Motile ghosts of the halophilic archaeon, volcanii

Yoshiaki Kinosita (木下 佳昭)a,b,1,2, Nagisa Mikami (三上 渚)b, Zhengqun Lib, Frank Braunb, Tessa E. F. Quaxb, Chris van der Doesb, Robert Ishmukhametova, Sonja-Verena Albersb, and Richard M. Berrya

aDepartment of Physics, University of Oxford, OX1 3PU Oxford, United Kingdom; and bInstitute for Biology II, University of Freiburg, 79104 Freiburg, Germany

Edited by Howard C. Berg, Harvard University, Cambridge, MA, and approved September 4, 2020 (received for review May 15, 2020) swim using the archaellum (archaeal flagellum), a revers- To test the above hypothesis of ATP-driven motility in ar- ible rotary motor consisting of a torque-generating motor and a chaea, we established a permeabilized ghost model that enables the helical filament, which acts as a propeller. Unlike the bacterial flagellar manipulation of intracellular contents. Ghosts in ATP solution swim motor (BFM), ATP (adenosine-5′-triphosphate) hydrolysis probably like intact cells. Their motor rotation speed increases with [ATP] in drives both motor rotation and filamentous assembly in the archael- a way consistent with negative binding cooperativity. Other nucle- lum. However, direct evidence is still lacking due to the lack of a ver- otide triphosphates (NTPs) support slow rotation but with ∼200× satile model system. Here, we present a membrane-permeabilized reduced binding affinity compared with ATP. Measured motor ghost system that enables the manipulation of intracellular contents, torque indicates the energetic requirement for at least 12 ATP analogous to the triton model in eukaryotic flagella and gliding My- molecules to be hydrolyzed per revolution. We also demonstrated coplasma. We observed high nucleotide selectivity for ATP driving that CheY increases motor switching from counterclockwise (CCW) motor rotation, negative cooperativity in ATP hydrolysis, and the en- to clockwise (CW) rotation, and we discuss different rotary models. ergetic requirement for at least 12 ATP molecules to be hydrolyzed per revolution of the motor. The response regulator CheY increased motor Results switching from counterclockwise (CCW) to clockwise (CW) rotation. Swimming Motility of Live Cells. In this study, we Finally, we constructed the torque–speed curve at various [ATP]s used strains of the halophilic archaeon Haloferax volcanii (FYK3, and discuss rotary models in which the archaellum has characteristics FYK5) (SI Appendix, Result S1 and Table S1)withgenetically BIOPHYSICS AND COMPUTATIONAL BIOLOGY of both the BFM and F1-ATPase. Because archaea share similar cell introduced cysteine substitutions on the surface of the archaeal division and chemotaxis machinery with other domains of life, our filament, ArlA1(A124C). Hfx. volcanii is rod shaped, with a di- ghost model will be an important tool for the exploration of the uni- ameter of 0.9 μm and length of 2 to 10 μm, and swims using versality, diversity, and evolution of biomolecular machinery. multiple polar archaella (Fig. 1B and Movie S1). We increased the fraction of swimming Hfx. volcanii cells from 20 to 30% (13) to ATPase | archaellum | rotary motor | membrane-permeabilized ghost | 80% by adding 20 mM CaCl2 (SI Appendix, Fig. S2A). The Michaelis–Menten kinetics swimming-speed distribution of cells wild type (w.t.) for chemo- − taxis (FYK3) had two peaks, at 1.7 and 2.7 μms 1 (SI Appendix, otility is seen across all domains of life (1). Prokaryotes Fig. S1D). We observed only the slower peak in a CheY deleted Mexhibit various types of motilities, such as gliding, swim- strain (FYK5) (SI Appendix,Fig.S3A). ming, and twitching, driven by supramolecular motility machin- ery composed of multiple different proteins (2). In archaea only Significance swimming motility is reported, driven by the archaellum (archaeal flagellum), which consists of a reversible rotary motor Molecular motors are natural molecular machines that convert and a helical filament acting as a propeller (3, 4). The archaellar chemical energy to directional mechanical motion (e.g., ATP motor has no homology with the bacterial flagellar motor (BFM) [adenosine-5′-triphosphate]-driven linear motors myosin, kinesin, but is evolutionarily and structurally related to bacterial type IV anddyneininEukaryotes;theion-coupled rotary bacterial fla- pili (T4P) for surface motility (3). In , the filament gellar motor in Bacteria). Reconstituted systems such as in vitro is encoded by two genes, arlA and arlB [recently, the nomen- motility assays are powerful tools for understanding the mecha- clature of archaellum genes was changed from fla to arl (5)], and nisms of these motors. In this study, we established a membrane- the motor is composed of eight, arlC to -J (ref. 3 has details in permeabilized ghost system that controls the ATP-driven arch- Crenarchaeota). Euryarchaeota encode the same chemotaxis aellar rotary motor in halophilic archaea. With this assay, we system as flagellated bacteria, cheA, B, C, D, R, W, and Y, which demonstrated high nucleotide selectivity and negative binding might have been acquired by horizontal gene transfer from cooperativity and measured the energetic requirements for rota- Bacillus/Thermotoga groups (6). tion. Our ghost model is also a platform for further studies of the Fig. 1A shows the current association of functions with motor archaellum. More generally, it is also a powerful tool for the in- genes, based on analysis of mutants and biochemical data: ArlC/ vestigation of other intracellular molecular functions in archaea. D/E as mediators of directional switching of archaellar rotation, coupled to chemotaxis signals (7); an ArlF/ArlG complex inter- Author contributions: Y.K. and R.M.B. designed research; Y.K. and N.M. performed re- acting with the surface layer (S-layer), with ArlF regulating ArlG search; N.M., Z.L., F.B., T.E.F.Q., C.v.d.D., R.I., and S.-V.A. contributed new reagents/ana- filament assembly (8, 9); ArlH as a regulator of the switch between lytic tools; Y.K. analyzed data; and Y.K. and R.M.B. wrote the paper. assembly of the archaella and rotation (10); ArlI as an adenosine The authors declare no competing interest. triphosphatase (ATPase) essential for both assembly and rotation This article is a PNAS Direct Submission. (11); and ArlJ as the membrane-spanning component. An inhib- Published under the PNAS license. itor of proton translocating ATP (adenosine-5′-triphosphate) 1Present address: Molecular Physiology Laboratory, RIKEN, Saitama 351-0198, Japan. synthases reduced both intracellular [ATP] and swimming speed 2To whom correspondence may be addressed. Email: [email protected]. in (12), suggesting that archaellar rota- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ tion is driven by ATP hydrolysis at ArlI. However, direct evidence doi:10.1073/pnas.2009814117/-/DCSupplemental. is lacking due to the lack of a reconstituted system.

