Bacteria switch only intermittently to motile planktonic lifestyles under favorable conditions. Under chronic nutrient deprivation, however, bacteria orchestrate a switch to stationary phase, conserving energy by altering metabolism and stopping motility. About two-thirds of bacteria use flagella to swim, but how bacteria deactivate this large molecular machine remains unclear. Here, we describe the previously unreported ejection of polar motors by γ-proteobacteria. We show that these bacteria eject their flagella at the base of the flagellar hook when nutrients are depleted, leaving a relic of a former flagellar motor in the outer membrane. Subtomogram averages of the full motor and relic reveal that this is an active process, as a plug protein appears in the relic, likely to prevent leakage across their outer membrane; furthermore, we show that ejection is triggered only under nutritional depletion and is independent of the filament as a possible mechanosensor. We show that filament ejection is a widespread phenomenon demonstrated by the appearance of relic structures in diverse γ-proteobacteria including Plesiomonas shigelloides, Vibrio cholerae, Vibrio fischeri, Shewanella putrefaciens, and Pseudomonas aeruginosa. While the molecular details remain to be determined, our results demonstrate a novel mechanism for bacteria to halt costly motility when nutrients become scarce.

In the face of starvation, bacteria must minimize their energy use. Here, we describe our unexpected finding that some bacteria take the drastic measure of ejecting their flagella in response to nutrient deficiency. Bacteria continually assemble flagella as propellers—unrelated to eukaryotic flagella—rotated by rotary motors embedded in the cell; continual rotation and assembly can consume up to 3% of a bacterium’s energy. Using electron cryo-tomography, a technique that provides high-resolution 3D images of intact bacteria, we were surprised to find partial flagellar motors in bacterial cells that were rare when nutrients were abundant but became common when nutrients were scarce. A variety of clues led us to hypothesize that these structures were relics of motors whose flagella had been ejected, which we confirmed using a genetic approach. Curiously, flagellar relics—which would otherwise be open portals through which the contents of the bacterial periplasm could leak—were plugged by an unidentified protein, presumably as a preservation measure. We speculate that flagellar ejection saves the bacterium from the costs of continuously assembling and rotating its flagella, as a last-ditch survival attempt. Our work provides a striking example of evolution arriving at a functional yet unintuitive solution to a problem.

Funding: This work was supported by a Medical Research Council grant MR/P019374/1 to MB, a Medical Research Council PhD Doctoral Training Partnership award grant number MR/K501281/1 to JLF, a Research fellowship of the German Research Foundation (DFG project number 385257318) to FMR, Grant TRR174 "Prokaryotic Cell Biology" from German Research Foundation (DFG) to KMT, Netherlands Organisation for Scientific Research (NWO) BBOL.737.016.004 to AB, a German Academy of Sciences Leopoldina (Fellowship Programme LPDS 2017-01) to SB, and NIH (AM, grants R01-GM051350 and R35-GM118108) to PBR. PBR is also supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001143), the UK Medical Research Council (FC001143), and the Wellcome Trust (FC001143). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2019 Ferreira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we describe work showing that γ-proteobacteria eject their polar Na + -driven flagella in response to nutrient depletion. The ejected flagella leave a “relic” of the ejected motor in the outer membrane composed of the P-, L-, H-, and T-rings and the basal disk. During this transition, a previously undescribed plug is incorporated into the relic. This is likely an active mechanism to block periplasmic leakage. We speculate on the nature of the novel flagellar plug and the significance of flagellar ejection. Note that this work was previously available as a preprint [ 13 ]; a similar preprint concluded that “relics” are instead outer-membrane assembly precursors [ 14 ].

