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

Identification of species-specific compounds in extracts of <i>S. arenicola</i> and <i>S. pacifica</i> by UHPLC-QToF-MS and PCA.

<p>(A) Total ion current (TIC) chromatograms of <i>S. arenicola</i> and (B) <i>S. pacifica</i> (C) TICs from the same (<i>S. arenicola</i>) subset of samples (three replicates) to highlight the reproducibility of acquisition. For ease of interpretation, chromatograms of 2 to 24 minutes are shown to illustrate the period of chromatography during which most compounds elute. Full chromatograms for both species are available in the Supplementary information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091488#pone.0091488.s006" target="_blank">Figure S6</a>). (D) Principal component analysis (PCA) scores plot, PC1 (t<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091488#pone.0091488-Paul1" target="_blank">[1]</a>) versus PC2 (t<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091488#pone.0091488-Phelan1" target="_blank">[2]</a>) showing the variation in the profiles of secondary metabolites from two species: <i>S. arenicola</i> (A, blue) and <i>S. pacifica</i> (P, red). Each symbol represents one bacterial strain described by all detected metabolites. (E) Inspection of the 2-D loadings plot for PC1 vs. PC2 reveals the variables responsible for the spatial arrangement of samples. (F) Extracted ion chromatogram (EIC) of <i>m/z</i> 754.3092 from both species, showing clear species differences in the abundance of this metabolite <i>S. arenicola</i> (blue) and <i>S. pacifica</i> (red). (G) MS spectrum for EIC peak. (H) Box-and-whisker plot of the abundance of the 754 ion in the two species (P<.0001).</p

Metabolic profiling distinguishes bacterial samples from two <i>Salinispora</i> species <i>S. arenicola</i> and <i>S. pacifica</i>.

<p>(<b>A</b>) OPLS-DA scores plot, predictive component t<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091488#pone.0091488-Paul1" target="_blank">[1]</a> versus orthogonal component (t₀<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091488#pone.0091488-Paul1" target="_blank">[1]</a>) showing the supervised separation between the two sample classes. The ellipse in A and B represents the Hotelling's T² 95% confidence interval for the multivariate data. (<b>B</b>) Loading S-plots derived from the LC-MS data set of two bacterial species. S-plot shows predictive component p<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091488#pone.0091488-Paul1" target="_blank">[1]</a> against the correlation p (corr) of variables of the discriminating component of the OPLS-DA model. (<b>C</b>) EIC of <i>m/z</i> 411 from all samples. (<b>D</b>) EIC of <i>m/z</i> 695 from two bacterial species. (<b>E</b>) Mass spectrum for <i>m/z</i> 411. (<b>F</b>) Box-and-whisker plot for <i>m/z</i> 411 – significantly present in <i>S. arenicola</i> but absent from <i>S. pacifica</i> (P<.0001) (<b>G</b>) Mass spectrum for <i>m/z</i> 695. (<b>H</b>) Box-and-whisker plot for <i>m/z</i> 695 – significantly present in <i>S. arenicola</i> but absent from <i>S. pacifica</i> (P<.0001).</p

Identification of six compounds from <i>S. arenicola</i> and <i>S. pacifica</i>.

<p>The proposed formula obtained after the PCA and OPLS-DA analysis according to high-resolution LC-QTOF-MS measurements.</p><p>*Overall sc°re calculated from the empirical formula match with the database search. ^**Neutral mass calculated for each compound.</p><p>^***Compound identification performed through the in-house accurate mass database match, MS/MS fragmentation and reference standards.</p

Chromatograms and mass spectra relating to the detection of rifamycin O.

<p>(<b>A</b>) LC-QToF-MS Total Ion Chromatogram (TIC) for <i>S. arenicola</i> strain MV0472 and (<b>B</b>) Mass spectrum of peak X and (<b>C</b>) Chromatogram for rifamycin O standard and (<b>D</b>) Mass spectrum of peak X (lower 2 panels). The retention times of X and rifamycin O standard are 24.5 and 24.55 min, respectively. The <i>m/z</i> of molecular ion of X and rifamycin O standards are 752.2985 and 752.2954.</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