Visualization and characterization of the pore formed by SpaP and SpaR.

<p>(A) Six selected class averages (4, 23, 29, 36, 55, 82) of negative-stained isolated SpaPR complexes imaged by electron microscopy. The length of the scale bar represents 50 Å. The two class averages at the top represent the SpaP₅ complex. Arrowheads in the class averages in the middle and at the bottom represent the anticipated position of SpaR on the SpaP₅ ring. The complete picture of all class averages can be seen in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006071#ppat.1006071.s009" target="_blank">S4 Fig</a>. (B) Fluorescent streptavidin detection of SDS PAGE-separated biotin maleimide-labeled proteins of whole cell lysates, cell culture supernatant, periplasmic fraction, or cytoplasmic fraction of <i>S</i>. Typhimurium Δ<i>prgHIJK</i>, <i>flhD</i>::<i>tet</i> moderately overexpressing indicated proteins from a medium copy number plasmid (pT12). (C) Blue native PAGE and immunodetection of a high molecular weight complex formed by EPEA-tagged SpaP alone.</p

SpaP-SpaP interactions analyzed by <i>in vivo</i> photocrosslinking and sequence co-variation.

<p>(A) Immunodetection of SpaP^FLAG on Western blots of crude membrane samples of <i>E</i>. <i>coli</i> BL21 (DE3) expressing SpaP_T15X^FLAG in the absence of all other T3SS components. The sample is shown with and without UV-irradiation to induce photocrosslinking of <i>p</i>Bpa to neighboring interaction partners. (B) Immunodetection of chromosome-encoded SpaP^FLAG on Western blots of crude membrane samples of <i>S</i>. Typhimurium expressing plasmid-encoded SpaP_T15X. (C) Immunodetection of SpaP^FLAG and the inner MS ring protein PrgK on Western blots of crude membrane samples of <i>S</i>. Typhimurium expressing indicated SpaP-<i>p</i>Bpa mutants separated by 2-dimensional blue native/SDS PAGE. Full 2D gels are only shown for SpaP^FLAG scanned in the 800 nm channel. The 2D gel showing SpaP_M187X^FLAG +UV has been re-probed with antibody for PrgK and scanned in the 700 nm channel. PrgK indicates the position of the assembled needle complex. An overlay of FLAG and PrgK signals is shown on the right. The relevant slice of the 700 nm image showing PrgK at 25 kDa and the overlay of both channels showing the needle complex-associated bands have been aligned to the corresponding 2D image. (D) Interaction map of SpaP. Lines indicate predicted interactions with a normalized coupling score > 0.8 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006071#ppat.1006071.s003" target="_blank">S3 Table</a>) at positions with experimentally identified SpaP-SpaP crosslinks (at least from one side). Positions with experimentally observed SpaP-SpaP interactions are shown in black, target positions only predicted are shown in light blue. Grey shading indicates TM helices. Only positions within or in close proximity to TM helices are shown. Abbreviations: chr—chromosomal.</p

Interactions among the export apparatus components SpaP, SpaQ, SpaR, and SpaS.

<p>(A) Immunodetection of SpaR^FLAG on Western blots of SDS PAGE-separated crude membrane samples of Δ<i>spaPQRS S</i>. Typhimurium expressing indicated SpaP-<i>p</i>Bpa mutants from a pT10-<i>spaPQR</i>^FLAG<i>S</i> plasmid. (B) Immunodetection of SpaP^FLAG on Western blots of SDS PAGE-separated crude membrane samples of Δ<i>spaPQRS S</i>. Typhimurium expressing indicated SpaR-<i>p</i>Bpa mutants from a pT10-<i>spaP</i>^FLAG<i>QRS</i> plasmid. (C) Immunodetection of SpaS_N258A^FLAG on Western blots of SDS PAGE-separated crude membrane samples of <i>S</i>. Typhimurium expressing indicated plasmid-complemented SpaP-<i>p</i>Bpa mutants. (D) As in (C) but assessing the SpaP-SpaS interaction in absence of the inner ring protein PrgK. (E) Immunodetection of SpaP^FLAG on Western blots of SDS PAGE-separated crude membrane samples of <i>S</i>. Typhimurium expressing chromosome-encoded indicated SpaP-<i>p</i>Bpa mutants in the presence or absence of the inner ring protein PrgK. (F) As in (E) but showing SpaR_M209X^FLAG. (G) Immunodetection of SpaP^FLAG on Western blots of crude membrane samples of <i>E</i>. <i>coli</i> BL21 (DE3) expressing indicated SpaP-<i>p</i>Bpa mutants together with SpaQRS to form the SpaPR complex. (H) As in (F) but expressing SpaP_V170XQR^FLAGS to reveal the SpaP-SpaR interaction in <i>E</i>. <i>coli</i>.</p

Screen of protein-protein interactions of SpaP and SpaR by <i>in vivo</i> photocrosslinking.