www.pnas.org/cgi/doi/10.1073/pnas.2009814117 PNAS Latest Articles | 1of7 Downloaded by guest on September 28, 2021 Live cell A ArlA,B B

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Fig. 1. Swimming ghosts. (A) The current model of the archaeallar motor in Euryarchaeota (details in the Introduction). (B, Left) Phase-contrast image of live cells. (B, Center) Fluorescent image of the same cells labeled with streptavidin-Dylight488, which binds to biotinylated filaments. (B, Right) Merged phase- contrast (magenta) and fluorescent (green) images. (Scale bars: 5 μm.) (C) Procedures to observe swimming ghosts. Live cells were permeabilized in a tube and then added to a flow chamber to observe swimming motility. (D) The same as B but for ghosts. (Scale bars: 5 μm.) (E) Sequential images of a change from a live cell to ghost (0.6-s intervals) (Movie S3). From 2.4 s, the ghost remains in focus and continues to swim. (Scale bars: 3 μm.) (F) Time course of swimming speed (triangles) and cell optical density (circles) from E. The arrow indicates the time of permeabilization. (G) Histograms of swimming speed in detergent before − − (Upper; 1.51 ± 0.34 μms 1) and after (Lower; 1.30 ± 0.21 μms 1) transformation into ghosts (mean ± SD, n = 24). B–G show data for Hfx. volcanii strain FYK3 (w.t. for chemotaxis).