Little is currently known about how the widespread polar-flagellated γ-proteobacteria modulate motility. This group includes a diverse set of pathogens occupying both sessile and planktonic niches, including human pathogens V. cholerae and P. shigelloides, opportunistic pathogens P. aeruginosa and S. putrefaciens, and nonpathogenic members including the squid symbiont V. fischeri. These γ-proteobacteria have one or more polar flagella whose motors are powered by Na + -ions; these motors are both faster [ 8 ] and provide higher torque [ 8 , 9 ] than the model peritrichous (positioned over the entire cell surface) flagellar motors from Salmonella and E. coli; polar Na + -driven motors assemble at only one pole until division. Na + -driven polar motors have a number of structural differences to the well-studied enteric-like motors [ 9 – 11 ]. Most striking in subtomogram averages is the addition of periplasmic (H-ring and T-ring) and outer membrane (basal) disks [ 2 , 9 ]. The T-ring, made up of MotX and MotY, contributes to the assembly and scaffolding of the stator complexes (PomA/PomB). Many γ-proteobacteria also incorporate a sheath, an outer membrane extension that encapsulates the flagellum. The H-ring and basal disk, composed of FlgO, FlgP, and FlgT, have recently been shown to assist in outer membrane penetration in these bacteria [ 12 ], although their function is likely broader than sheath formation, given that many unsheathed bacteria also retain an H-ring.

Methods for deactivation of flagella in different environments other than nutrient depletion are diverse across those bacteria studied to date. Rhodobacter sphaeroides has a unidirectional flagellum that is stopped by a “molecular brake” for navigation [ 3 ], while Bacillus subtilis uses a “molecular clutch” to stop flagellum rotation and swimming for biofilm formation [ 4 ]. The Salmonella enterica serovar Typhimurium (“Salmonella”) and Escherichia coli motors are proposed to be inactivated by a “backstop brake”, YcgR, a cyclic di-GMP (c-di-GMP) binding protein [ 5 , 6 ], while P. aeruginosa modulates its motility via a YcgR homologue, FlgZ [ 7 ]. The α-proteobacterium Caulobacter crescentus, meanwhile, actively ejects its single, polar flagellum upon surface sensing [ 6 , 7 ] in order to build an adhesive stalk for surface adhesion.

Many bacteria switch to a nonmotile lifestyle in stationary phase to conserve energy [ 1 ], but how this switch is accomplished is poorly understood. The most widespread motility device used by bacteria is the flagellum, found in approximately two-thirds of bacteria [ 2 ]. Bacterial flagellar filaments are helical propellers that extend several microns from the cell from a periplasm-spanning rotary motor; rotation of the filament by a transmembrane rotary motor exerts thrust that propels the bacterium forward. Motor torque is generated by harnessing ion flux across the inner membrane; this torque is first transmitted to a periplasm-spanning rod, then to a flexible extracellular hook, and finally to the filament. The rod exits the outer membrane through dedicated P- and L-rings that act as channels through the peptidoglycan layer and outer membrane, respectively. Although the flagellum is vital for migration to favorable environments, sites of biofilm formation, or sites of infection, it is counterproductive for the cell to retain a functional flagellum during nutrient depletion, and stopping motility is preferable to resource exhaustion.

