Assembly of the Type II Secretion System such as Found in Vibrio cholerae Depends on the Novel Pilotin AspS
(A) Alignment of a representative subset of the secretin sequences used in this study to demonstrate sequence conservation (darker to lighter shades of green represent higher to lower levels of sequence conservation). Accession numbers for all secretins investigated in this study are given in Table S2. (B) S-domain sequences from the secretins were subject to CLANS analysis . The position corresponding to each S-domain sequence from the Vibrio-type EpsD and GspD proteins is represented by red dots, ExeD by orange dots and the group circled in red. The position corresponding to each sequence from the Klebsiella-type PulD, OutD, EtpD and GspD proteins is colour-coded in blue. The connections shown represent an E-value cut-off of 1e−10. (C) E. coli BL21(DE3)(ΔgspD,ΔaspS) complemented with either pETDuet (GspD-C4+AspS) or pETDuet (GspDΔS-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 and incubated with a modified sample buffer containing Lumio reagent, analysed by SDS-PAGE and imaged by fluorimetry. (D) Size-exclusion chromatography profiles of the purified AspS (red), the purified MBP-S-domain fusion (green) and the complex of AspS and MBP-S-domain fusion (blue) on a Superdex200 column. An SDS-PAGE gel of the peak fractions of AspS-MBP-S-domain complex shows an approximately stoichiometric ratio of the two proteins. A280, absorbance at 280 nm; mAU, milli absorbance units. Figure S5 shows the results of the control experiment, where AspS and MBP without S-domain do not interact.