Fig 1.
Internal cores of M. pneumoniae observed by negative-staining EM.
(A) Untreated cell bound to a carbon-coated grid. The membrane protrusion at the right pole is the attachment organelle. (B) Cell treated with 1% Tween 20. The cell was treated on a grid. The cell membrane was partially damaged, and the internal core remained at the right pole of the cell. (C) Magnified image of (B). (D) Core fraction isolated through sucrose-gradient centrifugation after cells were treated by 1% extraction of Triton X-100. (E) The boxed area of (D) is magnified. The same magnification was applied to (A), (B), and (E).
Fig 2.
EM images of an internal core.
The core can be divided into three parts: a terminal button, paired plates, and a bowl (wheel) complex. Yellow arrows indicate the boundaries between parts. Bold (A), slim (B), fork (C), and branched (D) types are aligned as the cell front on the right. A less-dense area in (C) is marked by an asterisk. (E-G) Structural features of bowl complex. Two typical images are magnified for the bowl (E), the paired plates (F), and the terminal button (G). Yellow arrowheads indicate the boundaries between parts. The original and colored images are shown in the adjacent panels in (E) and (G). Bold and slim structures are shown in the upper and lower panels, respectively. (E) The bowl complex can be divided into two parts, an arch (dark yellow) and accessories (light yellow). (F) The plate in the slim type has a bend around 60 nm from the back end, as marked by the yellow triangle. (G) The terminal button can be divided into a small oval (dark yellow) and accessories (light yellow). (H) Schematics and dimensions of bold (left) and slim (right) types of internal core images. Each length shown is the average of 40 structures.
Fig 3.
Protein components of an internal core.
(A) Protein profiles of the core fraction. The cells were treated by Triton X-100, fractionated by sucrose-gradient centrifugation, subjected to SDS-PAGE, and stained by CBB. Gel images of 12% and 8.75% polyacrylamide are shown in the left and right panels, respectively. The amount of fraction applied to each lane was adjusted to derive from the same number of cells. The molecular mass is shown on the left. The protein bands identified by PMF are marked by an arrow with the gene ID (MPN number) and the annotation [13]. The asterisks indicate the bands that were not identified by PMF. Gray boxes highlight the identical protein bands identified from the gels with both acrylamide concentrations. The gene ID and annotations are colored according to their categories (see the text). (B) Cell images labeled by EYFP for MPN066, MPN332, and MPN387. Phase-contrast and fluorescence images are merged. The cell images labeled for other proteins are shown in S2 Fig. (C) ORFs encoding the components of the attachment organelle.
Fig 4.
Localization of attachment organelle components determined by fluorescence in a living cell.
(A) Distribution of peak positions of fluorescence relative to the cell front. The cell front was defined from phase-contrast images as shown in S5 Fig. Gene identification number (ID) and protein annotation are indicated on the left. The average from 20 cells is marked by a black triangle and shown with standard deviation (SD) in each panel. The histograms are colored according to their categories shown in Fig 3 and S1 Table. (B) Fluorescence of EYFP fused to the C terminus and of ECFP fused to the N terminus of HMW2 in a cell. Fluorescence and phase-contrast images are merged. (C) Measurement of relative positions of EYFP and ECFP signals. Signal intensities along the yellow dotted line in (B) were plotted and traced as Gaussian distribution. (D) Distribution of the distances between two different proteins. Gene ID and its annotation are presented on the left with colors showing their fluorescence; EYFP (green), ECFP (cyan), and Cy3 (red). P1 adhesin was detected by immunofluorescence (IF) microscopy using a monoclonal antibody [32]. The average of 20 cells is shown with SD and marked by a black triangle in each panel.
Fig 5.
Localization of attachment organelle components determined by immunogold EM.
The cells expressing N-terminus EYFP-fusion proteins were treated by 0.3% Triton X-100, chemically fixed, and sequentially labeled by antiserum against EYFP and the secondary antibody conjugated with a 5-nm gold particle. Bold and slim core structures are shown in the left and the right panels, respectively, for each protein. Histograms show the distributions of the gold positions measured along the axis and these positions are traced by Gaussian distribution shown by a black line. The bar of the histogram is colored according to the categories shown in Fig 3. The peak positions from the terminus and the number of examined cores are shown in each panel.
Fig 6.
Schematic illustrations of the attachment organelle.
(A) Upper: Positions of internal proteins along attachment organelle determined from EYFP fluorescence. The average position in individual cells and SD relative to the cell edge are plotted with a black diamond and a colored bar, respectively. The proteins are mapped onto three localization groups, the terminal button (pink), the paired plates (green), and the bowl complex (blue). Middle: Distance between two signals measured from fluorescence microscopy. The positions of the terminal button, paired plates, and bowl complex are colored magenta, green, and cyan, respectively. Lower: Positions of internal proteins along a core determined from immunogold EM. The calculated peak and SD are presented as a black diamond and a bar, respectively. (B) Relations among four types of core structures. The slim, the fork, and the branched types are aligned to the EM grid in the common way. A possible reproduction cycle of an internal core is suggested by the arrow. (C) The attachment organelle including component proteins. The force is generated at the bowl complexes, is transmitted through the paired plates as indicated by a white arrow, and reaches the P1 adhesin complex.