Summary
Motility is seen across all domains of life1. Prokaryotes exhibit various types of motilities, such as gliding, swimming, and twitching, driven by supramolecular motility machinery composed of multiple different proteins2. In archaea only swimming motility is reported, driven by the archaellum (archaeal flagellum), a reversible rotary motor consisting of a torque-generating motor and a helical filament which acts as a propeller3,4. Unlike the bacterial flagellar motor (BFM), adenosine triphosphate (ATP) hydrolysis probably drives both motor rotation and filamentous assembly in the archaellum5,6. However, direct evidence is still lacking due to the lack of a versatile model system. Here we present a membrane-permeabilized ghost system that enables the manipulation of intracellular contents, analogous to the triton model in eukaryotic flagella7 and gliding Mycoplasma8,9. We observed high nucleotide selectivity for ATP driving motor rotation, negative cooperativity in ATP hydrolysis and the energetic requirement for at least 12 ATP molecules to be hydrolyzed per revolution of the motor. The response regulator CheY increased motor switching from counterclockwise (CCW) to clockwise (CW) rotation, which is the opposite of a previous report10. Finally, we constructed the torque-speed curve at various [ATP]s and discuss rotary models in which the archaellum has characteristics of both the BFM and F1-ATPase. Because archaea share similar cell division and chemotaxis machinery with other domains of life11,12, our ghost model will be an important tool for the exploration of the universality, diversity, and evolution of biomolecular machinery.
The archaellar motor has no homology with the BFM, but is evolutionarily and structurally related to bacterial type IV pili (T4P) for surface motility3. In Euryarchaeota, the filament is encoded by two genes, flgA (flaA in Methanococcus) and flgB (flaB in Methanococcus), and the motor eight flaC-J (see Ref. 3 for details in Crenarchaeota). Euryarchaeota encode the full set of a chemotaxis system, cheA, B, C, D, R, W, and Y, like flagellated bacteria, which might have been acquired by horizontal gene transfer from Bacillus/Thermotoga groups11.
Figure 1a (top) shows the current association of functions with the motor genes, based on analysis of mutants and biochemical data: FlaC/D/E as switching proteins for the directional switch of archaellum rotation coupled with the signals from the chemotaxis system13; FlaG and FlaF complex interacting with the surface layer (S-layer), with FlaF regulating FlaG filament assembly14,15; FlaH as a regulator of the switch between assembly of the archaella and rotation16; FlaI as the ATP-driven motor for both assembly and rotation5; FlaJ as the membrane-spanning component. An inhibitor of proton translocating ATP synthases reduced both intracellular [ATP] and swimming speed in Halobacterium salinarum6, suggesting that archaellar rotation is driven by ATP hydrolysis at FlaI. However, direct evidence is lacking due to the lack of a reconstituted system. There is also no direct evidence as to which components are anchored to the cell and which rotate with the filament: Figure 1b illustrates possibilities which we discuss below.
Here we present an in vitro experimental system for the archaellum, similar to the Triton model for the eukaryotic flagellum7 and the permeabilized ghost model for gliding Mycoplasma mobile8,9 We use the halophilic archaeon Haloferax volcanii. Hfx. volcanii possesses multiple polar archaella and swims at 2-4 μm s-1 at room temperature, with CW rotation more efficient for propulsion than CCW (Fig. 1c top, Supplementary Result 1 and Supplementary Video 1)17 We increased the fraction of swimming Hfx. volcanii cells from 20-30 % to 80 % by adding 20 mM CaCl2 (Supplementary Figure 1a).