Results

γ-proteobacterial motility slows as cell density increases While tracking the swimming speeds of the γ-proteobacterium P. shigelloides, we noticed that bacteria with multiple Na+-driven polar flagella showed a significant decrease in swimming speed when grown to later growth stages (Fig 1A). Video tracking of individual bacterial cells revealed that P. shigelloides swam at 40 μm s−1 between optical density (OD) 0.2 and OD approximately 0.7 before swimming speeds sharply dropped at OD 0.8, down to 12 μm s−1 at OD 1.0. Furthermore, the percentage of active swimmers dropped from over 95% at early growth stage up to OD 0.6 to approximately 5% by OD 1.0. Another γ-proteobacterium, V. fischeri, displayed the same characteristic behavior (Fig 1A). Strikingly, however, the model enteric bacterium Salmonella that uses a different family of flagellar motors continued swimming as well as, if not faster than, Salmonella cells at OD 0.2 when cultured to higher cell densities (Fig 1A). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. γ-proteobacteria swimming slows at later growth stages due to loss of flagella. (A) Swimming speeds of P. shigelloides, V. fischeri, and S. enterica sv. Typhimurium at increasing cell density. Speed relative to initial speed at OD 600 0.2 are represented. Error bars indicate standard error. (B) Representative negative-stain EM images of cells grown to three different cell densities of P. shigelloides and V. fischeri, revealing loss of polar Na+-driven flagella yet no loss of peritrichous Salmonella flagella. Scale bars are 1 μm. (C) Mean number of flagella, counted from 150 cells (50 per biological replicate) at increasing cell densities suggests loss of polar flagella. The error bars indicate a 95% t-based confidence interval. (D) The absolute number of attached flagella in the population calculated from the mean number of flagella and CFU suggests flagellar ejection by P. shigelloides and V. fischeri. The error bars indicate a 95% t-based confidence interval. (E) Negative-stain EM images of flagella recovered from the supernatant of stationary phase P. shigelloides confirms consistent flagellar loss from the base of the hook. Inset shows close-up of a hook. Scale bar is 100 nm. (F) Negative-stain EM images of flagella recovered from the supernatant of stationary phase Salmonella. Inset shows close-up of a broken filament; no hooks were observed in isolated Salmonella flagella. Scale bar is 100 nm. (G) SDS-PAGE gel of P. shigelloides and Salmonella cells grown to stationary phase confirms ejection of polar Na+-driven flagella at the base of their hook. Polar FliC (filament) and FlgE (hook) proteins are seen in the S of the P. shigelloides culture. Underlying data in S1 Data. CFU, colony-forming unit; EM, electron microscopy; OD, optical density; P, pellet; S, supernatant. https://doi.org/10.1371/journal.pbio.3000165.g001

Decreased swimming speed is due to loss of flagella In the course of a previous study [9], we noted that flagellation levels in γ-proteobacteria are highest early in their growth stage (low cell density). To determine whether the decrease in swimming speed of γ-proteobacteria was due to differences in flagellation levels at different growth stages, flagella were counted in negative-stain electron microscopy (EM) images of cells to assess flagellation levels (Fig 1B). Time courses of both P. shigelloides and V. fischeri cells grown for longer periods of time (at increasing cell densities) showed decreasing numbers of flagella per cell (Fig 1B and 1C), and cells grown overnight into late stationary phase had no flagella. Salmonella, in contrast, had increasing or stable numbers of flagella at later growth stages (higher cell densities). We sought to distinguish whether solely flagellar synthesis is down-regulated or whether flagella are also actively disassembled in γ-proteobacteria at high cell densities. Plotting the absolute number of flagella in the entire population indicated that flagella are indeed lost. The number of cells in a population was determined by calculating colony-forming units (CFUs) and relating this to the mean number of flagella per cell as counted by EM (Fig 1D). The absolute number of flagella in the P. shigelloides and V. fischeri populations declined over time, demonstrating that bacteria are losing flagella faster than they are synthesizing them (Fig 1D). The Salmonella positive control, in contrast, showed an increase in the absolute number of flagella in the population in direct correlation with CFU count, as assembly continued and no ejection occurred in later growth stages (at higher cell densities).

Used γ-proteobacterial growth medium contains free flagella with hooks at one end To test our speculation that flagella are ejected, we determined whether supernatant of P. shigelloides cells grown to stationary phase contains released flagella. Polyethylene glycol (PEG) precipitation revealed that P. shigelloides growth medium indeed contained free flagella (Fig 1E). Curiously, 50% of all flagellar ends had an attached hook. Given that a flagellum only has a hook attached at one of its ends, we conclude that, effectively, all isolated P. shigelloides flagella have attached hooks, similar to ejected flagella from Caulobacter crescentus [15]. Flagellar filaments were also recovered from the supernatant of a Salmonella control (Fig 1F). Strikingly, however, no hooks were observed on any of the 70 randomly imaged Salmonella filament ends. This suggests random instead of determinate sites of filament breakage in Salmonella. As the number of flagella on Salmonella cells increases or remains stable at high density (Fig 1C), the broken flagellar filaments must regrow, which is in agreement with previous studies [16,17].