Construction of Swimming Ghosts. We present an in vitro experi- were not able to determine the direction of archaellar filament mental system for the archaellum, similar to the triton model rotation. We therefore established a ghost-bead assay for for the eukaryotic flagellum (14) and the permeabilized ghost measuring ATP-coupled motor rotation (Fig. 2A). We at- model for gliding Mycoplasma mobile (15, 16). In those exper- tached cells with sheared, biotinylated archaellar filaments iments, samples were immobilized, allowing rapid addition of nonspecifically to the cover glass surface and then introduced high concentrations of detergent and rapid removal after per- 500-nm streptavidin beads, whichattachedtothefilaments, meabilization. Because rapid buffer exchange is not possible allowing direct observation of the speed and direction of with swimming Hfx. volcanii cells, we instead suspended motile archaellar rotation (Materials and Methods and SI Appendix, − cells in buffers containing lower concentrations of detergent Fig. S6). Addition of 0.1 mg mL 1 streptavidin (which would (0.015% [wt/vol] sodium cholate) and 2.5 mM ATP (Fig. 1C). cross-link adjacent filaments in a rotating bundle) did not stop The detergent reduced the refractive index of cells, indicating bead rotation, indicating that shearing removed most fila- permeabilization of the cell membrane and corresponding loss ments and rotating beads are attached to a single archaellum of cytoplasm. Fluorescent imaging revealed that permeabilized (Movie S4)(17). cells still possessed archaellar filaments, the cell membrane, For the preparation of ghosts, live cells labeled with rotating and S-layer (Fig. 1D and SI Appendix,Fig.S4). Remarkably, the beads were treated in a flow chamber with detergent (0.03% permeabilized cells still swam (Movie S2 and SI Appendix,Fig. sodium cholate) for less than 10 s to permeabilize their cell S5A). We named them “ghosts,” as in similar experiments on membrane. This higher concentration of detergent increased the M. mobile (15). yield of ghosts compared with the swimming cell assay. ATP was Fig. 1E shows a typical example of a live swimming cell changing absent from the detergent buffer, allowing ghost formation to be to a ghost, marked by a sudden change of image density at 2.4 s detected as cessation of rotation. Immediate removal of detergent, (Movie S3). The solution contained 2.5 mM ATP, and the swimming speed did not change dramatically when this cell by replacement with a buffer containing ATP, minimized further became a ghost (Fig. 1F; SI Appendix,Fig.S5B shows more damage to ghosts (observed with prolonged exposure to deter- examples). Fig. 1G showshistogramsofswimmingspeedsof gent) (SI Appendix, Result S2 and Fig. S7) and reactivated rotation cells in detergent before and after permeabilization, indicating (Fig. 2B and Movie S5). To assess the effects of detergent, we that ghosts swim at the same speed as live cells in this saturating measured rotation of beads with chemotaxis wild-type ghosts ATP concentration (P = 0.421834 > 0.05 by t test, ratio 0.93 ± (FYK3) in the presence of detergent (SI Appendix, Fig. S7). The 0.24, n = 24) (SI Appendix,Fig.S5C).Theabsenceofthefaster rotation speed fluctuated, often to zero, and beads rotated ex- = swimming peak (compare Fig. 1 G, Upper with SI Appendix,Fig. clusively CCW (n 11). This result indicates that detergent S1 D, Lower for the same strain with no detergent) indicates inhibited CW rotation in ghosts, which led us to remove detergent suppression of the faster swimming mode by detergent, before in all other bead experiments. permeabilization. We investigated the effect of different NTPs in the chemotaxis w.t. strain (FYK3). We observed both directions of rotation in Characterization of ATP-Coupled Motor Rotation by a Ghost-Bead this assay (Fig. 2C). We did not see any differences between CW Assay. Although we succeeded in producing swimming ghosts, and CCW rotation rates (SI Appendix, Fig. S8) and therefore, we found them difficult to track due to their low contrast, and we analyzed speeds collectively. Previous in vitro experiments

2of7 | www.pnas.org/cgi/doi/10.1073/pnas.2009814117 Kinosita et al. Downloaded by guest on September 28, 2021 A ADP+Pi B +Sodium cholate +500 μM [ATP], DNase ATP

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25 10 25 Magnitude 500 nm grids 0 0 0 Live cell Ghost 0.0 0.5 1.0 35.0 35.5 36.0 52.0 52.5 53.0 0 5 10 0 5 10 0 5 10 20 Time (sec) Speed (Hz) C CW 2 mM D E 3 Only ATP 125 μM 8 +0.5 mM ATP-γ-S 10 +2 mM ADP +2 mM ADP+Pi 32 μM 6 2 0 200-nm grids 0 4 32 μM 1/Speed (s) Revolutions

Speed (Hz) 1 -10 2 125 μM

CCW 2 mM -20 0 -0.005 0 0.005 0.01 0.015 0 5 10 ATP GTP CTP UTP -1 200-nm grids Time (sec) [1/ATP] (μM )

Fig. 2. Visualization of motor rotation in ghosts via beads attached to archaellar filaments. (A) Schematic of experimental setup (Upper) and phase-contrast images of a live cell (Lower Left) becoming a ghost (Lower Right). (Scale bars: 5 μm.) (B) An example of ghost formation, with reduced flow rates to allow tracking of the bead without loss of focus. (Upper) The y coordinate of the rotating bead during ghost preparation (markers at 500-nm intervals). (Lower) Shaded sections (Upper) are expanded (Left); with corresponding speed distributions by Fourier transform analysis (Right) using the same colors as in Upper. Blue shows the live cell, orange shows the motor stopped after treatment with detergent, and red shows the motor reactivated after addition of ATP. Rotation cannot reliably be measured during media exchange time, ∼10 s (data from Movie S5). (C, Left) The x–y plots of locations of two different beads attached to archaeallar filaments. Green and blue represent CW and CCW rotation, respectively. (C, Right) Angle vs. time for several similar beads. The slopes increase with [ATP] in both rotation directions, indicating an ATP-coupled motor rotation. (D) Rotation rate for different NTPs at 10 mM. The means ± SD were 6.14 ± 1.48 Hz for ATP (n = 43), 0.69 ± 0.24 Hz for GTP (n = 30), 1.18 ± 0.36 Hz for CTP (n = 32), and 0.99 ± 0.32 Hz for UTP (n = 29). (E) Lineweaver–Burk plot of rotation rate and inhibitors. Blue, ocher, green, and red represent data with 0.5 mM ATP-γ-S (n = 118), 2 mM ADP (n = 140), 2 mM ADP + Pi (n = = 114), and without inhibitors (n 345), respectively. Data are representative of three independent experiments. BIOPHYSICS AND COMPUTATIONAL BIOLOGY