To prepare our experimental model system, we suspended motile cells in buffers containing detergent (0.015 % sodium cholate) and 2.5 mM ATP (Fig. 1d). Fluorescent imaging revealed that ghosts still possessed archaellar filaments, the cell membrane, and S-layer (Fig. 1c bottom and Supplementary Figure 2). The detergent reduced the refractive index of cells, indicating permeabilization of the cell membrane and corresponding loss of cytoplasm. Remarkably, the permeabilized cells still swam (Supplementary Video 2 and Fig. 1d lower right) We named them “ghosts,” as in similar experiments on Mycoplasma mobile9. Fig. 1e shows a typical example of a live swimming cell changing to a ghost, marked by a sudden change of image density at 8.75 sec. The solution contained 2.5 mM ATP and the swimming speed did not change dramatically when this cell became a ghost (Fig. 1f, see Supplementary Figure 3a for another example). Fig. 1g shows histograms of swimming speeds of cells, before and after adding detergent, indicating that ghosts swim at the same speed as live cells in this, saturating, ATP concentration (P = 0.421834 > 0.05 by t-test, ratio 0.93 ± 0.24, n = 24, Supplementary Figure 3b). Wild-type ghosts showed a single speed peak around 1.5 μm s-1 in detergent (Fig. 1g, bottom), in contrast to peaks at ~1.7 and 3 μm s-1 for the same cells without detergent (Supplementary Figure 1b). If CW rotation is associated with the faster peak17, and is suppressed by detergent, this is consistent with our lack of observation of CW rotation of beads in the presence of detergent (see below). The lack of the 3 μm s-1 peak in cells lacking CheY (Fig. 1g, top and Supplementary Figure 4) would then indicate that CheY is required for CW rotation, as in the bacterial flagellar motor18. However, we found it difficult to track swimming ghosts due to their low contrast, and were not able to determine the direction of archaellar filament rotation.
To overcome these difficulties, we established a ghost-bead assay for measuring ATP-coupled motor rotation (Fig. 2a). We attached cells with sheared, biotinylated archaellar filaments nonspecifically to the cover glass surface, and then introduced 500 nm streptavidin beads which attached to the filaments (Material and Methods and Supplementary Result 2). Addition of 0.1 mg ml-1 streptavidin (which would crosslink adjacent filaments in a rotating bundle) did not stop bead rotation, indicating that shearing removed most filaments and rotating beads are attached to a single archaellum19 (Supplementary Video 3). For the preparation of ghosts, live cells labelled with rotating beads were treated in a flow chamber with detergent (0.03 % sodium cholate, as for swimming cells) for less than 30 sec to permeabilize their cell membrane, and subsequently the detergent was replaced with buffer containing ATP. Motor rotation was stopped by permeabilization and reactivated by the addition of ATP (Supplementary Video 4, Fig. 2b). Although beads on ghost cells rotated only CCW in the presence of detergent (n = 11, see Supplementary Result 3 and Supplementary Figure 7), we observed both directions of rotation after detergent removal (Fig. 2c). We did not see any differences between CW and CCW rotation rates (Supplementary Figure 8) and therefore analyzed speeds collectively.
We next investigated the effect of different nucleotide triphosphates (NTPs). Previous in vitro experiments showed that purified FlaI hydrolyzes different NTPs at similar rates20. However, the archaellar rotational rates in ghosts in 10 mM GTP, CTP, and UTP were 5-10 times slower than in ATP (Fig. 2d). This suggests that the motor complex might increase the selectivity of FlaI for nucleotides and/or prevent extra energy consumption in vivo like the endopeptidase Clp (see Fig. 1B in Ref. 21). We also tested the inhibitory effects on rotation of ADP, ADP+Pi, and the non-hydrolysable ATP analog ATP-γ-S (adenosine 5’-[γ-thio]triphosphate). We saw no rotation with ATP-γ-S alone. We measured the rotation rates of 500 nm beads attached to archaella in ghosts over a range of [ATP] between 63 μM and 10 mM, with and without each of ADP (2 mM), ADP+Pi (each 2mM) and ATP-γ-S (0.5 mM). Figure 2e shows the results as a Lineweaver-Burk plot. All 3 caused large reduction of rotation rates at lower [ATP], but much smaller reductions of fmax, indicating competitive inhibition. The inhibitor constants, Ki, were estimated to be 1.94 mM for ADP (Ocher), 1.22 mM for ADP.Pi (Green), and 0.11 mM for ATP-γ-S (Blue). We also observed modest effects of pH, and ion concentration on rotation (Supplementary Result 4).
