DOI: https://doi.org/10.21203/rs.3.rs-1802191/v1
Many bacteria belonging to the phylum Bacteroidetes move on solid surfaces, which is called gliding motility. In our previous study with the Bacteroidetes gliding bacterium Flavobacterium johnsoniae, we proposed a helical loop track model, where adhesive SprB filaments are propelled along a left-handed closed helical loop on the cell surface. Attachment of SprB to the substratum results in cell movement. In this study, we observed the gliding cell rotating counterclockwise about its axis when viewed from the rear to the advancing direction of the cell, which was consistent with the helical loop track model. Total internal reflection fluorescence microscopy of SprB on a gliding cell revealed that one labeled SprB focus sometimes overtook and passed another SprB focus that was moving in the same direction, suggesting the presence of multiple lanes in the helical loop track. Several electron microscopic analyses revealed the presence of a multi-rail structure underneath the outer membrane, which was associated with SprB filaments and contained GldJ protein. A similar structure was observed in the distantly related marine gliding Bacteroidetes Saprospira grandis. These results provide new insights into the mechanism of Bacteroidetes gliding motility, in which the SprB filaments are propelled along the multi-rails underneath the outer membrane.
Bacteroidetes gliding motility on solid surfaces is unrelated to other bacterial motilities. Left-handed helical movement of adhesive SprB filaments on the cell surface is required for gliding motility but the nature of the gliding machinery in the cell was unclear. Here we show that the helical movement of SprB on the cell surface causes cell gliding with rotation on solid surfaces and presence of the multi-rail structure on the underneath of the outer membrane that may be a part of helical loop track for SprB. These findings provide insights for understanding the mechanism of Bacteroidetes gliding motility.
Cells of many bacterial species belonging to the phylum Bacteroidetes move over surfaces at approximately 1–5 µm/sec in a process called gliding motility 1. Gliding motility requires cell contact with a solid surface, since cells suspended in liquid do not actively move. Genetic analyses have suggested that the Bacteroidetes gliding motility is unrelated to other well-studied bacterial motility mechanisms such as flagellar motility, type IV pilus-mediated twitching motility, myxobacterial gliding motility, and Mycoplasma gliding motility, but instead relies on a novel machinery consisting of Gld and Spr proteins that are confined to members of the large and diverse phylum Bacteroidetes such as Flavobacterium johnsoniae 2–6. Some Gld and Spr proteins are not only involved in gliding motility but are also components of the type IX secretion system (T9SS) 7. T9SSs were first identified and studied in the nonmotile oral pathogen Porphyromonas gingivalis and in F. johnsoniae 8–11. Recent studies have revealed that T9SS is a unique system that is clearly different from other secretion systems in terms of its supramolecular structure and secretion mechanism 12–14. F. johnsoniae cells use the T9SS to secrete dozens of proteins including soluble extracellular enzymes and motility adhesins that reside on the cell surface 15–17.
F. johnsoniae has been studied as a model organism to understand the molecular mechanism of Bacteroidetes gliding motility. Genetic and molecular analyses demonstrated that SprB, a huge filamentous 6,497 amino acid protein, is a primary cell-surface adhesin of F. johnsoniae 18. Immunofluorescence microscopic analysis using antiserum against SprB revealed that the SprB filaments are propelled at approximately 2 µm/sec with a ~ 19 degree tilt with respect to the long axis of the cell. Mathematic analyses suggested a “helical loop track” model in which gliding 'motors' act on SprB filaments that have attached to a surface generating rotation and translocation of the cell body19. A recent cryo-electron microscopic study has revealed that GldL and GldM, a transmembrane core complex for gliding motility has a structural organization similar to that of the bacterial flagellar stator complex consisting of MotA and MotB, which acts as a proton channel for torque generation 20–22. GldLM complex shows relatively static localization distributed in many foci along the cell body, and fueled by the proton gradient to drive the helical motion of SprB adhesin 23,24. However, the structural basis for why SprB moves along a helical track is still unknown.
In this paper, detailed analysis of SprB movement on the F. johnsoniae cell surface by immunofluorescence microscopy and morphological analysis of the gliding machinery by electron microscopy suggest the presence of a multi-rail structure as a component of the helical loop track involved in SprB movement. Similar multi-rail structures were seen in the distantly related gliding Bacteroidetes Saprospira grandis, suggesting that this may be a general feature of the Bacteroidetes gliding machinery. The results provide new insight into the mechanism of this common form of bacterial gliding motility.
