Proposed cell organisations of planctomycetes.

<p><b>A</b>) The cell plan for <i>G.obscuriglobus</i> proposed in the current publication. This cell plan mostly follows the established view in past publications (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091344#pone.0091344-Fuerst2" target="_blank">[4]</a>) including ribosome-less paryphoplasm and intracytoplasmic membrane (ICM), except for presentation of riboplasm, which now appears as multiple vesicles surrounding the nuclear body. Cell wall is indicated in red; cytoplasmic membrane - in dark blue; paryphoplasm - in yellow; riboplasm and nuclear body interior - in light blue; intracytoplasmic membrane - in green; inner nuclear body membrane - in brown; nucleoid DNA - in black; ribosomes – grey circles. <b>B</b>) The cell plan for <i>Pirellula</i>, which is considered as “simplest” among planctomycetes. A major internal compartment defined by an intracytoplasmic membrane (internal to cytoplasmic membrane bounding the protoplast) encloses a naked nucleoid. Unlike <i>G. obscuriglobus</i>, this bacterium thus does not contain a membrane-bounded nuclear body within the major internal pirellulosome compartment. Designation of the structures the same as for (<b>A</b>).</p

Distribution of the FtsK protein in <i>G. obscuriglobus</i> (A) and <i>E.coli</i> (B) cells.

<p>Immunogold labelling was performed on high-pressure frozen, cryosubstituted, and then thin-sectioned cells. <b>A</b>) In <i>G. obscuriglobus</i> cells FtsK is localised mostly to the interior of nuclear body (NB) and in riboplasm (R) compartments, but not to paryphoplasm (P). <b>B</b>) Instead, in <i>E.coli</i> cells FtsK is distributed along the cell periphery (arrowheads). Bar marker, 500 nm. Arrowheads indicate gold particles. Bar marker, 500 nm.</p

Tomographic reconstruction of a <i>G. obscuriglobus</i> cell and 3D models for nuclear envelope and riboplasm.

<p><b>A</b>) Transmission electron micrographs of thick-sectioned cryosubstituted (high-pressure frozen) cells showing internal <i>G. obscuriglobus</i> compartments. Nuclear body (NB) contains the nucleoid DNA (N), areas of riboplasm (R) contain ribosomes only and no fibrillar nucleoid DNA, and paryphoplasm (P) is ribosome-free. Areas where nuclear body envelope is surrounded by a single membrane are indicated by arrowheads, and where areas of this envelope surrounded by a double membrane by arrows. Numbers 1-4 indicate the order of appearance of a particular image within the tilt-series. Double-membrane nuclear envelope conformation in successive tilt-series is consistent with a continuous surface of double-membrane sheet in these regions, and consistency with membrane continuity is preserved also where single membrane appears to comprise the envelope in certain regions. Stars indicate the riboplasm vesicles used for the 3D model generation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091344#pone-0091344-g001" target="_blank">Figure 1C</a>). The whole cell reconstruction can be viewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091344#pone.0091344.s003" target="_blank">Movies S1</a>. Bar, 1 µm. (<b>B</b>) and (<b>C</b>) 3D models based on the results of electron tomography, from the cell viewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091344#pone-0091344-g001" target="_blank">Figure 1A</a> (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091344#pone.0091344.s003" target="_blank">Movies S1</a>), with extrapolations and manual adjustments every 10 slices. <b>B</b>) Nuclear body shown from two different angles. Bar marker, 500 µm. <b>C</b>) Riboplasm compartment (R) in the form of vesicles, completely surrounded by membranes, from front and back side (180°) views. Bar markers, 500 µm and 200 µm for the back-side view figure.</p

A model for mechanism of cell division of <i>G. obscuriglobus</i> cells.

<p>Step 1, the bud appears as a hump on the surface of a cell (Figures S5 and S6 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091344#pone.0091344.s001" target="_blank">File S1</a>). The nuclear body is divided, before or during the formation of a bud, forming two fully enveloped structures, as shown in step 2. Finally, one of the nuclear bodies migrates into a newly formed cell (step 3). Other riboplasm vesicles not containing nucleoid DNA are also transferred into the newly formed cell (Figure S6B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091344#pone.0091344.s001" target="_blank">File S1</a>). Cell wall is indicated in red; plasma membrane – blue; ICM – green; paryphoplasm – yellow; riboplasm – light blue; nucleoid – black; ribosomes – grey circles.</p

3-D reconstructions of the pore complex.