Although we expected bi-directional rotational to be mediated by the response regulator CheY10, live cells without CheY were observed to rotate in either direction, without switching during our typical recording time of 30 s (n = 5 for CW rotation, n = 76 for CCW rotation). To observe the role of archaeal CheY in motor switching, we extended our recording time to 300 s. Switching from CCW to CW rotation was frequent in wild type live cells, but rare in ΔCheY live cells even during 5 min recordings (Fig. 3 and Supplementary Figure 10). Wild type ghosts still switched, but the bias and fraction of switching cells were changed, suggesting the chemotaxis system was still active, but altered (Supplementary Table 3).
Figure 4a shows the dependence of rotation speed f, revs per second) of 200, 500, and 970 nm beads upon [ATP] in the range 8 μM to 10 mM. Michaelis-Menten fits to the data (solid lines) are poor below 30 μM ATP. Figure 4b shows the relationship between log([ATP]) and log(f / (fmax-f)), where fmax is estimated by Michaelis-Menten plot (Fig. 4a), and the slope represents Hill coefficients of 0.63, 0.82 and 0.89 for 200 nm, 500 nm, 970 nm beads respectively. This result indicates negative cooperativity in ATP-driven archaellar rotation, (see below for discussion). Figure 4c shows the relationships between torque, speed and [ATP] for archaellar rotation. The maximum motor torque was estimated to be ~200 pN nm for live cells and ~170 pN nm for ghosts, comparable to Hbt. salinarum live-cell experiments (160 pN nm)22.
Our estimated maximum torque of ~170 pN nm corresponds to the motor doing work for a single rotation (2πT, ~1000 pN nm) equivalent to the free energy of hydrolysis of ~12 ATP molecules per revolution, assuming the free energy of 80-90 pN nm per ATP molecule. Conservation of energy therefore sets a lower limit of 12 /rev/motor on the ATP hydrolysis rate, ~15 times higher than that measured in vitro for FlaI23. This indicates that motor assembly enhances ATPase activity in the archaellum, as observed in other systems; for example the PilC-PilT interaction in T4P24 and β- and γ-subunit interaction in F1-ATPase25. Hydrolysis of 12 ATP molecules per revolution is consistent with previous reports22 and with models of a 2-fold FlaJ rotor rotating within a 6-fold FlaI ATPase22,26.
Negative cooperativity in archaellar rotation at low [ATP] (Fig. 4a-b) might be explained by a mechanism similar to that proposed for F1-ATPase, where bi-site and tri-site hydrolysis correspond to nucleotide occupancy of the three catalytic sites alternating between 1 and 2 or between 2 and 3 or three, respectively27, and the hydrolysis rate is slower when only 1 site is occupied. In this scenario, negative cooperativity would be the result of the same interactions within the FlaI hexamer that power rotation22,26. Negative cooperativity could also arise from communication between FlaI and FlaH rings, similar to inter-ring effects in chaperonins28. Although FlaH has only the Walker A motif, ATP binding is known to modulate the interaction between hexametric rings of FlaI and FlaH 16
Our finding that the time-averaged motor torque decreases with increasing speed at low loads (Fig. 4c) differs from a previous report22, which assumed constant torque irrespective of viscous load and speed, and explained the observed speed variations by assuming an extra contribution to the viscous drag from an unseen remnant of the filament. The required length of these remnants (ξ,0.8 pN nm s) would be about 4 μm22, which seems unlikely given our observation that most filaments are removed by shearing. The curves in figure 4c are qualitatively similar to those reported for the BFM with varying ion-motive force29 By contrast, the equivalent data for F1-ATPase correspond to Michaelis-Menten kinetics and torque that decreases linearly with increasing speed (see Fig. 2 in Ref.30). Simple models for the torque-speed relationships, similar to those applied to the BFM29,31,29,31 and high-resolution detection of steps in rotation32 using gold nanoparticles30,33 may reveal the details of the rotation mechanism of the archaellum in future.
