) were cultured to an OD600 of ∼0.6 and IPTG was added to the culture (0.1 mM, final concentration). At the indicated time-point cell extracts were prepared from the cultures using Lumio analysed by SDS-PAGE and imaged by fluorimetry. (C) The strains described above were cultured to an OD600∼1, extracted and then fractionated by sucrose density centrifugation. Replicate samples were analysed by SDS-PAGE for detection of GspD multimers with Lumio reagent and immunoblotting for BamA and F1β.