Cytoskeletal components can turn wall-less spherical bacteria into kinking helices

Abstract

Bacterial cell shape is generally determined through an interplay between the peptidoglycan cell wall and cytoplasmic filaments made of polymerized MreB. Indeed, some bacteria (e.g., Mycoplasma) that lack both a cell wall and mreB genes consist of non-motile cells that are spherical or pleomorphic. However, other members of the same class Mollicutes (e.g., Spiroplasma, also lacking a cell wall) display a helical cell shape and kink-based motility, which is thought to rely on the presence of five MreB isoforms and a specific fibril protein. Here, we show that heterologous expression of Spiroplasma fibril and MreB proteins confers helical shape and kinking ability to Mycoplasma capricolum cells. Isoform MreB5 is sufficient to confer helicity and kink propagation to mycoplasma cells. Cryoelectron microscopy confirms the association of cytoplasmic MreB filaments with the plasma membrane, suggesting a direct effect on membrane curvature. However, in our experiments, the heterologous expression of MreBs and fibril did not result in efficient motility in culture broth, indicating that additional, unknown Spiroplasma components are required for swimming.

Fig. 1. Morphology and motility of Spiroplasma citri GII-3 and Mcap transformants with genome insertions of mreB and fib genes of S. citri.

a Large darkfield images of cultures of Mcap^control and Mcap^mreB1-5-fib cells, imaged area approximately 62 µm wide. Scale bar: 10 µm. These micrographs are representative of at least 3 independent experiments. b Darkfield microscopy images showing representative morphologies of short rod-shaped Mcap^control cells (1), and of Mcap^mreB1-5-fib cells: long helical (2) and nonhelical (3) cells; a branched helical cell (4); a straight, branched filament showing helical or straight lateral extensions (5); a short helical cell (6); and a long wavy cell (7). These micrographs are representative of at least 3 independent experiments. Scale bar: 2 µm. c Box plot display of the cell length and helical pitch in S. citri, Mcap^control transformed with the empty vector and Mcap^mreB1-5-fib cell populations measured in darkfield microscopy images. The central line corresponds to the median. The lower and upper hinges of the boxes correspond to the 25th and 75^th percentiles, and the whiskers represent the 1.5×interquartile range extending from the hinges. Points are outliers. The top left insert represents the helical pitch p and the length L of a representative helical cell. Helical pitch measurements were restricted to helical cells, and were made after 4 passaging in liquid broth. Data were collected for 150 cells (cell length) or 50 cells (helical pitch) from 3 biological replicates for each cell population and compared by the two-tailed Student’s t test; *** indicates a significant difference between populations at p < 0.001. Source data are provided as a Source Data file.

Fig. 2. Time-lapse images showing the kink-based cell movements.

Darkfield microscopy images were recorded in S. citri (top) and in Mcap^mreB1-5-fib (bottom). White arrows point to kinks. Note the helicity shift upon propagation of the kink along the cell body. These micrographs are representative of at least 3 independent experiments. Scale bar: 5 µm.

Fig. 3. Helicity of Mcap transformants bearing different combinations of mreB and fib genes observed using darkfield microscopy.

a Box plot display of the helical pitch in Mcap transformants. Data were collected for 50 cells from 3 biological replicates for each transformant population. The central line corresponds to the median. The lower and upper hinges of the boxes correspond to the 25th and 75^th percentiles, and the whiskers represent the 1.5×interquartile range extending from the hinges. Points are outliers. Source data are provided as a Source Data file. b Representative helical cells observed in Mcap transformed with the different plasmid constructs. These micrographs are representative of at least 3 independent experiments. Scale bar: 5 µm.

Fig. 4. Cell movements in Mcap transformants.

Left: Darkfield microscopy time-lapse images showing helicity changes due to the propagation of membrane deformations in Mcap transformants bearing all mreB genes (top), mreB5 only (middle) or fib only (bottom). The micrographs are representative of at least 3 independent experiments. Right: Proposed simplified models for morphology changes starting from movies from which darkfield images on the left were extracted. Models were built using Blender-3D. Examples of left- (L) and right- (R) handed helices are indicated; k stands for kinks. Note that Mcap transformants having mreB5 only or fib only lose their helicity upon propagation of the kink-like membrane deformation. Scale bar: 1 µm.

Fig. 5. 2D cryo-electron microscopy reveals the formation of cytoskeletal filaments in Mcap transformants bearing S. citri cytoskeleton genes.

a Spiroplasma citri cytoskeletal fibers are localized in the cytoplasm next to negatively curved areas of the plasma membrane. Note the tapered (duckbill-shaped) tip of the cell indicated by the white arrow. b Mcap^control cells transformed with the empty vector do not show any cytoplasmic cytoskeleton fibers. c–h Cytoskeletal fibers were imaged in Mcap^mreB1-5-fib c, Mcap^mreB1-5 d, Mcap^mreB1 e, Mcap^mreB1-fib f, Mcap^mreB5 g, and Mcap^mreB5-fib h cells. i Internalized vesicle (indicated by *) observed in some Mcap^mreB5 cells. Inset, magnified view of the region outlined by the white box in each image. Scale bars: 100 nm and inset 20 nm. These images are representative of at least 3 independent experiments, except with Mcap^mreB5, for which a clear cytoskeleton was visible in a single cell (see main text).