Cryo transmission electron micrographs of the purified PorK/N complex.

<p>(A) First column -top views of the complex, second column- side views, third column—tilted views. The side views show two major rings and within each major ring two distinct sub-rings were observed, shown by the black arrow heads. Note the thin electron dense band between the two major rings, shown by the white arrow head, which may represent the lipid tails overlap of the PorK lipoprotein. (B) Purified native PorK/N complex from the wild type has the same structure as from the mutant (W50ABK*WbaP) at that resolution. (C) LDAO treated complex, the association between the two major rings has been ablated and single complexes are now noticeable. Scale bars 20 nm. (D) A 3-D reconstruction of the PorK/N ring. (E) Schematic representations of the PorK/N rings. The double major ring form (<i>in-vitro</i>) is proposed to be an artifact of the two major rings interacting via the PorK lipid shown in purple. Whereas the <i>in-vivo</i> form is proposed to be a single major ring composed of PorN (dark blue) and PorK (light blue) with the PorK lipid (purple) involved in anchorage to the OM which is represented by two white parallel lines.</p

A proposed model for the roles and interactions of PorK, PorL, PorM, PorN and PG0189 in the T9SS.

<p>PorK and PorN interact to form a ring-shaped structure that is localised in the periplasm and tethered to the outer membrane via the PorK lipid moieties (lipid shown as black line). This structure may be further stabilised by its association with the PG0189 outer membrane protein. It is proposed that the PorK and PorN rings assemble around the periplasmic extensions of the unknown OM secretion pore. Both PorL and PorM have transmembrane spanning domains and are proposed to transduce energy from the inner membrane (PMF) or cytosol (ATP) and power secretion of the T9SS substrates through the transient interactions with the PorK/N complex. The topology of the PorL and PorM inner membrane proteins is not known.</p

Electron micrographs of negatively stained PorK/N complex.

<p>Complexes in fraction 6 of CsCl density gradients were stained with uranyl acetate and observed under TEM. (A) Homogenous ring-shaped structures of PorK/N complex. Scale bar 100 nm (B) Top view of the complex forming a ring with 32–36 subunits (white arrow heads). Scale bar 20 nm. (C) The first two columns show side views of the complex where two major rings were observed (black arrowheads). Scale bar 20 nm. The third column shows the tilted view of the complex verifying the presence of two rings. (D) A higher resolution image obtained by virtual section (0.76 nm) through an electron tomogram of a negatively stained complex. At this resolution individual subunits (32) were observed. Scale bar 20 nm.</p

PorK/N complex isolated from <i>P</i>.<i>gingivalis</i>.

<p>(A) An attempt at the purification of PorKLMN complexes from W50ABK*WbaP using a published method (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005820#sec015" target="_blank">methods</a> section). All fractions from CsCl density gradients were resolved by SDS-PAGE and only fraction 6 had protein bands which is shown in the figure. Indicated bands were identified by mass spectrometry. (B) The PorK and PorN complex was purified from W50ABK*WbaP using a modified protocol (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005820#sec015" target="_blank">methods</a> section). Fractions from CsCl density gradients were resolved by SDS-PAGE and visualized by Coomassie stain. PorK and PorN were identified by mass spectrometry (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005820#ppat.1005820.t001" target="_blank">Table 1</a>). (C) Blue native PAGE analysis of purified PorK/N complexes. Lane S- is bovine heart mitochondria solubilized in 1% digitonin. The protein bands corresponding to molecular masses of ≥ 10000 kDa and 2400 kDa bands were identified by mass spectrometry as pyruvate dehydrogenase complex and the tetrameric form of ATP synthase, respectively.</p

Identification of PorK and PorN protein bands by mass spectrometry.

<p>Identification of PorK and PorN protein bands by mass spectrometry.</p

Absence of EDSL on <i>porK</i>, <i>porN</i>, <i>porL</i> and <i>porM</i> mutants.

<p>Cryo-EM micrographs of whole cells (A) Wild type (B) <i>porK</i> mutant (C) <i>porN</i> mutant (D) <i>porL</i> mutant and (E) <i>porM</i> mutant. A magnified section is shown on the bottom of each image. Arrows point to the electron-dense surface layer (EDSL) that is absent in the mutants. Scale bars 100 nm.</p

Electron micrographs showing presence of large (50 nm) rings in <i>porL</i>, <i>porM</i> and <i>porP</i> mutants but not in <i>porK</i> and <i>porN</i> mutants.

<p>Preparations of large protein complexes were isolated from wild type lacking gingipains (33277ABK), <i>porK</i>, <i>porN</i>, <i>porL</i>, <i>porM</i> and <i>porP</i> mutants. The samples were negatively stained and viewed under the electron microscope. White arrows indicate ring structures that are consistent with the PorK/N complex. Black arrows indicate a smaller ring (~ 30 nm) that was distinct from the PorK/N complex and present in all samples. Scale bars, 200 nm.</p

Identification of PorL and PorM protein bands by mass spectrometry.

<p>Identification of PorL and PorM protein bands by mass spectrometry.</p

Determination of subcellular localization using proteinase K accessibility.

<p><i>P</i>. <i>gingivalis</i> cells (W50ABK*WbaP) were either lysed using a French Pressure cell or were left unlysed. Cells were treated with proteinase K and aliquots were collected at 0 (10s), 1, 4 h and after overnight incubation. Samples were subjected to SDS-PAGE followed by immunoblot analysis using antisera against the protein indicated on the right. The Coomassie blue stained gel shows the relative loading amount.</p

Chemical cross-linking of PorK/N complexes.

<p>Purified PorK/N complexes were cross-linked with BS3 for 15 min at room temperature. SDS gel loading dye was added to the samples and boiled for 5 minutes. Proteins in the samples were separated on SDS-PAGE gel, transferred to a nitrocellulose membrane and probed with PorK and PorN antibodies. The PorK and PorN monomers together with putative cross-linked dimers are labeled.</p