Counterclockwise Rotation of a Gliding Cell. In our previous study, we proposed a helical loop track model for gliding motility of F. johnsoniae where SprB adhering to a substratum was propelled along a helical loop track on the cell surface, resulting in rotation and translocation of the cell 19. Here we investigated whether gliding cells actually rotate as proposed. To visualize cell rotation, we attempted to find a cell surface protein that did not change position with respect to the long axis during gliding, and observe its behavior by total internal reflection fluorescence (TIRF) microscopy using fluorescently-labeled antiserum. Cell surface proteins were isolated and collected by centrifugation. The isolated cell surface proteins were separated by SDS-PAGE (Fig. S1A). SprB with molecular mass greater than 250 kDa was detected by immunoblot analysis using anti-SprB antiserum (Fig. S1B). Major protein bands on the gel were identified by peptide mass fingerprinting analysis using MALDI-TOF-MS, which revealed that the cell surface protein fraction contained proteins with putative outer membrane protein (OMP) domains (Fjoh_0697, Fjoh_1311 and Fjoh_3514) and putative TonB-dependent receptors (Fjoh_0403, Fjoh_0736, Fjoh_4221 and Fjoh_4559) (Fig. S1A and Table S1). Amino acid sequence analyses for predicting signal peptides by SignalP 4.0 software25 and for predicting protein localization by CELLO v2.5 subcellular localization predictor26,27 suggested that most of the proteins were located at the outer membrane (Table S1). We generated rabbit antiserum against the cell surface protein fraction. Immunofluorescence microscopy using this antiserum and Alexa Fluor-555-conjugated secondary antibodies showed that signals were dispersed on the cell surface (Fig. S1C). TIRF microscopy with a translocating cell revealed that there were two types of signal movement (Movie S1). Type I signals, the most common, were located at a position a fixed distance away from a cell pole and periodically appeared on the surface with respect to the short axis when the cell moved on a substratum, indicating cell rotation. TIRF analysis showed that the type I signals always appeared from one side with respect to the short axis of the moving cell, demonstrating rotation in a counterclockwise (CCW) direction when viewed from the rear to the advancing direction of the cell (Movie S2). Type II signals, which comprised less than 20 % of otal signals, moved on the cell surface with respect to both axes like the SprB signal (Movie S3). This type of signal behavior was expected since SprB, which migrates from one pole to the other along an apparent helical track 19, was present in the surface protein fraction that was used to generate the polyclonal antiserum. We also generated antiserum against purified OMP domain-containing protein Fjoh_0697, which appeared to be a major cell-surface protein (Fig. S1A). TIRF analysis using anti-Fjoh_0697 antiserum revealed that the Fjoh_0697 signal did not migrate from pole to pole but was instead located at a position a fixed distance from a cell pole. In a translocating cell Fjoh_0697 periodically appeared on the surface with respect to the short axis, demonstrating CCW rotation of the gliding cell as observed from the rear of the cell (Fig. 1A, Movie S4).
SprB is the primary motility adhesin on the F. johnsoniae cell surface. Left-handed helical flow of immunolabeled SprB on a gliding cell was previously observed 19. In this study, for detailed analysis of the individual SprB signal movement on a gliding cell surface, limited numbers of SprB molecules on a cell were labeled with highly diluted antiserum (see Materials and Methods) to reduce signals and track SprB signals easily, and movement of signals on the surface of gliding cells was observed by fluorescence microscopy. Under these conditions, about 58% of cells had 1 to 10 SprB signals and the rest had none. Consistent with the previous report 19, the SprB signals were propelled between cell poles along an apparently helical loop track on gliding F. johnsoniae cells (Fig. 1B, Movie S5). Cells migrated 6.1 ± 1.1 µm (mean ± standard deviation, N = 63) when the Fjoh_0697 signal made one revolution in the direction of the short axis of a cell (helical pitch of Fjoh_0697) (Fig. 1C left and D), while the moving distance of SprB on the substratum during one revolution of SprB in the direction of the short axis of a cell (apparent helical pitch of SprB) (Fig. 1C right and D) was 4.55 ± 1.1 µm (N = 22). The rotational rate of Fjoh_0697 signals was directly proportional to the cell velocity (Fig. 1E). To show the relationship between movements of a cell and a labeled protein, velocities of labeled proteins and cells were determined. SprB (50 signals) and Fjoh_0697 (25 signals) from 12 cells were plotted on a graph (Fig. 1F). Plots showed that the velocity of Fjoh_0697 signals was positively correlated with the cell velocity. These data demonstrated that Fjoh_0697 signals are located at fixed positions on the cells. In contrast, SprB signals moved in both directions on the cells and the velocity of each SprB was not necessarily related to the cell velocity (Fig. 1F). In SprB as well, it should be more proportional, assuming that all SprB molecules move on a helical loop track of the cell surface at the same pace and that the movement of SprB causes the cell to move. The difference may be accounted for by the that the velocity of SprB varies from molecule to molecule, and SprB that advances the cell is only a part of SprB molecules. SprB, whose value on the horizontal axis is 0 in Fig. 1F, appears to actually move the cell. It is suggested from Fig. 1F that the other SprB molecules exist away from the substratum and move at varied velocities to some extent. The movement of SprB on a cell was then examined in more detail.
