2024 Volume 21 Issue 2 Article ID: e210015
Mycoplasma mobile is a parasitic bacterium that forms gliding machinery on the cell pole and glides on a solid surface in the direction of the cell pole. The gliding machinery consists of both internal and surface structures. The internal structure is divided into a bell at the front and chain structure extending from the bell. In this study, the internal structures prepared under several conditions were analyzed using negative-staining electron microscopy and electron tomography. The chains were constructed by linked motors containing two complexes similar to ATP synthase. A cylindrical spacer with a maximum diameter of 6 nm and a height of 13 nm, and anonymous linkers with a diameter of 0.9–8.3 nm and length of 14.7±6.9 nm were found between motors. The bell is bowl-shaped and features a honeycomb surface with a periodicity of 8.4 nm. The chains of the motor are connected to the rim of the bell through a wedge-shaped structure. These structures may play roles in the assembly and cooperation of gliding machinery units.
Mycoplasma mobile, a parasitic bacterium, glides on solid surfaces at speeds of up to 4.0 μm per second through a specialized mechanism. The gliding machinery, located at one pole of the cell, is composed of surface legs and internal motor structures. The force generation unit within the internal structure evolved from ATP synthase. This study aimed to clarify the entire architecture of the gliding machinery using electron tomography.
Mycoplasma mobile is a parasitic bacterium found on the gills of freshwater fish [1,2], forming a gliding machinery on the cell pole and glides on the solid surface in the direction of the cell pole at a maximum speed of 4 μm/s (Fig. 1). This gliding mechanism, observed only in M. mobile and its related species, repeatedly grabs and pulls sialylated oligosaccharides [3–6] on the host cell surface via ATP hydrolysis [7–10]. Although this mechanism appears similar to that of Mycoplasma pneumoniae, they do not share the proteins required for gliding [2,11–18]. The gliding machinery of M. mobile and its related species consists of internal and surface structures [19–23]. The main protein components of the surface structures are Gli123, Gli349, Gli521, and Gli42 [24–27]. Gli123, Gli349, and Gli521 are involved in complex assembly, binding to sialylated oligosaccharides, and transmitting gliding forces, respectively [3,25–28]. The structures of Gli349, Gli521, and Gli123 have been clarified to be rods with some hinges of total lengths, 97, 121, 34 nm, respectively by using electron microscopy (EM) [29–32]. The internal structure consists of a bell at the front and chains of twin motor connected to the bell [22,23]. The bell is a rigid structure featured by honeycomb structure. The individual units of twin motor show similarities with F-type ATP synthase in sequence and overall structures, suggesting its evolutional origin [2,19,22]. However, the structure of the bell and the entire internal structure remain unclear [19,21,22], which are necessary to understand the gliding mechanism. In this study, the internal structures prepared under several conditions were analyzed using negative staining and electron tomography.
M. mobile 163K (ATCC 43663) with an OD600 at 0.08 was inoculated to nine-fold volume of fresh Aluotto medium and grown at 25°C for two days [28,33].
Sample PreparationCultured cells were collected by centrifugation at 10,000×g at 25°C for 5 min and suspended in Aluotto medium for a 30-fold concentration of the culture [28,33]. A cell suspension of 5 μL was placed on a carbon-coated electron microscope (EM) grid for 10 min. The EM grids were treated by a hydrophilizer (PIB-10, Vacuum Devices Co., Ltd. Mito, Japan) at 10 mA for 30 s. The suspension was removed, and the grids were treated twice with 5 μL of a detergent solution (0.1% (v/v) Triton X-100, 0.1 mg/mL DNase I, 5 mM MgCl2 and 5 mM CaCl2 in PBS consisting of 75 mM sodium phosphate (pH 7.3), and 68 mM NaCl) for 1 min at 25°C. The grids were then washed with 10 μL PBS and stained with 2% phosphotungstic acid (pH 7.0) and air-dried. Permeabilized cells were prepared by a single treatment with detergent solution. Chemically fixed cells were prepared by treating twice with the detergent solution including 0.2% (v/v) Triton X-100, and fixed with 2% (v/v) glutaraldehyde for 5 min at 25°C.
Image Acquirement and AnalysisThe samples were observed using a Talos F200C G2 transmission electron microscope (Thermo Fisher Scientific, Waltham, MA, USA) at 200 kV, 28000x (3.76 Å/pix) with a 4k×4k Ceta 16M CMOS detector (Thermo Fisher Scientific). Single-axis tilt series were collected covering an angular range from –65° to +65° or –60° to +60° at 6- to 8-μm underfocus using the Tomography software package (Thermo Fisher Scientific). Subtomogram averaging was done using the EMAN 2.99 software package. Tomographic reconstruction was performed using IMOD 4.11.8 software package. The fast Fourier transform (FFT) was calculated using ImageJ v1.53g. Finally, 3D-rendering and segmentation processes were performed using UCSF Chimera 1.15 software package.