Shed hook–filament structures accumulate in P. shigelloides supernatant To verify that Salmonella filaments are sheared midfilament, whereas P. shigelloides filaments are cleaved at the base of the hook, flagella were recovered from cell cultures and analyzed by SDS-PAGE. Cells grown to high OD were removed by pelleting, and flagella were recovered from the remaining supernatant by PEG precipitation before SDS-PAGE analysis. Similar samples were collected from Salmonella cultures and volumes adjusted to match P. shigelloides cell count. The Plesiomonas supernatant had significant bands at molecular weights corresponding to both the polar flagellin (FliC) and hook (FlgE), as well as a faint band at the expected size of the distal rod (FlgG). The Salmonella supernatant contained a weaker flagellin band but lacked bands at the molecular weights corresponding to the Salmonella hook (FlgE) or rod (Fig 1G). This confirms that Plesiomonas cells consistently lose their flagella at the base of the hook, while Salmonella flagella accumulation in the supernatant is due to shearing midfilament. We conclude that this consistent behavior in response to a specific environment, i.e., late growth stage and high cell densities, is best explained by an ejection mechanism.

Partial relics of flagella remain at the cell pole at high OD The hypothesis that γ-proteobacteria eject their flagella led us to speculate that a partial flagellar motor structure would remain after ejection of the filament and hook. Cryo-electron tomograms were collected of P. shigelloides cells grown to either early or late growth stages (low or high cell density). Consistent with our hypothesis, partial flagellar structures were seen to remain at the pole of cells grown to high OD, with fewer in cells grown to low OD (Fig 2A, left column). In all cases, partial flagellar structures were only observed at the flagellated cell pole. Our swimming speed and flagellar ejection results led us to speculate that these structures are the “relics” of once fully-functioning flagella that were subsequently ejected in late-growth stage (high OD) cultures, leaving only the outer-membrane rings (the P-, L-, H-, and T-rings and the basal disk) assembled. The P- and L-rings, which are integral parts of the relic structure, require a rod and chaperone for assembly; the P- and L-rings are incapable of assembling in the absence of an assembled rod [18], further consistent with these structures being relics of previously full flagella. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. Old γ-proteobacterial cell poles bear the relics of ejected flagella. (A) Top row: slices through tomograms showing flagella and relic structures in five γ-proteobacteria. (Left to right): P. shigelloides, V. cholera, V. fischeri, S. putrefaciens, and P. aeruginosa. Top row: black arrows point to full flagella, and white arrows point to relics. Scale bar is 100 nm. Middle row: example subtomograms of full flagella in the five species. Scale bar is 50 nm. Bottom row: example subtomograms of relics in the five species. Scale bar is 50 nm; empty red arrowheads highlight open relic structures from unsheathed bacteria, filled red arrowheads highlight membrane-capped relic structures from sheathed bacteria. (B) Assembly and disassembly states are seen in P. shigelloides tomograms, including precursors that differ from relic structures. Scale bar is 50 nm. (C) Subtomogram average of the P. shigelloides polar flagellar motor. (Left to right): central slice through the subtomogram average, interpretive colored overlay relating subtomogram average to isosurface representation, cutaway isosurface rendering of subtomogram average, and whole isosurface rendering. Dotted line represents likely peptidoglycan location; red arrow highlights where the plug density sits in the relic. Scale bar is 15 nm. The structure is deposited to the Electron Microscopy Data Bank as EMDB-4570. (D) Corresponding subtomogram average (left) with isosurface (right) of the P. shigelloides polar relic structure. Scale bar is 15 nm. The structure is deposited to the Electron Microscopy Data Bank as EMDB-4569. https://doi.org/10.1371/journal.pbio.3000165.g002 The two data sets demonstrated that at high OD, relics are enriched while flagella are depleted, correlating with our observations of flagellar ejection (Table 1). In cells imaged at OD 0.25, flagella were predominant: 43% of cell poles had exclusively intact flagella, 48% had both intact flagella and relics, and 9% had no structures; the average number of intact flagella per pole was 5.5; if the cells had relics, the average number of relics was 2. In cells imaged at OD 1.0, relics were predominant: none of the cells had intact flagella alone; 36% had both relics and intact flagella, 12% had exclusively relics, and 52% had neither relics nor flagella; and in contrast to cells grown to low OD, if the cells had intact flagella, the average seen was 1.3, while the average number of relics was 3.3. Note that because relics are harder to detect in the cryo-tomograms since they are relatively small and lack a flagellum, we may not have identified all relics that were present in the imaged cells. Consistent with this, imaging lysed cells grown to OD 1 revealed substantially more relics than we could detect in intact cells (up to 20 at a pole) (S1 Fig). This data set was not used in Table 1, however, as we cannot deconvolute any effect of cell lysis on relic appearance. Nevertheless, this result confirms that many relics accumulate at the cell pole. The results in Table 1 link OD to presence of the relic structures and demonstrate that not only are flagella being ejected, but assembly is also halted, as many cells harvested at high OD have neither flagella nor relics, indicating newly formed poles that have not yet assembled a single flagellum. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Appearance of outer membrane relic structures is related to cell density. Number of filaments and outer membrane relics counted from tomograms of P. shigelloides cells grown to OD 600 0.25 (N = 44) or OD 600 1.0 (N = 25). https://doi.org/10.1371/journal.pbio.3000165.t001