Multiple Lanes in Helical Loop Track for SprB Movement. Some SprB signals exhibited unexpected movements such as mid-cell U-turns. Figure 2A and Movie S6 show an SprB signal that moved toward the anterior (right) pole, looped around the anterior pole at approximately 1.8 sec and then moved toward the posterior pole. Interestingly, before the signal reached the posterior pole, it made a U-turn and moved toward the anterior pole (3.5–4.4 sec). The SprB signal then looped around the anterior pole and migrated all of the way to the posterior pole without conducting another mid-cell U-turn (5.2–9.5 sec). The frequency of mid-cell U-turns was about 0.07 per min per SprB, suggesting that the U-turn events do not happen frequently. Multiple SprB signals on a cell did not always move at the same speeds. In Fig. 2B and Movie S7, a cell that had two SprB signals traveling in the same direction is shown. The low intensity signal moved more slowly (about 1.2 µm/sec) than did the high intensity signal (about 2.4 µm/sec). The high intensity signal thus overtook and passed the low intensity signal while traveling to the anterior (right) pole. These results suggest that the track on which SprB traveled had multiple lanes.
Alteration of SprB Velocity during Translocation. Since we observed that some SprB signals moved with different speeds on the cell surface, we asked if SprB can change velocity during pole-to-pole movement. Since it was difficult to track individual SprB signals on cells of normal size for long periods of time, we observed SprB movement on filamentous cells generated by inhibiting cell septation with the antibiotic cephalexin. Addition of 20 µg/ml cephalexin to growth media resulted in filamentous cells that were about 100 µm long. The localization of SprB on glutaraldehyde-fixed cells as observed by immunofluorescence microscopy showed similar distributions on filamentous (cephalexin-treated) cells and on non-elongated control cells that had not been exposed to the antibiotic (Fig. S2). In both cases cells had about 1.5 SprB signals/µm on the cell surface under the labeling condition noted in the legend to Fig. S2. The average velocity of SprB on a filamentous cell was 2.3 ± 0.6 µm/sec (N = 96) which was similar to SprB movement on a normal-length cell (2.1 ± 0.6 µm/sec, N = 128) (Fig. 2G). We then determined whether the velocity of SprB along the cell surface varied. SprB movements on filamentous cells that were not themselves translocating on a glass surface are depicted by the kymographs in Fig. 2. On these nontranslocating cells the SprB molecules were apparently not attached to the substratum, since otherwise the action of the motility motors against these would have resulted in cell movement. The movement of four SprB signals over 9.4 sec on a filamentous cell is shown in Fig. 2C (the kymograph from Movie S8). Distance between signals marked with “b” and “c” did not change during the observation, resulting in parallel lines in the kymograph indicating that these signals moved with similar velocity. In contrast, signal “a” approached and overtook signal “b” within the first 5 sec of the recording. Similar to the observation of overtaking movement on a normal cell (Fig. 2B), this result suggests that SprB signals move with different velocities on a track with multiple lanes. Two other examples of SprB movement on individual filamentous cells are shown in Fig. 2D and Fig. 2E, which correspond to Movies S9 and S10 respectively. Parallel oblique lines appeared in these kymographs; however, some lines were not parallel, although SprB signals were moving in the same direction. SprB signals “d” and “e” moved toward the right with similar velocities of approximately 3.0 µm/sec, resulting in parallel oblique lines for about the first 8 sec of observation (Fig. 2D and Movie S9). After this point, signal “d” moved more slowly and eventually stopped, as indicated by the vertical line in the kymograph after 11 sec. As a result, the distance between signals “d” and “e” increased after 8 sec. In addition to this “slowdown and stop" behavior, we also observed ‘stay and go’ movement of SprB signals (Fig. 2E, Movie S10). Signal “f” did not move until about 4 sec after start of observation, as indicated by the vertical line. Subsequently, signal “f” began to move toward the right and drew an oblique line in the kymograph. In addition, the time course of SprB movement along the long axis of a cell by tracking analysis showed the alteration of SprB velocity during translocation (Fig. 2F). These results indicate that the velocity of SprB signals can change during translocation.