showed that purified ArlI hydrolyzes different NTPs at similar ΔCheY live cells (FYK5) even during 5-min recordings (Fig. 3B). rates (18). However, the archaellar rotational rates in ghosts in FYK3 ghosts also switched very rarely (Fig. 3C), but their proba- 10 mM GTP (guanosine-5′-triphosphate), CTP (cytidine-5′- bility of CW rotation (P = 0.27) was similar to that of live cells with triphosphate), and UTP (uridine-5′-triphosphate) were 5 to 10 times CheY (P = 0.25); both were greater than live cells with CheY de- slower than in ATP (Fig. 2D). Other NTPs supported rotation at leted (P = 0.07) (Fig. 3 and SI Appendix,TableS3). We observed speeds similar to those in ∼200× lower ATP concentrations (SI CheY-GFP (green fluorescent protein) in Hfx. volcanii FYK7 using Appendix,Fig.S9). This result suggests that ArlI might specifically fluorescence microscopy (SI Appendix,Fig.S12). Live cells (SI recognize the adenine base of ATP, rather than distinguishing only Appendix,Fig.S12A, Upper) showed CheY-GFP distributed between purines and pyrimidines as in F1-ATPase (19), and/or throughout, with additional polar clusters, as in our previous report prevent extra energy consumption in vivo like the endopeptidase (13). By contrast, we could detect no CheY-GFP in ghosts (SI Clp (figure 1B of ref. 20). Appendix,Fig.S12A, Lower). These results indicate that CheY We also tested the inhibitory effects on rotation of ADP increases the probability of, but is not absolutely required for, CW (adenosine-5′-diphosphate), ADP + Pi (inorganic phosphate), rotation and switching in live cells. CheY is lost during ghost for- and the nonhydrolyzable ATP analog ATP-γ-S (adenosine-5′- mation, but the CW probability remains at the live cell level: motors [γ-thio]triphosphate). We saw no rotation with ATP-γ-S alone. stop switching, and one in three remains CW even though CheY is We measured the rotation rates of 500-nm beads attached to missing. We discuss this further in SI Appendix,ResultS4. archaella in ghosts over a range of [ATP] between 63 μM and 10 mM, with and without each of ATP-γ-S (0.5 mM), ADP (2 mM), ATP and Load Dependence of Motor Rotation. Fig. 4A shows the and ADP + Pi (each 2 mM). Fig. 2E shows the results as a dependence of rotation speed (f; revolutions per second) of 200-, Lineweaver–Burk plot. All three caused large reduction of ro- 500-, and 970-nm beads upon [ATP] in the range 8 μMto10mM. tation rates at lower [ATP] but much smaller reductions of the Michaelis–Menten fits to the data (solid lines) are poor below μ speed at saturating [ATP] (fmax), indicating competitive inhibi- 30 MATP(Movies S6 and S7; SI Appendix,Fig.S9shows more tion. The inhibitor constants, Ki, were estimated to be 0.11 mM examples). Fig. 4B shows the relationship between log([ATP]) and for ATP-γ-S (blue), 1.94 mM for ADP (ocher), and 1.22 mM for log(f/(fmax − f)), where fmax is estimated from the Michaelis– ADP.Pi (green). We also observed modest effects of pH and ion Menten fit (Fig. 4A), and the slope represents Hill coefficients of concentration on rotation (SI Appendix, Result S3 and Fig. S10). 0.63, 0.82, and 0.89 for 200-, 500-, and 970-nm beads, respectively. This result indicates negative cooperativity in ATP-driven arch- Archaeal CheY-Mediated CW Rotation. Bidirectional rotation of the aellar rotation (see below for discussion). Fig. 4C shows the re- flagellar motor in bacteria is mediated by the response regulator lationships between torque, speed, and [ATP] for archaellar CheY, which induces CW rotation (21). Live Hfx. volcanii cells rotation. The maximum motor torque was estimated to be ∼200 pN with CheY deleted (FYK5) were observed to rotate in either nm for live cells and ∼170 pN nm for ghosts, comparable with Hbt. direction, without switching during our typical recording time of salinarum live cell experiments (160 pN nm) (22). 30 s (n = 5 for CW rotation, n = 76 for CCW rotation). To observe the role of archaeal CheY in motor switching, we ex- Discussion tended our recording time to 300 s (Fig. 3). Switching from CCW Our estimated maximum torque (T) of ∼170 pN nm corresponds to CW rotation was frequent in chemotaxis wild-type live cells to the motor doing work for a single rotation (2πT, ∼1,000 pN (FYK3) (Fig. 3A and SI Appendix, Fig. S11) but very rare in nm) equivalent to the free energy of hydrolysis of ∼12 ATP