Diverse γ-proteobacteria retain relics of ejected flagella In order to determine whether this pattern is widespread amongst the γ-proteobacteria beyond P. shigelloides, we collected cryo-tomograms from four additional γ-proteobacterial species: V. cholerae, V. fischeri, S. putrefaciens, and P. aeruginosa (Fig 2A, right four columns). Markedly, in all species, we observed similar relic structures in the outer membrane of the cell poles, alongside intact flagella (Fig 2A). This was particularly unexpected in V. cholerae, S. putrefaciens, and P. aeruginosa, given that they only ever assemble one flagellar motor at the pole. Indeed, in one tomogram, we observed four relics alongside a single intact flagellum in V. cholerae. Relics from sheathed and unsheathed γ-proteobacteria had different outer-membrane morphologies. Relics from tomograms of sheathed V. cholerae and V. fischeri were capped by a continuous sealed outer membrane. Ejection of the flagellum in these bacteria must include shearing of the sheath from the outer membrane and subsequent closure of the torn membrane. Evidently, the unsheathed species P. shigelloides, S. putrefaciens, and P. aeruginosa maintain a portal through the outer membrane at all times with their L-ring that is unperturbed upon flagellar ejection. This suggests an as-yet unidentified difference between the L-rings from sheathed and unsheathed bacteria.

Multiple assembly intermediate states, distinct from relics, are observed in cryo-tomograms Alongside relics and intact flagella, multiple intermediate states were captured in our cryo-tomograms (Fig 2B). Early precursor structures include fully formed C-rings and rods but lack P- and L-ring–based outer-membrane disks and external structures. However, by far the most common structures observed were fully formed flagella and relic structures.