Visualization of Multi-Rail Structure for Gliding Machinery. Observation of SprB movement on the cell surface led us to hypothesize the presence of a multi-rail structure for the SprB “trains”. We visualized such a structure by electron microscopy. Cells were burst by osmotic shock to reduce cell thickness and were then negatively stained with 0.5% uranyl acetate. Multi-rail structures which formed bundles of 2–12 fibers were observed (Fig. 3A-D). The thickness of a fiber in a bundle was 7.5 ± 0.9 nm (N = 58). The multi-rail structure was easily peeled from the cell after osmotic shock. Thin filaments appeared to be attached to the multi-rail structure (Fig. 3D). Mutants deficient in gliding-related proteins (Gld proteins, Spr proteins and RemA protein) were examined for the presence of the multi-rail structure. The multi-rail structures were observed in each of the spr and remA mutants at more than 30% of osmotically shocked cells, but the structures were not observed in any of the gld mutants when at least 50 osmotically shocked cells of each gld mutant were examined (Fig. S3 and Table S2). These results suggest that the Gld proteins are required for formation of the multi-rail structure. F. johnsoniae gld mutants are completely deficient in gliding 28 whereas spr mutants retain limited ability to glide on some surfaces 29,30. The absence of the rails in the gld mutants suggested that these structures played an important role in gliding.
Association of the Multi-Rail Structure with SprB Filaments. For more detailed structural analysis of the multi-rail structure, the supernatant of an osmotically shocked sample was centrifuged and the resulting precipitate was suspended and analyzed by TEM. Consistent with the observation of multi-rail structures on osmotically shocked cell surfaces, thin filaments were attached to the multi-rail structures released from wild type cells, whereas there were no filaments attached to the multi-rail structure from the sprB mutant (Fig. 3E and F). Immuno-electron microscopic analysis with anti-SprB antiserum revealed that the thin filaments were SprB (Fig. 3G). These results suggest that the multi-rail structure is associated or interacted with the SprB filaments and forms a part of the gliding machinery.
The Multi-Rail Structure Underneath the Outer Membrane. The multi-rail structure was visualized by quick-freeze deep-etch electron microscopy, which provides images of membrane-attached or -embedded structures on a fractured surface. Figure 3H-J shows images of quick-freeze deep-etch replica with the wild type and gldNO mutant treated with osmotic shock. In the wild-type cells, multi-rail structures were observed on the periplasmic surface of the outer membrane and filaments (presumably SprB) extended from the outer membrane (Fig. 3H and I), as previously observed 31. In contrast, neither multi-rail structures nor SprB filaments were observed in cells of the gldNO mutant (Fig. 3J). To determine the presence of the multi-rail structure in an intact cell, plunge-frozen F. johnsoniae cells were analyzed by cryo-electron tomography. Similar to the previous observation by Liu et al. 32, section images from two representatives of 3D reconstruction of the wild-type cells showed SprB filaments extending from the outer membrane (Fig. 4A and B, Movie S11 and S12). In addition, long thin electron-dense structures were observed, apparently in the periplasm. Importantly, these structures seemed to form parallel lines. Analysis of stacks of section images confirmed the presence of apparently left-handed parallel lines, which may correspond to the multi-rail structure (Fig. 4C and D). Neither cell-surface SprB filaments nor the electron-dense rail structures were observed in cells of the gldJ, gldK, gldL, gldM or gldNO mutants (Movie S13-17). These mutants are completely nonmotile and are also completely deficient in secretion of SprB protein 15,16,33. Taken together, the electron microscopy results suggest that the multi-rail structure was located on the periplasmic surface of the outer membrane.
Components of the Multi-Rail Structure. Since the mutant study revealed that Gld proteins are required for formation of the multi-rail structure, we examined whether Gld proteins were components of the multi-rail structure by immunoelectron microscopy of osmotically shocked cells using antibodies against Gld lipoproteins (GldB, GldD, GldH and GldJ). Using anti-GldJ antibody, gold particles were accumulated on the multi-rail structure (Fig. 5A), suggesting that GldJ is a component of this structure. The other antibodies did not react to the multi-rail structure (data not shown).
Cells of the gldA, gldB, gldD, gldF, gldG, gldH, gldI, gldJ, gldK, gldL, gldM, and gldNO mutants showed no multi-rail structures (Fig. S3). GldJ was reported to be unstable in gldA, gldB, gldD, gldF, gldG, gldH, and gldI mutants 33. In contrast, GldJ was apparently stable in cells with mutations in gldK, gldL, gldM, and gldN 28. We reexamined the amount of GldJ in gldK, gldL, gldM, and gldNO mutants and obtained similar results, except for a partial reduction of GldJ levels in a gldK mutant (Fig. 5B). We also demonstrated that sprB mutant cells retained wild type levels of GldJ protein (Fig. 5B). Blue native PAGE analyses suggested that GldJ was part of a large complex in wild type and sprB mutant cells, whereas it appeared to be part of a smaller complex in cells of gldK, gldL, gldM and gldNO mutants (Fig. 5C). These results suggest that GldK, GldL, GldM and GldNO contribute to multimerization and/or complex formation of GldJ, and may explain the absence of multi-rail structures in cells lacking these proteins.