So far, there is no direct evidence as to which components of the archaellum are fixed relative to the cell (“stator”) and which rotate with the filament (“rotor”). FlaF and G are most likely part of the stator, anchored to the S-layer. Previous reports indicate interaction between FlaF and the S-layer and a deficiency in swimming motility of S-layer deleted cells14,15. Our observations of increased motility with [CaCl2] and the speed fluctuations at low [CaCl2] (Supplementary Figures 1 and 9), given that calcium stabilizes S-layer34, support this hypothesis. Homology to F1-ATPase and T4P are generally taken to favour a model where rotation of a FlaJ dimer within the central core of the FlaI hexamer is driven by cyclic changes in the conformation of the FlaI hexameric ATPase, coupled to ATP hydrolysis22,26. In this model, FlaJ is the rotor and all other motor components are the stator (Figure 1b, left). For switching, changes caused by CheY binding, presumably somewhere on FlaC/D/E, would have to propagate all the way to the core of FlaI, which would need separate mechanochemical cycles for CW and CCW rotation. Figure 1b, right, illustrates the other extreme possibility, most similar to the BFM. In this model, conformational changes in FlaI would push on FlaF/G, either directly or via FlaC/D/E. In the latter case, the switch mechanism could reside within FlaC/D/E and FlaI need not have separate modes for CW and CCW rotation. Intermediate models are also possible (Figure 1b, middle). Our ghost model may allow labelling of archaellar components to observe directly which are part of the rotor35,36, analysis of rotational steps as in isolated F1 and other molecular motors30,37,38, and direct investigations of the role of CheY.
Our finding that archaeal CheY increases CW bias (Fig. 3) is inconsistent with previous reports10 that the role of archaeal CheY in Hbt. salinarum enhances switching from CW to CCW rotation, as revealed by a dark-field microscopy of swimming cells10,39. This study measured static filaments to be right-handed in Hbt. salinarum M175, and inferred from this that CW rotation propels a cell forward, CCW backwards. We speculate that the contradictory result might be due to misinterpretation of filament helicity caused by errors in accounting for reflections in the microscope optics - cryo-EM data show left-handed helicity in Hbt. salinarum M175, supporting our conclusion40. With due care to account for reflections in our microscope (Supplementary figure 6), our bead assay is a direct observation of the rotational direction of the motor.
Our ghost assay represents the first experimental system that allows manipulation of the thermodynamic driving force for an archaeal molecular motor, following previous examples including eukaryotic linear motors41, the PomAB-type BFM38, and Mycoplasma gliding motor9. We anticipate that this assay will be helpful for other biological systems. Archaea display chemotactic and cell division machinery acquired by horizontal gene transfer from bacteria11,12. Although the archaellum and bacterial flagellum are completely different motility systems, they share common chemotactic proteins. Theoretically, only our ghost technique allows monitoring of the effect of purified CheY isolated from different hosts on motor switching. Similarly, our ghost cells offer the potential to manipulate and study the archaeal cell division machinery as with in vitro ghost models of Schizosaccharomyces pombe42 Ghost archaea offer the advantages of both in vivo and vitro experimental methods and will allow the exploration of the universality, diversity, and evolution of biomolecules in microorganisms.
Material and Methods
Strain and Cultivation
Strains, plasmids, and primers are summarized in Supplementary Table 1, 2. Haloferax. volcanii (HfX. volcanii) cells were grown at 42°C on a modified 1.5 % Ca agar plate (2.0 M NaCl, 0.17 M Na2SO4, 0.18 M MgCl2, 0.06 M KCl, 0.5% (wt/vol) casamino acid, 0.002% (wt/vol) biotin, 0.005% (wt/vol) thiamine hydrochloride, 0.01% (wt/vol) L-tryptophane, 0.01% (wt/vol) uracil, 10 mM HEPES-NaOH (pH 6.8) and 1.5% (wt/vol) Agar). Note that 20 mM CaCl2 should be added (Supplementary Result 1). Colonies were scratched by the tip of a micropipette and subsequently suspended in 5ml of Ca liquid medium. After 3h incubation at 37°C, the culture was centrifuged at 5,000 r.p.m and concentrated to 100 times volume. The 20 μl culture was poured into 25-ml fresh Ca medium and again grown for 21 h with shaking of 200 r.p.m at 40°C. The final of an optical density would be around 0.07.
Gene manipulation based on selection with uracil in ΔpyrE2 strains was carried out with PEG 600, as described previously43. For the creation of KO strains, plasmids based on pTA131 were used carrying a pyrE2 cassette in addition to ~1000-bp flanking regions of the targeted gene. flgA1(A124C) was expressed by tryptophane promotor (Supplementary Result 2).