Kinosita et al. PNAS Latest Articles | 3of7 Downloaded by guest on September 28, 2021 Wild type live cell cooperativity could also arise from communication between ArlI A and ArlH rings, similar to interring effects in chaperonins (30). 4 CW Although ArlH has only the Walker A motif, ATP binding is 2 known to modulate the interaction between hexametric rings of ArlI and ArlH (10). 0 Our finding that the time-averaged motor torque decreases -2 with increasing speed at low loads (Fig. 4C) differs from a pre- Speed (Hz) vious report (22), which assumed constant torque irrespective of -4 CCW viscous load and speed and explained the observed speed vari- ations by assuming an extra contribution to the viscous drag from 0 100 200 300 0 150 an unseen remnant of the filament. The required length of these Time (sec) Counts remnants (ξ; 0.8 pN nm s) would be about 4 μm (22), which B seems unlikely given our observation that most filaments are ΔCheY live cell removed by shearing. The apparent trend of increasing torque 4 with speed, at low speeds in live cells, is within the error bars and 2 may be no more than experimental noise (SI Appendix, Fig. S13). Negative cooperativity (Fig. 4B) suggests that the hexametric 0 ATPase ArlI, in which two ATP molecules can be bound (25, -2 27), may have two distinct mechanisms for torque generation,

Speed (Hz) depending on [ATP]. At low [ATP], only one catalytic site would -4 be occupied, the second site having too-low binding affinity. At high [ATP], two ATP molecules could be bound and simulta- 0 100 200 300 0 150 neously hydrolyzed. In this scenario, the maximum torque would Time (sec) Counts increase with [ATP], consistent with Fig. 4C. The curves in C Fig. 4C are qualitatively similar to those reported for the BFM Wild type ghost with varying ion-motive force (31). By contrast, the equivalent 10 data for F1-ATPase correspond to Michaelis–Menten kinetics and torque that decreases linearly with increasing speed (figure 2 5 of ref. 32). Simple models for the torque–speed relationships, 0 similar to those applied to the BFM (31, 33) and high-resolution detection of steps in rotation (34) using gold nanoparticles (32, 35), may reveal the details of the rotation mechanism of the

Speed (Hz) -5 archaellum in future. -10 With due care to account for reflections in our microscope (SI 0 100 200 300 0 400 Appendix, Fig. S6), the rotational direction of the motor was directly determined by our bead assay. Together, our results Time (sec) Counts (Fig. 3 and SI Appendix, Fig. S6) demonstrate that in our ex- ’ Fig. 3. Archaeal CheY-mediated motor switching. (Left) Representative periments, at temperatures 20 °C below those of the organism s 5-min recordings of single motors using 970-nm beads in wild-type (FYK3; A) natural habitat, CheY greatly increases the probability of, but is and ΔCheY (FYK5; B) live cells and 500-nm beads in wild-type ghosts (C). not absolutely required for, CW rotation and switching in the Positive and negative speeds represent CW and CCW rotation, respectively. archaellum. The same is true in the flagellar motor, which re- (Insets) The y–x plots of bead rotation. Grids represent 500 nm. (Right) His- quires CheY for CW rotation at room temperature, but not tograms of speeds for each cell. below ∼6 °C (36). This similarity seems remarkable given the lack of evolutionary relation between the two motors but perhaps not so given that they use the same chemotaxis proteins to molecules per revolution, assuming a free energy of 80 to 90 perform the same function. Deletion of CheY also removed the pN nm per ATP molecule (23). Conservation of energy therefore fast-swimming mode (SI Appendix, Fig. S3), consistent with previous reports that linked fast swimming to CW rotation in sets a lower limit of 12 per revolution per motor on the ATP — hydrolysis rate, ∼15 times higher than that measured in vitro for other archaeal with right-handed helical filaments pushing cells (“forwards”) when rotating CW and pulling when ArlI (24). This indicates that motor assembly enhances ATPase CCW (SI Appendix, Fig. S3B) (37, 38). The absence of the fast- activity in the archaellum, as observed in other systems: for ex- swimming (CW) peak in cells in detergent before per- ample, the PilC–PilT interaction in T4P (25) and β- and γ meabilization (Fig. 1G) indicates a possible direct or chemotactic -subunit interaction in F1-ATPase (26). Hydrolysis of 12 ATP effect of detergent on the switch. Our findings appear to con- molecules per revolution is consistent with previous reports (22) tradict a previous study reporting CW-only rotation in a CheY and with models of a twofold ArlJ rotor rotating within a sixfold deletion strain of Hbt. salinarium (39). However, that study did ArlI ATPase (22, 27). not measure rotation direction directly, instead inferring it from < Hill coefficients 1 in fits to the data of Fig. 4B indicate forward swimming under the assumption that filaments were negative cooperativity in archaellar rotation at low [ATP]: sub- right handed in their strain, Hbt. salinarium M175, as in ref. 37. sequent ATP molecules bind the multimeric motor with lower Subsequent work revealed that filaments are in fact left handed rate constants than previous ones (28). Negative cooperativity in in Hbt. salinarium M175 (40), and thus, that rotation in the CheY the archaellar motor might be explained by a mechanism similar deletion was CCW—in agreement with our direct observations. to that proposed for F1-ATPase (29), where each subsequent When CheY is lost during ghost formation, cells stop switching, nucleotide that binds changes the conformational state of the many remaining “locked” in CW rotation. This indicates that entire complex, and the binding affinity at each site is lowered other factors also control switching of the archaellum. when the other two sites are occupied. In this scenario, negative There is, to date, no direct evidence published that answers cooperativity would be the result of the same interactions within the fundamental question of which components of the arch- the ArlI hexamer that power rotation (22, 27). Negative aellum are fixed relative to the cell (“stator”) and which rotate