Helical Organization of GldJ in the Cell Envelope. The trajectory of SprB movement suggested the presence of a left-handed closed helical loop track spanning the cell from pole to pole. To elucidate the organization of GldJ in the cell envelope, we examined the localization of GldJ in fixed permeabilized cells by immunofluorescent TIRF microscopy. Images of wild-type cells showed multiple parallel oblique lines (stripe pattern) spanning the cell from pole to pole. (Fig. 6A). The average distance between the fluorescent lines in the direction of the long axis was 0.70 ± 0.14 µm (N = 126) (Fig. S4A). In contrast, the stripe pattern was not observed in the gldJ mutant cells (Fig. 6B). Due to TIRF microscopy that illuminates molecules near the contact regions with the glass, the stripe pattern, which tilts from the upper left to the lower right direction, represents the surface of the left-handed helical structure. Considering the trajectory of one molecule of SprB 23, the number of stripes observed per cell was not consistent with a single closed helical loop but suggested approximately four parallel closed helical loops from pole to pole for a typical 6 µm-long cell (Fig. 7A). The GldJ’s stripes observed here, which may form the four parallel closed helical loops, seem to be consistent with the trajectories of multiple SprB molecules in a nontranslocating cell in our previous study 19. F. johnsoniae wild-type and gldJ mutant cells expressing a GldJ-mCherry fusion protein were subjected to TIRF microscopy. The fusion protein appeared to form stripes, similar to the results using the GldJ immunofluorescent labeling (Fig. S4).
Presence of a Multi-Rail Structure in the Marine Gliding Bacterium S. grandis. Aizawa 34,35 reported that bundle fibers are found in osmotically shocked cells of the marine gliding Bacteroidetes S. grandis. S. grandis glides on solid surfaces at 5 µm/sec 35,36. S. grandis, a member of the class Sphingobacteriia 37, is not closely related to F. johnsoniae, a member of the class Flavobacteriia. Orthologs of F. johnsoniae Gld and Spr proteins were identified by BLAST search with the S. grandis genome 37 (Table S3). S. grandis had orthologs of gliding motility proteins (GldA, GldB, GldD, GldF, GldG, GldH, GldJ, GldK, GldL, GldM, GldN, SprA, SprB, SprC, SprD, SprE, and SprT). However, orthologs of the periplasmic lipoprotein, GldI and the cell-surface lectin, RemA 38 (which is not essential for gliding) were not found in S. grandis. TEM analysis revealed the presence of abundant bundle fibers in osmotically shocked cells as previously reported 34,35 (Figs. 3K and L). Negative staining with PTA revealed thin filaments, which may be SprB ortholog filaments, attached to the bundle fibers (Fig. 3L). The thickness of each fiber of the bundle fibers was 7.5 ± 0.8 nm (N = 73). Cryo-electron tomography also revealed the bundle fibers in intact S. grandis cells (Movie S18). Structural similarity of the multi-rail structures (bundle fibers) between F. johnsoniae and S. grandis suggest that the multi-rail structure may be a common component of the gliding machinery in diverse members of the phylum Bacteroidetes.
In a previous study, we proposed a model for gliding motility of F. johnsoniae where SprB is propelled along a closed helical loop track on the cell surface 19. Many of these SprB molecules are not engaged with the substratum and thus they move along the helical track without causing cell movement. In contrast, SprB molecules that attach to the substratum and are acted on by the motor result in rotation and translocation of the cell. Rotation of the gliding cells observed in this study support this model. CCW rotation of the gliding cells observed by the OMP domain-containing protein Fjoh_0697 (viewed from behind the rear of forward translocating cells) is consistent with the cell rotation generated by the left-handed helical movement of SprB. We observed left-handed helical movement of SprB and left-handed helical gliding of cells. The handedness of the stripes observed by cryo-EM tomography and TIRF microscopy (Figs. 4 and 6, Fig. S4) is consistent with those of SprB and cell movements. Shrivastava et al. 39 reported right-handed movements of SprB and cell movements. We do not have an explanation for this difference. Regardless of the handedness of the movements, it seems clear that SprB movement along helical tracks results in rotation and forward movement of gliding cells.