A novel protein plug in flagellar relics suggests a method for preventing periplasmic leakage To better understand the relationship of relics to intact motors, we determined the structures of both by subtomogram averaging of the P. shigelloides structures (Fig 2C and 2D). Clear 13-fold symmetry was observed in the stator complexes and MotXY ring of the full motor (S2 Fig), consistent with previous studies [9,11]. The relic structure resembled the outer-membrane flagellar structure, although it lacked bound stator complexes, suggesting that the relic is composed of the same proteins as the outer membrane–associated structures from the flagellar motor. In both structures, the distance between the MotXY ring (T-ring) and the outer membrane was 19 nm, and the diameter of the MotXY ring was 44 nm. In the motor, the diameter of the rod exit hole in the outer membrane was 15 nm, which is the same as in the relic outer membrane that is held open by the L-ring, despite lack of the rod. To confirm that relic structures really are remnants of old flagellar motors, we sought to confirm that they are composed of the same proteins as the outer-membrane portion of the flagellar motor. We purified a His 6 - and mCherry tagged version of S. putrefaciens MotX, the outermost protein from the T-ring, from membranes by affinity purification. Negative-stain EM and 2D classification of our affinity-purified, MotX-containing particles revealed homogenous top-views. These were comprised of a series of concentric rings, filled in the middle, consistent with top-views of relic subtomogram averages (S3 Fig). The sizes of the rings matched the basal disk (50 nm), T-ring (37 nm), and MotY-ring (29 nm) of S. putrefaciens. Based on these observations, we conclude that relics are composed of flagellar outer-membrane proteins yet lack flagellar inner membrane, rod, and hook components. Notably, an extra density in the relic structure plugged the P-ring aperture in the position that was previously occupied by the trans-periplasmic rod. Comparison of the flagellar and relic subtomogram averages confirmed that this density was unambiguously an additional protein absent from intact flagellar motors: while the rod was absent from the relics, the additional relic density observed did not correspond to loss of density elsewhere in the relic, which would result from a conformational change in a previously assembled component protein. Furthermore, this plug appeared to be an obligate part of the relic, as no relics were observed without a plug upon detachment of the axial structure—made of part of the rod, the hook, and filament—from the relic structure. The relics from both sheathed and unsheathed bacteria contained the plug protein regardless of the sealing of the outer membrane in the sheathed flagella. Bioinformatic predictions of potential novel flagellar components based on operon co-occurrence and phylogenetic profiling techniques [19], however, failed to identify suitable candidates, and we were unable to refine relic purification sufficiently for mass spectrometry analysis. We conclude that the plug is an as-yet unidentified protein that plugs the P-ring aperture concomitantly with flagellar ejection to prevent periplasmic leakage.