Cultured M. mobile cells that adhered to an EM grid were extracted using a detergent and subsequently stained. This process removes the cell membrane and cytoplasm, thereby exposing the internal structures. Bell and chain parts were observed (Fig. 2A–C). Tilt images were captured in increments of either 1° or 3°, reaching 60° or 65° (Movies 1 and 2). The tomograms were then reconstructed from these tilt series (Fig. 2D–F). The features evident in the 2D images are consistent with those reported previously [20,22,23]. The reconstruction revealed an internal structure with a maximum height of 50.4 nm in the desiccated state (Fig. 2G).
The bell is known to have a honeycomb structure; however, its overall architecture remains unclear [23]. Thus, we focused on the bells that had been exposed to two rounds of detergent treatment on an EM grid (Fig. 3). Tilt series of the exposed bells were acquired across a range of –65° to +65° and subsequently reconstructed into tomograms. The resulting reconstructed bell has a height of approximately 50 nm. The FFT of the horizontal image revealed three distinct directional periodicities (Fig. 3H). Each periodicity is oriented at a 60-degree interval. The periodicity in all three directions was 8.4 nm (Figs. 1 and 3H).
Determining whether the holes within the honeycomb structure permeated through the entire bell height or were limited to its surface remained challenging, owing to the inadequately adjustable staining conditions (Fig. 3A–D). The bell was deformed due to drying during sample preparation. To observe the bells under more natural conditions, we applied glutaraldehyde fixation before drying (Fig. 4A–C). This approach visualizes the rim of a bowl-shaped bell (Fig. 1). The chains and bell rims were characterized by distinct densities (Fig. 4D). The bells exhibited bowl structures with some distortions with major axis of 437.4±18.2 nm (n=7) (Fig. 4E and F). A chemically fixed cell appeared to retain the bell shape with a height of approximately 100 nm and a major axis of approximately 490 nm. In this cell, 46 chains of 432±76 nm (n=153) extend radially outward from the bell at intervals of approximately 24 nm. Additionally, to gain insight into the structure of the intracellular gliding machinery, we analyzed specimens that preserved some cell membranes with reduced detergent extraction (Fig. 4G and H). The focused cell was approximately 80 nm high, and the bowl was better retained than in the fully extracted specimens (Fig. 4I and J). The chains extend from the bell in a uniform direction, with a distinct orientation from the fully extracted state.
We focused on chain structures. Small densities were observed between the two particles, i.e. twin motors (Fig. 5A). Particle images were obtained from four tomograms. Each particle contained two twin motors that formed a chain. A structure with a resolution of 31.0 Å (Fourier shell correlation [FSC]=0.143) was obtained through subtomogram averaging of 718 particles (Fig. 5B). Ribbon models of the F1-ATPase from Bacillus sp. PS3 [PDB ID: 7XKQ] fitted well into the reconstructed chain model, indicating that the negatively stained chain maintained its original structure (Fig. 5C). Several novel features were found here (Fig. 1). A cylindrical structure was observed at the connecting part of the twin motors. Its height and the maximum diameter were approximately 13 nm and 6 nm, respectively based on an image averaged from 1342 original ones. The amorphous filaments were visualized as structures linking neighboring chains. Each filament had a length of 14.7±6.9 nm (n=12) and width of 0.9–8.3 nm. Multiple filaments are extended from a single twin motor (Fig. 5D). Subtomogram averaging of the single-unit twin motors was performed using 1342 particles collected from five tomograms. Averaged structure with a resolution of 24.3 Å (FSC=0.143) revealed several protrusions extending from the side of F1-like hexamers, as previously reported (Fig. 5E) [19].
Terminal structures of the chains were observed at the edge of the bell in the chemically fixed specimens (Figs. 1 and 6A). These structures had wedge-like shapes and extended along the inner surface of the bell (Fig. 6B and C). The length of each wedge was distributed 58.3±8.5 nm (n=22) (Fig. 6D).
The structure of the gliding machinery is being clarified, focusing on surface proteins such as Gli349 and internal bells and chains [10,19,21,22,24–27]. However, little is known about the structures that assemble this machinery, even though they are essential for clarifying the gliding mechanism. The results obtained in this study may provide clues for elucidating their structures (Fig. 1).
The name “bell” is derived from the first name for the internal structure of the gliding machinery, namely the “jellyfish” structure [23]. The entire bell is shown as a bowl of uniform height (Fig. 4). The honeycomb-repeating structure of the bell suggests that it is a small protein, possibly a polymer of MMOB4860 [21,23]. M. mobile cells always have one or more gliding machinery, suggesting that the formation of this machinery is tightly coupled with cell proliferation [34,35]. We speculate that the controlled polymerization of bell component proteins is the first step in the formation of de novo gliding machinery that couples with cell division.