A detailed analysis of the SprB movement in this study shows that SprB can turn around in the middle, SprB can catch up with and overtake another SprB, and SprB can change its speed, suggesting that SprB does not always move at a constant speed on a single track. We observed the multi-rail structure associated with the SprB filaments in the F. johnsoniae cells that were burst by osmotic shock to reduce cell thickness and were then negatively stained with 0.5% uranyl acetate. The location of the multi-rail structures on the periplasmic side of the outer membrane of F. johnsoniae was revealed by quick-freeze deep-etch electron microscopy. Considering this location, lipoproteins could be components of the structure. Immuno-electron microscopic analysis revealed that GldJ lipoprotein was associated with the multi-rail structures. It was previously reported that GldJ is organized in discrete bands that appear to form a helical structure 33, which is consistent with the present results. GldJ lipoprotein shares 30% identity with GldK lipoprotein over 382 amino acids 33. P. gingivalis PorK (GldK homolog) with PorN makes a ring structure of ~ 8 nm in thickness and 52 nm in diameter 14,40. It is worthy of note that the thickness of fiber in the multi-rail structure involving GldJ is almost the same as that of the PorK/PorN ring. Various gld mutants in addition to the gldJ mutant lacked the multi-rail structures. GldJ was reported to be unstable in gldA, gldB, gldD, gldF, gldG, gldH, and gldI mutants 33. The lack of GldJ in these mutants may explain the absence of the multi-rail structures. GldJ appeared to be part of a large complex in wild-type cells, whereas it was present in lower molecular weight form in cells of gldK, gldL, gldM, or gldNO mutants. GldK, GldL, GldM, and GldN may be required for the assembly or stability of a GldJ complex associated with the multi-rail structures.
The marine gliding bacterium S. grandis had a multi-rail structure similar to that of F. johnsoniae (Fig. 3E and L). The structure in S. grandis seemed to be continuous fibers and covered the entire cell body (Fig. 3K, Movie S18). The presence of multi-rail structures in both F. johnsoniae and S. grandis cells provides new insights into the mechanism of gliding motility employed by members of the phylum Bacteroidetes. We propose a helical multi-rail model for gliding motility in which the SprB filaments span the outer membrane, interacting with the substratum at the cell surface, and interacting directly or indirectly with the multi-rail structure in the periplasm (Fig. 7). The SprB filaments are propelled along the left-handed helical multi-rail structure. Sometimes the SprB filaments change direction even in the middle of a pole-to-pole path by shifting to another rail (Fig. 7B). When motors act on SprB filaments that are firmly attached to the substratum, the filaments are propelled along the rails but remain stationary to the substratum. This results in rotation and forward movement of the cell body.
Gliding motility of the d proteobacterium Myxococcus xanthus has been extensively studied, and it also involves cell rotation during migration 41–47. The rotation of M. xanthus cells is CCW (viewed from the rear) as also observed here for F. johnsoniae. In M. xanthus, cytoplasmic and cytoplasmic membrane proteins migrate along a helical track in the cytoplasm, resulting in cell movement 44,47. The cytoplasmic membrane motor proteins form a complex with periplasmic and outer membrane proteins that exert force on cell-surface adhesins that are attached to the substratum 46, 48–50, which is similar to the F. johnsoniae motility model.
Although the helical movement of motility proteins drives the helical movement of both F. johnsoniae and M. xanthus cells 51, there are numerous differences between the two systems. Many proteins have been identified that are involved in the gliding motility of both organisms but there is little if any similarity between them 2,3,52. For F. johnsoniae, the cell-surface adhesin SprB appears to be propelled along a helical track associated with the periplasmic face of the outer membrane that may be comprised of the lipoprotein GldJ. There is no evidence for helical movement of components in the cytoplasm or cytoplasmic membrane and the current model involves motors, stationary on the cell, that propel SprB along the cell surface 23,24. In contrast, for M. xanthus movement of cytoplasmic membrane proteins along a helical cytoplasmic track is proposed 44, resulting in movement of surface proteins. Significant gaps in our understanding of the F. johnsoniae and M. xanthus motility machines remain. For M. xanthus the cytoplasmic membrane motor proteins (AglR, AglQ, AglS) are responsible for force generation. In contrast, for F. johnsoniae the cytoplasmic membrane proteins GldL and GldM forms a nanoscale electrochemical motor 20, 53–55, which drives both gliding motility and protein secretion. Control of the motors in response to the environment is also distinct for each organism. The M. xanthus motors are controlled by the frz chemotaxis system 56 whereas F. johnsoniae and other members of the Bacteroidetes lack critical chemotaxis proteins 2 and must use another mechanism to control cell movement.
Shrivastava et al.57 suggested that the gliding motor in F. johnsoniae is rotary because F. johnsoniae cells rotated when tethered to glass by anti-SprB. We do not yet know how a rotary motor results in the helical movement of SprB. One possibility is that the rotating motor pushes on a tread carrying SprB filaments and propels it along the helical GldJ track 4,24. Alternatively, the SprB filaments could be anchored on the tracks, and the tracks could be propelled by the motor 24,58.