4of7 | www.pnas.org/cgi/doi/10.1073/pnas.2009814117 Kinosita et al. Downloaded by guest on September 28, 2021 A B 2 2 200 nm 10 500 nm 6 970 nm 1 4 - f ) max 2 f 0 1 v /( Speed (Hz)

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250 Live cell (n =212) [ATP] = 10 mM (n =126) 250 μM (n = 66) 200 32 - 40 μM (n = 58)

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Fig. 4. ATP- and load-dependent archaeal motor rotation. (A) Rotation rates of 200-nm (green), 500-nm (pink) and 970-nm beads (purple) attached to 3[ ] archaellar filaments vs. [ATP]. The solid lines show fits to the Michaelis–Menten equation fmax ATP , where f and K are 10.3 Hz and 188 μM for 200-nm Km +[ATP] max m beads (n = 287), 6.8 Hz and 249 μM for 500-nm beads (n = 438), and 2.6 Hz and 132 μM for 1,000-nm beads (n = 303). (Right) Corresponding rotation rates of live cells: 18.47 ± 6.86 Hz for 200-nm beads (n = 19), 10.17 ± 2.39 Hz for 500-nm beads (n = 32), and 3.22 ± 1.27 Hz for 970-nm beads (n = 72). (B) A Hill plot of the same data. The Hill coefficient, determined from the slope of the plots, was 0.63 for 200-nm beads, 0.82 for 500-nm beads, and 0.89 for 970-nm beads. (C) Torque vs. speed (mean ± SD) of live cells and ghosts at various [ATP]. Torque was estimated as T = 2πfξ, where f is rotation speed and ξ = 8πηa3 +6πηar2 is the viscous drag coefficient of the bead. ξ was varied by using bead size (d = 2a = 200, 500, and 970 nm) and viscosity (η = 2.5, 3.9, and 7.2 mPa·s in buffer, 5% Ficoll, and 10% Ficoll, respectively). r is the major axis of the ellipse describing the orbit of the bead center. [ATP] and number of cells are indicated.