<p><b>(<i>A</i> and <i>B</i>)</b> Views of the 3-D reconstructions based on one spiral membrane from fraction 3 membranes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169432#pone.0169432.g004" target="_blank">Fig 4A</a>). Pore complexes (arrows) are visible as embedded structures in the surface of the envelope, shown as viewed from the inner side of the spiral in Fig <b>5A</b> and from the outer side in Fig <b>5B</b>. Fig <b>5C</b> shows the basket structure of one of these pores projecting from the inner side of the membrane spiral. Bars, 20 nm. <b>(<i>D</i> and <i>E</i>)</b> Reconstruction of architecture of a single pore seen from two different angles. In panel <b><i>D</i></b>, a side view of the pore displays the basket structure with its distal ring (arrowhead) and a series of struts (arrow) connecting with the main pore rings. In panel <b><i>E</i></b>, a top view shows the ring-like element (arrowhead) of the main part of the pore and a central plug structure is visible within the pore connected to the ring’s inner rim via spokes.</p

Pores in the membranes of <i>Gemmata obscuriglobus</i> released via sonication.

<p><b>(<i>A</i></b>) Transmission electron micrograph of a membrane fragment released from a lysed cell via sonication and negatively stained with ammonium molybdate. Large pores (arrows) with relatively electron-dense pore centers surrounded by a thin lighter inner ring and a thicker outer ring are seen. Smaller pore structures (arrowheads) are also visible and may represent either another class of pores or a result of a reverse view of the same large pores resulting from overlapping folds in the membrane (evidence for such structures is not derived from other microscopy methods). Bar, 100 nm. <b>Inset:</b> enlargement of boxed large pore in main Fig where a pore centre (PC), an inner ring (IR) and an outer ring (OR) can be distinguished. Bar, 50 nm. (<b><i>B</i></b>) TEM of a pore seen in negatively stained membrane fraction isolated from sonication-lysed cells, showing pore complex structure including outer ring (OR), inner ring (IR), spokes connecting inner and outer rings (S) and central plug (CP). Bar, 30 nm. <b>(<i>C</i>)</b> Enlarged view of the inner ring (IR) and central plug (CP) of the boxed pores in Fig 3A, the octagonal shape of the rings (especially visible if the outer edge of the outer ring is traced) is consistent with an eight-fold symmetry. Bar, 15 nm.</p

Protein composition of <i>Gemmata obscuriglobus</i> pore-containing membrane.

<p><b>(<i>A</i>)</b> SDS-PAGE gel showing that <i>G</i>.<i>obscuriglobus</i> cells have three different types of membranes. Exclusively pore-containing membranes (fraction 3) display a characteristic protein profile distinct from that of membrane fractions which do not possess pore structures. <b>(<i>B</i>)</b> Venn diagram showing the number and distribution of proteins among the fractions and (in brackets) the number of proteins with the beta-propeller folds. The members of the beta-propeller cluster belong either exclusively to fraction 3 (4 proteins), or to fractions 3 and 2 (2 proteins), and to fractions 2, 3 and 6 (2 proteins). No beta-propeller containing proteins were found exclusively in fractions 2 or 6. <b>(<i>C</i>)</b> A beta-propeller family found in fraction 3 (pore-containing membranes), including some exclusive to fraction 3. Cluster analyses revealed a set of proteins with conserved C-terminal regions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169432#pone.0169432.s017" target="_blank">S13</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169432#pone.0169432.s020" target="_blank">S16</a> Figs) that model beta-propeller folds with high (>95%) confidence. Models 3 (for protein ZP_02737072), 4 (ZP_02736670), 5 (ZP_02734776) and 6 (ZP_ZP_02734577) were deduced from proteins found exclusively in fraction 3 (pore-containing fraction); models 2 (for ZP_02737073) and 7 (for ZP_02733245) were deduced from proteins found in fractions 3 and 2 only; models 1 (for ZP_02737797) and 8 (for ZP_02731113)–for proteins found in fractions 3, 2, and 6 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169432#pone.0169432.s027" target="_blank">S4 Table</a>).</p

Model of the pore complex of <i>Gemmata obscuriglobus</i>.