Various unanswered questions regarding the Bacteroidetes gliding machinery remain to be solved. The proton gradient across the cytoplasmic membrane is required for SprB movement and cell gliding 19. However, it is not known how the motor(s) transmit force to the SprB filaments on the cell surface. The apparent linkage of SprB filaments with the outer membrane-associated periplasmic rails described here may explain part of this transduction. Further studies including the clarification of the force-transducing mechanism are needed for a more complete understanding of this common form of the Bacteroidetes motility.
Bacterial Strains and Growth Conditions. Bacterial stains are listed in Table S2. F. johnsoniae cells were grown in Casitone-yeast extract (CYE) medium at 25 oC with shaking 59. To observe gliding motility, F. johnsoniae cells were grown in motility medium (MM; 3-fold diluted CYE) to an optical density of around 1.0 at 600 nm. S. grandis cells were grown in Marine medium (Marine broth 2216 (BD, NJ, USA) with 0.5% tryptone (BD)) at 25 oC with shaking to an optical density of around 1.5 at 600 nm.
Immunolabeling of Cell Surface Proteins. For immunofluorescence microscopy of live cells, cells were harvested by centrifugation at 5,000 × g for 1 min, and the pellet was suspended in fresh MM with 1/500 to 1/1000 dilution of antiserum against SprB, against pooled cell surface proteins or against Fjoh_0697 and incubated for 5 min. Cells were collected by centrifugation at 5,000 × g for 1 min and washed with fresh MM. The cells were suspended in fresh MM with 1/1000 dilution of Alexa Fluor 555-conjugated antibody against rabbit IgG (Abcam, Cambridge, UK). After incubation for 5 min cells were washed and suspended in fresh MM.
Optical Microscopy. F. johnsoniae cells were poured into a tunnel slide assembled by using double-sided tape to attach a coverslip onto a glass slide 19,60, and were observed using an inverted microscope (Olympus IX83; Olympus, Tokyo, Japan). TIRF images were acquired with UApoN 100×OTIRF objective lens (Olympus) using a 561 nm laser. Images were recorded with an iXon3 897 EMCCD camera (Andor Technology PLC, Northern Ireland, UK) using MetaVue software (Molecular Device, CA, USA). Time-lapse montage images and kymographs were processed with ImageJ 1.48r software (http://imagej.nih.gov/ij/).
Isolation and Identification of Cell Surface Proteins. F. johnsoniae was grown in MM at 25°C for 6 h. The cells were suspended in PBS (Sigma-Aldrich, MO, USA) and passed several times through a 26 G-1/2 inch needle to shear cell surface structures. After removal of the cells by centrifugation at 9,000 × g for 10 min, a fraction of surface proteins was collected by ultracentrifugation at 66,000 × g for 60 min. Proteins were separated by SDS-PAGE and stained with Coomassie Brilliant blue R250. Protein bands were excised and digested by trypsin. Proteins were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; Urtraflex III, Bruker Daltonics, MA, USA).
Osmotically Shocked Cells. F. johnsoniae was grown in 5 ml of MM at 25°C for 3 h. Cells were suspended in 100 µl of ice-cold sucrose solution (0.5 M sucrose, 0.15 M Tris-HCl, pH 7.5). The cells were placed on ice for 15 min and then mixed rapidly with 1.4 ml of ice-cold ultrapure water. Intact cells were removed by low speed centrifugation at 9,000 × g for 3 min. Osmotically shocked cells were collected from the supernatant by high-speed centrifugation at 20,000 × g for 5 min. For S. grandis, cells were first fixed with 1.5% paraformaldehyde for 30 min at room temperature, and then washed twice with ice-cold ultrapure water or 50 mM MgCl2 solution. Intact S. grandis cells were removed by centrifugation at 2,000 × g for 3 min and osmotically shocked cells were collected by centrifugation at 20,000 × g for 5 min. Centrifugation was done at 4°C.
Electron Microscopy. Samples were negatively stained with 2% PTA (pH 7.0) or 0.5% uranyl acetate on a Butvar B-98 (Sigma-Aldrich) coated copper grid, and observed by transmission electron microscopy (JEM-1230NT; JEOL, Tokyo, Japan). Micrographs were taken at an accelerating voltage of 80 kV. For immunogold electron microscopy, osmotically shocked cells were treated with 1,000-fold diluted antiserum against SprB protein in PBS containing 2% BSA and incubated on ice for 20 min. The cells were washed three times with PBS and treated on ice with 20-fold diluted goat anti-rabbit IgG conjugated to 5 nm diameter gold particles (BBI solutions, Cardiff, UK) in PBS containing 2% BSA for 20 min, washed three times, and then stained with 0.5% uranyl acetate. For anti-GldJ immunostaining, osmotically shocked cells were fixed with 1.5% paraformaldehyde before treatment with primary antibody.