The twin motors, which are force generation units in the internal structure, are vertically connected to form a chain. In the image reconstructed by cryotomography, these chains were aligned, facing the same plane as that of the membrane [22]. Because the isolated chain and bell complex contained no obvious membrane proteins, the chain was expected to bind to an unidentified membrane protein. The cylinders identified in this study may have been linked to this membrane protein. Negative-staining EM images of the isolated internal structure revealed sheets of individual chains that were almost equally spaced, suggesting a lateral linkage of the chains (Fig. 2，5D) [22,23]. This structure may be the linker that connects the chains identified in this study. We revealed that the bell is a bowl with a honeycomb structure and that the novel wedge anchors the chains to the bell. The number of chains, 46, was larger than that in the previous analysis, which ranged from 15 to 35 [22]. This difference suggests that the number and length of chains may change with the cell sizes which are influenced by serum lot, growth stage, and others.
Component ProteinsHere, we discuss the possible components of the three novel structures identified in this study [19,21,36]. Open reading frames (ORFs), MMOBs 1620–1670, 0150, 4530, 4860, and 5430 were previously identified to code for component proteins of isolated internal structures [23]. Excluding the ORFs identified as twin motor component proteins, MMOBs 1640, 1650, 0150, 4860, and 5430 remain unknown [19]. Previous immunofluorescence studies have shown localization of MMOB4860 at the cell front, suggesting that this protein corresponds to the wedge [23]. MMOBs 1640, 1650, and 5430 have been shown to localize to the cell neck based on fluorescent protein fusion results, making them candidates for novel structural components [21].
The results obtained in this study suggest that the bell serves as the starting point for the formation of the gliding machinery and as a structural anchor. When gliding machinery is formed, the component proteins form a honeycomb structure by polymerization, which serves as the starting point of gliding machinery formation, and when in motion, it serves as an anchor for the polarity of the machinery to work cooperatively.
The authors declare that they have no conflict of interest.
MF did experiments. MF, TT, YOT, DN did data analyses. MF and MM wrote a draft. All discussed the story and completed the manuscript.
We thank Hideki Nakagawa and Junko Shiomi at Osaka Metropolitan University for their technical help, and the Equipment Sharing Center for Advanced Research and Innovation for the use of the Talos F200C G2 transmission electron microscope and related software (HN). This study was supported by a MEXT, Grants-in-aid for scientific research (A) (JP17H01544), a JST CREST grant (JPMJCR19S5) to MM, and JST, the establishment of university fellowships for the creation of science technology innovation, Grant Number JPMJFS2138 to MF.
Tomograms are available in J-STAGE Data with the DOI of https://doi.org/10.34600/data.biophysico.25808272, as listed below. Movie 1. Tomography of isolated internal structures, shown as Fig. 2A, D, E, F, and G. Images every 3° from –60° to +60° are assembled. Horizontal edge of movie is 1540 nm. Movie 2. Slice movie of isolated internal structure reconstructed from a tilt series, as shown in Fig. 2A and D–G. Slice images 75.2 nm high are stacked for 50 slices. Horizontal edge of movie is 1540 nm. Movie 3. Slice movie of isolated internal structure reconstructed from a tilt series, as shown in Fig. 2B. Slice images 76.7 nm high are stacked for 51 slices. Horizontal edge of movie is 1540 nm. Movie 4. Slice movie of isolated internal structure reconstructed from a tilt series, as shown in Fig. 2C. Slice images 10.8 nm high are stacked for 67 slices. Horizontal edge of movie is 1540 nm. Movie 5. Slice movie of isolated internal structure reconstructed from a tilt series, as shown in Fig. 3 A–F. Slice images 81.2 nm high are stacked for 54 slices. Horizontal edge of movie is 1540 nm. Movie 6. Slice movie of isolated internal structure reconstructed from a tilt series, as shown in Fig. 3G. Slice images 67.7 nm high are stacked for 45 slices. Horizontal edge of movie is 1540 nm. Movie 7. Slice movie of chemically fixed and isolated internal structure reconstructed from a tilt series, as shown in Fig. 4A–F. Slice images 153.4 nm high are stacked for 102 slices. Horizontal edge of movie is 1540 nm. Movie 8. Slice movie of isolated internal structure reconstructed from a tilt series, as shown in Fig. 4G–I. Slice images 90.2 nm high are stacked for 60 slices. Horizontal edge of movie is 1540 nm. A preliminary version of this work, DOI: https://doi.org/10.1101/2024.02.08.578511, was deposited in the bioRxiv on February 09, 2024.