with the filament (“rotor”). ArlF and G are most likely part of Our ghost model may allow labeling of archaellar components to the stator, anchored to the S-layer. Previous reports indicate observe directly which are part of the rotor (42, 43), analysis of interaction between ArlF and the S-layer and a deficiency in rotational steps as in isolated F1 and other molecular motors (32, swimming motility of S-layer deleted cells (8, 9). Our observa- 44, 45), and direct investigations of the role of CheY. tions of increased motility with [CaCl2] and the speed fluctua- Our ghost assay represents an experimental system that allows tions at low [CaCl2](SI Appendix, Figs. S2 and S8), given that manipulation of the thermodynamic driving force for an archaeal calcium stabilizes S-layer (41), support this hypothesis. Homol- molecular motor, following previous examples including eukary- ogy to F1-ATPase and T4P is generally taken to favor a model otic linear motors (46), the PomAB-type BFM (45), and the My- where rotation of an ArlJ dimer within the central core of the coplasma gliding motor (15). We anticipate that this assay will be ArlI hexamer is driven by cyclic changes in the conformation of helpful for other biological systems. Archaea display chemotactic the ArlI hexameric ATPase, coupled to ATP hydrolysis (22, 27). and cell division machinery acquired by horizontal gene transfer In this model, ArlJ is the rotor, and all other motor components from bacteria (6, 47). Although the archaellum and bacterial fla- are the stator (SI Appendix, Fig. S14, Left). For switching, gellum are completely different motility systems, they share changes caused by CheY binding, presumably somewhere on ArlC/D/E, would have to propagate all of the way to the core of common chemotactic proteins. Theoretically, only our ghost ArlI, which would need separate mechanochemical cycles for technique allows monitoring of the effect of purified CheY iso- CW and CCW rotation. SI Appendix, Fig. S14, Right illustrates lated from different hosts on motor switching. Similarly, our ghost the other extreme possibility, most similar to the BFM. In this cells offer the potential to manipulate and study the archaeal cell model, conformational changes in ArlI would push on ArlF/G, division machinery as with in vitro ghost models of Schizo- either directly or via ArlC/D/E. In the latter case, the switch saccharomyces pombe (48). Ghost archaea offer the advantages of mechanism could reside entirely within ArlC/D/E, and ArlI need both in vivo and vitro experimental methods and will allow the not have separate modes for CW and CCW rotation. Interme- exploration of the universality, diversity, and evolution of biomolecules diate models are also possible (SI Appendix, Fig. S14, Center). in microorganisms.