Preparation of Quick-Freeze Deep-Etch Replica Specimens. Bacterial cells, which were partly disrupted by osmotic shock, were mounted onto a cover glass and quickly frozen by metal contact at liquid nitrogen temperature. Frozen samples were subjected to freeze-fracture deep-etch replication, according to previous protocols 61,62. After knife-fracture and deep-etching at − 104 oC, samples were rotary-shadowed with Pt/C at an angle of 20o and backed with pure carbon in a freeze-fracture device (JFD-V: JOEL). Replicas were floated off the cover glass onto the surface of full-strength hydrofluoric acid. Household bleach was occasionally used to remove remaining debris from the replica. Replicas were rinsed with three changes of water and picked up onto copper grids for electron microscopic examination.
Cryo-Electron Tomography. Quantifoil molybdenum 200 mesh R0.6/1.0 grids (Quantifoil Micro Tools GmbH, Großlöbichau, Germany) were glow discharged and pretreated with a solution of 10 nm colloidal gold particles concentrated 1.5 times before use (MP Biomedicals, CA, USA) for tomogram alignment. A 3 µl sample was applied to the grid, blotted by filter paper, and plunged into liquid ethane using Vitrobot (FEI, OR, USA). Images were collected at the liquid-nitrogen temperature using a Titan Krios FEG transmission electron microscope (FEI) operated at 300 kV on FEI Falcon 4 k × 4 k direct electron detector (FEI). The magnification was calibrated by measuring the layer-line spacing of 23.0 Å in the Fourier transform of images of tobacco mosaic virus mixed in the sample solution. The pixel size on the specimen was 0.57 nm. Single-axis tilt series were collected covering an angular range from − 70 to 70 with a nonlinear Saxton tilt scheme at 4–10 µm underfocus using the Xplore 3D software package (FEI). A cumulative dose of 200 e−/Å2 or less was used for each tilt series. Images were generally binned two-fold and 3D reconstructions were calculated using the IMOD software package 63. Surface-rendering images were obtained using the three-dimensional modeling software Amira 5.2.2 (Visage Imaging, San Diego, CA).
Blue Native Gel Electrophoresis. Cells were sonicated in lysis buffer containing 1% (w/v) n-dodecyl-β-D-maltoside (DDM), 0.5 M sucrose, 10 mM Tris-HCl (pH 7.5), 1 mg/ml DNase, and protease inhibitor cocktail (Sigma-Aldrich). Soluble fractions were mixed with 10× Blue native sample buffer (5% w/v CBB G-250, 500 mM 6-aminocaproic acid, 100 mM Bis-Tris-HCl, pH 7.0), and loaded on a native gel (3–12% Bis-Tris gel; Thermofisher scientific, MA, USA). Proteins on the gel were blotted on PVDF membrane and subjected to immuno-detection with anti-GldJ rabbit polyclonal antiserum 33.
Permeabilization of F. johnsoniae Cells.
Cells were fixed with 1% paraformaldehyde for 20 min at 25 oC. Fixed cells were permeabilized in 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1% Triton X-100 for 30 min at 25 oC. Permeabilized cells were washed three times with PBS (Sigma-Aldrich, MO, USA). For immunofluorescence microscopy of GldJ, permeabilized cells were treated with 100-fold diluted antibody against GldJ in PBS containing 2% BSA and incubated at 4 oC for 16 h. Cells were washed with fresh PBS. The cells were suspended in PBS containing 2% BSA with 1/1000 dilution of Alexa Fluor 555-conjugated antibody against rabbit IgG. After incubation for 30 min, cells were washed three times with PBS and observed by TIRF microscopy. For TIRF microscopy using cells expressing a GldJ-mCherry fusion protein, the cells were fixed and permeabilized before observation as described above except that 2% Triton X-100 was used because significant amounts of free mCherry, which were presumably generated by proteolysis, were found in the cells, revealed by Western blot analysis (data not shown). Permeabilized cells were washed three times with PBS to remove free mCherry from the cells and subjected to TIRF microscopy
Competing interests: The authors declare no competing interests.
ACKNOWLEDGMENTS We thank S. Aizawa for supplying S. grandis strain, T. Hamaguchi for helping to draw the gliding model in Fig. 7, and the general supporting team at Osaka City University for Scientific Research on Innovative Areas Harmonized Supramolecular Motility Machinery and Its Diversity supported by the Japan Society for the Promotion of Science (JSPS) Kakenhi Grant (Grant ID 25117501), directed by M. Miyata, for technical help with electron microscopy. This work was supported by the JSPS Kakenhi Grants (Grant IDs 24117006 and 25293375 to KN) and by National Science Foundation Grant MCB-1516990 to MJM.