<p>The pore complex is composed of at least two concentric upper rings (blue), and a lower ring (light blue) connected by struts to a distal ring (green) to form a basket structure. The central plug (purple) rests within the inner ring and spans the length of the pore. The whole pore complex rests within membrane (orange). The structure and dimensions are based on available data from all EM methods applied, from both whole cells and fraction 3 isolated membranes, and with minimal extrapolation, so that although the pore is probably not a hollow structure the space within the pore has not been filled in.</p

<i>Gemmata obscuriglobus</i> internal membrane pores as seen in freeze-fractured cells.

<p><b>(<i>A</i>)</b> Transmission electron micrograph of a platinum/carbon (Pt/C)-shadowed replica of a whole cell of <i>G</i>. <i>obscuriglobus</i> which has been prepared via the freeze-fracture technique. Bar, 100 nm. Inside the cell, a large spherical internal organelle consistent with the nuclear body organelle surrounding the nucleoid has been fractured (split) along and through the surface membranes of its envelope. Pores with a central core and at least one surrounding ring are visible on one region of one of the membranes of this organelle surface. Insets represent successive enlarged views of the boxed region in the main image displaying the pores at higher magnification. Bars, 100nm. At the highest enlargement the substructure of each of several pores can be resolved including central core and surrounding inner dark and outer light rings (right inset). (<b><i>B</i></b>) This micrograph of the whole cell reveals an apparently cross-fractured major internal organelle compartment and a membrane surface (boxed) representing a fracture through the membrane surrounding the organelle. Bar, 200 nm. <b>(<i>C</i>)</b> An enlarged view of the boxed region of the freeze-fractured cell seen in Fig 2B showing a region of a membrane surface where roughly circular pore structures (arrowheads) are visible, in some cases with two light rings surrounding a dark centre,. Bar, 50 nm.</p

Pores are inserted into the internal membranes of <i>Gemmata obscuriglobus</i> cells.

<p>(<b><i>A</i></b>) Transmission electron micrograph of a thin-section of a cryosubstituted cell of <i>G</i>. <i>obscuriglobus</i>, showing a portion of the nuclear body envelope, apparently consisting of two closely apposed membranes enclosing the fibrillar nucleoid DNA (N) (for evidence of DNA fibrillar nature in <i>G</i>. <i>obscuriglobus</i> see [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169432#pone.0169432.ref009" target="_blank">9</a>]. The membranes (arrows) are interrupted by a disc-like structure (indicated by arrowhead within the boxed region) consistent with a pore complex inserted between the membranes on either side. Bar, 50 nm. <b>(<i>B</i>)</b> Enlargement of the sectioned cell of <i>G</i>. <i>obscuriglobus</i> seen in Fig 1A, showing a disc structure (arrowhead) seen <i>en face</i>, situated between the folded double membranes of the nuclear body envelope on either side (arrows). Bar, 50 nm. <b>(<i>C</i>)</b> Transmission electron micrograph of cell lysed by grinding in liquid N₂, followed by negative staining of thawed cells with uranyl acetate. An internal membrane fragment (IM) possibly representing the nuclear body envelope or other internal compartment membranes appears to have been released from a lysed cell, and the mostly intact cell wall (CW) can also be seen. The membrane displays numerous evenly distributed pore structures on its surface, enlarged views of which can be seen in the inset. Bar, 500 nm. Inset shows enlargement of pore structures, which display a dense core surrounded by a light ring further surrounded by a dense ring. Bar, 50 nm. (<b><i>D</i></b>) Transmission electron micrograph of negatively stained preparation of a completely released internal compartment from cells lysed as in C. Pore structures are widely distributed over the membrane surface including within the boxed region. The ‘canoe’ shape is typical for pore-containing membranes. Bar, 500 nm. <b>(<i>E</i>)</b> An enlarged view of the boxed region in Fig 1D showing the large pore structures (arrows), each displaying dark pore centre regions, and lighter inner and outer ring structures, distributed densely on the membrane surface. Bar, 100 nm.</p