<p>(A) Protter visualization of SpaP presenting predicted TM topology, positions analyzed by <i>in vivo</i> photocrosslinking (thick stroke), and identity of interactions (colored). (B) As in (A) but showing SpaR. (C) Immunodetection of SpaP^FLAG on Western blots of crude membrane samples of <i>S</i>. Typhimurium expressing indicated plasmid-complemented SpaP-<i>p</i>Bpa mutants separated by SDS PAGE. <i>p</i>Bpa mutations are denoted as “X”. Each sample is shown with and without UV-irradiation to induce photocrosslinking of <i>p</i>Bpa to neighboring interaction partners. Since the running behavior of crosslinked proteins often deviates from the calculated mass due to incomplete unfolding and since membrane proteins like SpaP often show an aberrant running behavior, the position of a crosslink on a gel does not easily allow drawing direct conclusions on the size of the crosslinked adduct. Crosslinked proteins identified by mass spectrometry or Western blotting are indicated. Other highlighted interactions shown in A and B were based on comparable SDS PAGE band pattern. (D) As in (C) but showing SpaR complemented from a low-copy number plasmid expressing SpaPQR^FLAGS. (E) As in (C) but expression of SpaP-<i>p</i>Bpa mutants from their chromosomal location. (F) As in (D) but expression of SpaR-<i>p</i>Bpa mutants from their chromosomal location. Abbreviations: J—PrgJ, P—SpaP, Q—SpaQ, S—SpaS.</p

Isolation and stoichiometry analysis of the SpaPR subcomplex of the needle complex.

<p>(A) Elution profile of the purified SpaPR^FLAG complex run on a Superdex 200 10/300 GL column. The peaks corresponding to the SpaPR^FLAG complex and 3xFLAG peptide are indicated. (B) Coomassie-stained SDS PAGE gel of purified SpaPR^FLAG complex and of its FLAG-deficient control (left). Immunodetection of SpaP (green) and SpaR^FLAG (red) on Western blot from purified SpaPR^FLAG complex separated by SDS PAGE (right). (C) Traces of indicated detector signals from size exclusion chromatography—multi angle laser light scattering of purified SpaPR^FLAG complex (left). ASTRA-calculated mass profile of total components of peak of purified SpaPR^FLAG complex (polypeptides and detergent, middle). ASTRA-calculated mass profile polypeptide components of peak of purified SpaPR^FLAG complex (right). (D) Native mass spectrum of the SpaPR^STREP complex. Peak series corresponding to the SpaP:SpaR^STREP complex in a 5:1 ratio is marked in red, with the most abundant charge state (14+) indicated. The peak series marked in blue corresponds to the same SpaPR complex bound to a ligand with a mass of approximately 710 Da, indicative of an associated phospholipid. Note that the measured mass for SpaPR heterohexamer (157.882 kDa) is heavier than the theoretically calculated mass (157.280 kDa). Abbreviations: Coo: Coomassie stained, WB: Western blot, RI: refractive index, LS: light scattering.</p

Models of SpaP, SpaR, SpaQ, SpaS, and PrgJ in the T3SS needle complex and its assembly.

<p>(A) Model of the central SpaP complex with surrounding export apparatus components SpaQ, SpaR, and SpaS, and direct connection to the inner rod formed by PrgJ. These results suggest that SpaP, SpaR, and PrgJ form the socket structure on the periplasmic side of the inner membrane patch of the base. (B) Model of a view of the membrane patch of the needle complex from the cytoplasmic side highlighting SpaP, SpaQ, SpaR, and SpaS. (C) Model of needle complex assembly. The unified Sct nomenclature [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006071#ppat.1006071.ref023" target="_blank">23</a>] is shown in parenthesis.</p