Preparation of biotinylated cells
The culture of HfX. volcanii Cys mutant was centrifuged and suspended into buffer A (1.5 M KCl, 1 M MgCl2, 10 mM HEPES-NaOH pH 7.0). Cells were chemically modified with 1 mg ml-1 biotin-PEG2-maleimide (Thermo Fischer) for 1 h at room temperature, and excess biotin was removed with 5,000 g centrifugation at R.T for 4 min.
Motility assay on soft-agar plate
A single colony was inoculated on a 0.25% (wt/vol) Ca-agar plate and incubated at 37°C for 3-5 days. Images were taken with a digital camera (EOS kiss X7; Canon).
Microscopy
All experiments were carried under an upright microscope (Eclipse Ci; Nikon) equipped with a 40× objective (EC Plan-Neofluar 40 with Ph and 0.75 N.A.; Nikon) or 100× objective, a CMOS camera (LRH1540; Digimo). Images were recorded at 100 fps for 10-30 sec. For a motility experiment at 45°C, a phase-contrast microscope (Axio Observer; Zeiss) equipped with a 40× objective (EC Plan-Neofluar 40 with Ph and 0.75 N.A.; Zeiss), a CMOS camera (H1540; Digimo), and an optical table (Vision Isolation; Newport) were used.
For fluorescent experiment, a fluorescent microscope (Nikon Eclipse Ti; Nikon) equipped with a 100× objective (CFI Plan Apo 100 with Ph and 1.45 N.A.; Nikon), a laser (Nikon D=eclipse C1), an EMCCD camera (ixon+ DU897; Andor), and an optical table (Newport) were used. The dichroic mirror and emitter were Z532RDC (C104891, Chroma) and 89006-ET-ECFP/EYFP/mcherry (Chroma) for an FM4-64 experiment, Z442RDC (C104887, Chroma) and 89006-ET-ECFP/EYFP/mcherry for an S-layer experiment, and Z442RDC and ET525/50m (Chroma) for a Dylight488 experiment.
Construction of swimming ghosts
The flow chamber was composed of a 22×22 coverslip and slide glass. Two pieces of double-sided tape, cut to a length of ~30 mm, were used as spacers between coverslips17 Two tapes were fixed with a ~5 mm interval, and the final volume was ~15 μl. The glass surface was modified with a Ca medium containing 5 mg ml-1 bovine serum albumin (BSA) to avoid cells attaching to a glass surface.
To construct swimming ghosts, 10 ul of cell culture in Ca medium and buffer B (2.4 M KCl, 0.5 M NaCl, 0.2 M MgCl2, 0.1 M CaCl2, 10 mM HEPES-NaOH pH 7.2) containing 1 mg ml-1 DNase, 5 mM ATP (A2383, Sigma Aldrich), and 0.03 % sodium cholate (Sigma Aldrich) was mixed in an Eppendorf tube. Subsequently, the 20 ul mixture was infused into the flow chamber.
Phase-contrast images were captured at 20 frames s-1 for 15 sec. Swimming trajectories were determined by the centroid positions of cells and subjected to analysis using Igor pro. Given the trajectory of cells, r(t) = [x(t), y(t)], the swimming velocity v(t) was defined as .