Verification that the outer-membrane structure is a flagellar relic and not an assembly precursor We sought to verify whether our so-called relic partial flagellar structures were indeed flagellar relics and not flagellar assembly precursors by testing the prediction that relics would require prior assembly of the rod while assembly precursors would not. To abolish rod assembly, we developed an S. putrefaciens ΔflhA mutant incapable of assembling the rod due to disruption of the intrinsic flagellar type III secretion system. The mutant was nonmotile (Fig 3A), and of 68 cell poles imaged by ECT, no relic structures were seen (Figs 3B and S4), confirming that they require a functional flagellar type III secretion system despite being general secretory pathway (Sec) exported. Normal flagellar gene expression was confirmed by complementation with an flhA over-expression plasmid for which polar flagellation, motility, and relics were regained (Fig 3A and 3B). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. Flagellar partial structures are relics, not assembly intermediates. (A) Soft-agar plates confirm loss of motility upon deletion of flhA, regained upon complementation. (B) Representative tomograms of (top to bottom) wild-type S. putrefaciens with partial structures, ΔflhA mutant without partial structures, and return of partial structures upon complementation with pBTOK-flhA. (C) Ejection caught in the process. A slice through a tomogram of S. putrefaciens immediately after an ejection event. The break point of ejection appears to be between the proximal and distal rod. Scale bar is 50 nm. (D) A close-up view of the recently ejected filament, containing the filament, hook, and distal rod. Scale as in next panel. (E) A close-up view of the components remaining in the cell immediately after ejection of the filament, hook, and distal rod. The plug density is already in place, and the proximal rod remains, along with the MS-ring and cytoplasmic components. Scale bar is 25 nm. (F) Periplasmic distance at position of relics or motors. Box is first and third quartile, the line in the box is the median, and the cross is the mean. (G) Rod, hook, and relic genes are interspersed together in an operon. Colors as in Fig 2C and 2E. Underlying data in S1 Data. HBB, hook-basal body. https://doi.org/10.1371/journal.pbio.3000165.g003 Further confirming that flagellar partial structures are relics, we observed flagellar ejection “caught in the act” in one tomogram of wild-type S. putrefaciens, with a filament, hook, and rod structure adjacent to a flagellar partial structure (Fig 3C). The ejected filament retained both the hook and distal rod (Fig 3D), which correspond to the only structures missing from the motor; notably, the P-ring of the partial structure was already plugged with a plug protein (Fig 3E). An orthogonal test for whether partial structures were relics or assembly intermediates was to inspect for anticipated interactions with the inner membrane because partial structures and intact flagella colocalize to the pole, suggesting a common localization mechanism. In the case of assembly intermediates, localization would likely be mediated via linkage to the inner membrane; in the case of relics, flagellar placement would have preceded ejection, and no connection to the inner membrane would be required. To test this, the distance between the inner- and outer-membrane disk was measured in fully formed motors as well as in partial structures. Periplasmic distances varied in both cases, as expected from previous work in V. alginolyticus [20]. Nevertheless, variation in membrane distance at the position of partial structures was far greater than at the intact flagella (13.9 nm and 7.9 nm range, respectively), suggesting that partial structures do not contain a connection with the inner membrane (Fig 3F). These results, combined with the mixture of relic, rod, and hook genes interspersed throughout the same operon (Fig 3G), lead us to conclude that these structures are indeed relics of ejected flagellar motors made of an outer-membrane subset of flagellar proteins and not flagellar assembly intermediates.

Motor and relic placement are not random and follow the same placement pattern We observed that intact flagella and relics appear spaced on a grid at cell poles, suggesting that placement is not random and that both structures share a common grid. The nearest neighbor distances for all intact flagella and relics were determined and found to be on average 64 nm, approximately 20 nm greater than would be expected if distances were constrained by steric clashes of approximately 45-nm diameter C-rings. To assess whether the placement of both relics and intact flagella is the same, which would suggest a common mechanism for placement, histograms were plotted for all distances to nearest neighbors of relics, intact flagella, and random data (Fig 4A). The relics and intact flagella shared identical curves, with a much tighter distribution than the randomized data set, consistent with a common placement mechanism. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. Relic positioning is the same as full flagella, indicating a common assembly placement. (A) Histograms for all nearest neighbors of relics (N = 114), motors (N = 424), and random synthesized data (N > 500). (B) Clark–Evans distribution of a random synthesized data set (N ≥ 500) compared to data from tomograms of P. shigelloides (N = 74). (C) 2D positions of relics (red circles) and motors (black circles) on six individual cells. Underlying data in S1 Data. https://doi.org/10.1371/journal.pbio.3000165.g004 To quantify the dispersal of intact flagella and relic structures independent of the number of structures per cell, Clark–Evans distribution analysis [21] was calculated for each cell pole with more than four intact flagella or relics (N = 71) and compared to a synthesized random data set (Fig 4B). Values between 1 and 2.15 suggest a uniform grid, while values below 1 suggest clustering. A value close to 1 is a random dispersion. The approximately 500 synthesized random poles (constrained by the same number of structures and pole area as the real data) had a mean of 1, as expected. The Plesiomonas data set, however, had a mean close to 2. This shows that structures at the pole are placed in a nonrandom distribution in a grid-like arrangement and that the relics and intact flagella are part of the same grid (Fig 4C). Peritrichous flagella in B. subtilis have previously been shown to have a Clark–Evans ratio greater than 1 [22]. How new flagella avoid assembling beneath existing relics, however, is unclear.