FtsZ-mediated fission of a cuboid bacterial symbiont

Abstract

Less than a handful of cuboid and squared cells have been described in nature, which makes them a rarity. Here, we show how Candidatus Thiosymbion cuboideus, a cube-like gammaproteobacterium, reproduces on the surface of marine free-living nematodes. Immunostaining of symbiont cells with an anti-fimbriae antibody revealed that they are host-polarized, as these appendages exclusively localized at the host-proximal (animal-attached) pole. Moreover, by applying a fluorescently labeled metabolic probe to track new cell wall insertion in vivo, we observed that the host-attached pole started septation before the distal one. Similarly, Ca. T. cuboideus cells immunostained with an anti-FtsZ antibody revealed a proximal-to-distal localization pattern of this tubulin homolog. Although FtsZ has been shown to arrange into squares in synthetically remodeled cuboid cells, here we show that FtsZ may also mediate the division of naturally occurring ones. This implies that, even in natural settings, membrane roundness is not required for FtsZ function.

Keywords: Bacterial cell division; Bacterial cell shape; Microbial symbiosis.

Graphical abstract

Figure 1

Ca. T. cuboideus cells are cuboid (A) A scanning electron micrograph (SEM) showing the top view of the bacterial coat (left) and a transmission electron micrograph (TEM) showing the bacterial coat overlying a transverse section of the nematode (right). (B) SEM images display three representative Ca. T. cuboideus cells arranged from the youngest to the oldest (three leftmost panels) and a cell that is attached to the worm cuticle (rightmost panel). Arrowhead points at filamentous structures. (C) 3D-structured illumination microscopy (SIM) images of cells stained with fluorescent wheat germ agglutinin. The top row shows a non-dividing and a dividing cell in front view (yx view, left) and 90° shifted side view (yz view, right; a black rectangle has been placed in the lower right corner to cover a neighboring cell for clarity), and bottom panel shows multiple Ca. T. cuboideus cells in different stages of the cell cycle (cyan). Scale bars (A–C) correspond to 1 μm. See also Figures S1 and S3 and Tables S1 and S2.

Figure 2

Morphometry of Ca. T. cuboideus (A) Schematic representation axes of a Ca. T. cuboideus cell indicating its three axes, the proximal (nematode-attached) pole, and the distal (free) cell pole. Length is defined as the axis parallel to the host surface and perpendicular to the division plane, Width as perpendicular to the host surface, and Depth as parallel to the host surface and the division plane. (B) Boxplot shows the Length, Width, and Depth of 15 cells imaged with 3D SIM microscopy. Box is the interquartile range (IQR), where the lower edge is 25^th percentile (first quartile [Q1]) and the upper edge the 75^th percentile (third quartile [Q3]). Whiskers show the range between the lowest value (Min) and the highest value (Max). Line inside each box indicates the median. (C) Scatterplot shows the axes measurements of 256 Ca. T. cuboideus cells, grouped into the categories dividing (red, n = 44), non-dividing (blue, n = 212), and non-dividing, squared (green, n = 122). In non-dividing cells Length and Depth are undiscernible. See also Table S3.

Figure 3

Ca. T. cuboideus cells localize FtsZ at their septum (A–C) (A) Three representative cells immunostained with an anti-fimbriae antibody (B) and four cells with a PG metabolic probe EDA-DA, or (C) an anti-FtsZ antibody. (A-C) Phase contrast image (upper panels) and corresponding fluorescence images are shown (lower panels). White dotted cell outline is the cell shape deduced from phase contrast images. Scale bars correspond to 1 µm. (D) Quantitative analyses of the different fluorescence patterns corresponding to the different cell cycle stages. Plots show the normalized fluorescence emitted by Ca. T. cuboideus cells (a.u.) stained with an anti-fimbriae antibody (left plot; n = 63), a PG metabolic probe EDA-DA (middle plot; n = 48), or an anti-FtsZ antibody (right plot; n = 43) plotted against their cell width (%). In the middle and right plots cells were split into early (stage 1, full line) or late (stage 2, dotted line) septation stages. (E) Model of Ca. T. cuboideus growth and division for non-dividing (stage 0) and dividing (stage1 and 2) cells. See also Figures S2 and S3 and Tables S4 and S5.

Figure 4

Ca. T. cuboideus FtsZ forms either foci or straight or sharp-cornered filaments 3D SIM images of cells immunostained with an anti-FtsZ antibody. No membrane indentations appear in the two top cells, whereas the two bottom cells are invaginated. The left column shows the front view (yx) and the right column a 90° shifted side (yz) view. Dotted lines represent the cell outline of the respective cells (left panels) or the shape of the septum (right panels), deducted from the shape of the increased fluorescence background signal. Scale bar corresponds to 1 μm. See also Figure S2 and Table S5.