Bead assay
For the observation of a rotational bead attached to an archaellar filament, archaellar filaments were sheared by 30 times pipetting with 200 μl pipette (F123601, Gilson), infused into a flow chamber and kept for 10 min. Streptavidin-conjugated fluorescent beads (200 nm (F6774, Molecular probes), 500 nm (18720, Polysciences) or 970 nm (PMC 1N, Bangs lab)) in buffer B (2.4 M KCl, 0.5 M NaCl, 0.2 M MgCl2, 0.1 M CaCl2 10 mM HEPES-NaOH pH 7.2, 0.5 mg ml-1 BSA (Sigma Aldrich)) were added into the flow chamber, incubated for 15 min, and then rinsed with buffer to remove unbound beads. The solution was replaced to buffer B containing 0.03 % sodium cholate hydrate (C1254, Sigma Aldrich) and 1 mg ml-1 DNase. When the optical density of cells was decreased, buffer B was replaced with buffer A containing ATP. Rotary ghosts were prepared within 1 min (Fig. 2b). For pH measurements, the following buffer was used: Bis-Tris HCl for pH5.7 and 6.1 experiments; HEPES-NaOH for a pH8.0 experiment; and Tris-HCl for pH8.6 and pH9.3 experiments (Supplementary Figure 9). For nucleotides experiments, ADP (A2754, Sigma Aldrich), ATP-γ-S (A1388, Sigma Aldrich), GTP (ab146528, Abcam), CTP (R0451, Thermo Fischer Scientific), and UTP (R0471, Thermo Fischer Scientific) were used. Most data were collected at 100 frames s-1 for 10 sec.
Bead position was determined by centroid fitting, giving cell trajectories, r(t) = [x(t), y(t)]. The rotation rate was determined from either Fourier transform analysis (Fig. 2b) or a fit with a linear function to time course of bead rotation. (Fig. 2c). The rotational torque against viscous drag was estimated as T = 2πfξ, where f is rotational speed and = 8πηa3+6πηar2 the viscous drag coefficient, with r the radius of rotation (major axis of ellipse), a the bead radius, and η the viscosity. We neglected the viscous drag of filaments22, which is expected to be negligible compared to these beads44.
To measure the viscosity of the medium, we tracked diffusing fluorescent beads for 30 sec at 100 fps and performed an analysis of their mean-squared displacement versus time. From this analysis, the viscosities are estimated to be 0.0025 Pa·s in buffer, 0.0039 Pa·s in buffer + ficoll 5 %, and 0.0072 Pa·s in buffer + ficoll 10 % at 25 °C, which are slightly higher than a previous estimate22. We inferred that this discrepancy might be due to the proximity of the glass surface45.
Fluorescent experiments
For visualization of archaellar filaments, biotinylated cells were subsequently incubated with 0.1 mg ml-1 Dylight488-streptavidin (21832, Invitrogen) for 3 min, washed by centrifugation, and resuspended.
FM4-64 (F34653, Life Sciences) was used to stain the archaeal cell membrane. The powder was dissolved by buffer B (1.5 M KCl, 1 M MgCl2, 10 mM HEPES-NaOH pH 7.0), and the cells were incubated for 30 min. The extra dye was removed by centrifugation. For microscopic measurements, the glass surface was cleaned using a plasma cleaner (PDC-002; Harrick plasm).
Quantum dots 605 (Q10101MP, Invitrogen) was used to stain the archaeal cell surface, S-layer17 Cells were biotinylated with biotin-NHS-ester (21330, ThermoFisher) and incubated for 15 min at R.T. Extra biotin was washed by centrifugation. Biotinylated cells were subsequently incubated with the buffer containing QD605 at a molar ratio of 400:1 for 3 min, washed by centrifugation, and resuspended.
Author Contributions
Y.K. and R.M.B designed the research. Y.K. performed all experiments and obtained all data; N.M. helped genetics, biochemistry, and preparation of figures; Z.L, F.B., T.EF.Q., C.v.d.D and S.-V. A. helped genetics; R.I helped the ghost experiments; N.M. and R.M.B helped microscope measurements; Y.K., and R.M.B. wrote the paper.
Conflict of interest
The authors declare no competing interests.
Acknowledgements
We thank Prof. Achillefs Kapanidis and Dr. Abhishek Mazumder for sharing chemicals, Dr. Nariya Uchida for sharing his useful information in the torque calculation, and Dr. Mitsuhiro Sugawa for the technical advice in the microscope measurement. This study was supported in part by a grant from the Funding Program for the Biotechnology and Biological Sciences Research Council (to R.M.B), Collaborative Research Center Grant from the Deutsche Forschungsgemeinschaft (to S-V.A.). Y.K was recipient of the Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad and the Uehara Memorial Foundation postdoctoral fellow, and N.M. was recipient of the Yoshida Scholarship Foundation.
Footnotes
We changed the format from a brief communication into letter and added new results about the switching behaviors.