Kinosita et al. PNAS Latest Articles | 5of7 Downloaded by guest on September 28, 2021 Materials and Methods 500 nm [18720; Polysciences], or 970 nm [PMC 1N; Bangs Laboratory, Inc.]) in buffer B (2.4 M KCl, 0.5 M NaCl, 0.2 M MgCl2,0.1MCaCl2 10 mM Hepes-NaOH, Strains and Cultivation. Strains, plasmids, and primers are summarized in SI −1 Appendix, Tables S1 and S2. Hfx. volcanii cells were grown at 42 °C on a pH 7.2, 0.5 mg mL BSA) were added into the flow chamber, incubated for 15 min, and then rinsed with buffer to remove unbound beads. The solution modified 1.5% Ca agar plate (2.0 M NaCl, 0.17 M Na2SO4, 0.18 M MgCl2, −1 0.06 M KCl, 0.5% [wt/vol] casamino acid, 0.002% [wt/vol] biotin, 0.005% [wt/vol] was replaced with buffer B plus 0.03% sodium cholate hydrate and 1 mg mL DNase. Immediately after the optical density of cells decreased (typically ∼5s thiamine hydrochloride, 0.01% [wt/vol] L-tryptophane, 0.01% [wt/vol] uracil, 10 mM [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]-NaOH after buffer exchange), buffer B plus 0.03% sodium cholate hydrate was [Hepes-NaOH] [pH 6.8], 1.5% [wt/vol] Agar). Note that 20 mM CaCl should replaced with buffer B plus ATP. This process for preparing ghosts typically 2 ∼ be added (SI Appendix,Fig.S2A). Colonies were scratched by the tip of a ended 10 s after the addition of detergent solution. For Fig. 2B and Movie S5, micropipette and subsequently suspended in 5 mL of Ca liquid medium. which aimed to record bead rotation for demonstration purposes, we ex- After a 3-h incubation at 37 °C, the culture was centrifuged at 5,000 rpm and changed buffers slowly and carefully to avoid the cell going out of focus. This ∼ concentrated to 100 times volume. The 20-μL culture was poured into 25 mL increased the duration of the process to 30 s. fresh Ca medium and again grown for 21 h with shaking of 200 rpm at 40 °C. For pH measurements, the following buffer was used: bis(2-hydroxyethyl) · · The final optical density would be around 0.07. iminotris(hydroxymethyl)methane HCl (Bis-Tris HCl) for pH 5.7 and 6.1 ex- Gene manipulation based on selection with uracil in ΔpyrE2 strains was periments, Hepes-NaOH for a pH 8.0 experiment, and tris(hydroxymethyl) · · carried out with polyethylene glycol 600 (PEG 600), as described previously aminomethane HCl (Tris HCl) for pH 8.6 and 9.3 experiments (SI Appendix, γ (49). For the creation of knockout strains, plasmids based on pTA131 were Fig. S10). For nucleotides experiments, ADP (A2754; Sigma Aldrich), ATP- -S used carrying a pyrE2 cassette in addition to ∼1,000-bp flanking regions of (A1388; Sigma Aldrich), GTP (ab146528; Abcam and R0461; Thermo the targeted gene. arlA1(A124C) was expressed by tryptophane promotor Fischer Scientific), CTP (R0451; Thermo Fischer Scientific), and UTP (R0471; (SI Appendix, Fig. S1). Thermo Fischer Scientific) were used. Most data were collected at 100 fps for 10 s. Bead position was determined by centroid fitting, giving cell trajectories Preparation of Biotinylated Cells. The culture of Hfx. volcanii FYK3 cells was r(t) = [x(t), y(t)]. The rotation rate was determined from either Fourier centrifuged and suspended into buffer A (1.5 M KCl, 1 M MgCl2,10mM −1 transform analysis (Fig. 2B) or linear fits to bead angle vs. time. (Fig. 2C). The Hepes-NaOH, pH 7.0). Cells were chemically modified with 1 mg mL biotin- rotational torque against viscous drag was estimated as T = 2πfξ, where f is PEG2-maleimide (Thermo Fischer) for 1 h at room temperature, and excess rotational speed and ξ = 8πηa3 +6πηar2 the viscous drag coefficient, with r biotin was removed with 5,000 × g centrifugation at room temperature the radius of rotation of the bead center (estimated as the major axis of the (R.T.) for 4 min. observed elliptical projections of circular bead orbits), a the bead radius, and η the viscosity. We neglected the viscous drag of filaments (22), which is Motility Assay on Soft-Agar Plates. A single colony was inoculated on a 0.25% expected to be negligible compared with these beads (50). (wt/vol) Ca-agar plate and incubated at 37 °C for 3 to 5 d. Images were taken To measure the viscosity of the medium, we tracked fluorescent beads with a digital camera (EOS kiss ×7; Canon). diffusing near the coverslip surface for 30 s at 100 fps and performed an analysis of their mean-squared displacement vs. time. From this analysis, the Microscopy. All experiments were carried under an upright microscope viscosities are estimated to be 0.0025 Pa·s in buffer, 0.0039 Pa·s in buffer + (Eclipse Ci; Nikon) equipped with a complementary metal oxide semicon- Ficoll 5%, and 0.0072 Pa·s in buffer + Ficoll 10% at 25 °C, which are slightly ductor (CMOS) camera (LRH1540; Digimo) and a 40× objective (EC Plan- higher than previous estimates (22). We inferred that this discrepancy might Neofluar 40 with Ph and 0.75 numerical aperture [N.A.]; Nikon) or 100× be due to the proximity of the glass surface (51). objective (CFI Plan Apo 100 with Ph and 1.45 N.A.; Nikon). Images were − recorded at 100 frames s 1 (fps) for 10 to 30 s. For a motility experiment at Fluorescence Experiments. For visualization of archaellar filaments, bio- 45 °C, a phase-contrast microscope (Axio Observer; Zeiss) equipped with a − tinylated cells were subsequently incubated with 0.1 mg mL 1 Dylight488- 40× objective (EC Plan-Neofluar 40 with Ph and 0.75 N.A.; Zeiss), a CMOS streptavidin (21832; Invitrogen) for 3 min, washed by centrifugation, and camera (H1540; Digimo), and an optical table (Vision Isolation; Newport) resuspended. were used. FM4-64 (F34653; Life Sciences) was used to stain the archaeal cell mem- For fluorescence observations, a fluorescence microscope (Nikon Eclipse Ti; brane. The powder was dissolved in buffer B (1.5 M KCl, 1 M MgCl ,10mM Nikon) equipped with a 100× objective (CFI Plan Apo 100 with Ph and 1.45 2 Hepes-NaOH, pH 7.0), and the cells were incubated for 30 min. The extra dye N.A.; Nikon), a laser (Nikon D = eclipse C1), an electron-multiplying charge- was removed by centrifugation. For microscopic measurements, the glass coupled device (EMCCD) camera (ixon+ DU897; Andor), and an optical table surface was cleaned using a plasma cleaner (PDC-002; Harrick plasm). (Newport) were used. The dichroic mirror and emitter were Z532RDC (C104891; Chroma) and 89006-ET-ECFP/EYFP/mcherry (Chroma) for an FM4- Quantum dots 605 (QD605) (Q10101MP; Invitrogen) was used to stain the 64 experiment, Z442RDC (C104887; Chroma) and 89006-ET-ECFP/EYFP/ archaeal cell surface, S-layer (38). Cells were biotinylated with biotin-NHS-ester mcherry for an S-layer experiment, and Z442RDC and ET525/50m (Chroma) (21330; Thermo Fischer) and incubated for 15 min at R.T. Extra biotin was for a Dylight488 experiment. 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. Construction of Swimming Ghosts. The flow chamber was composed of a 22 × 22-mm coverslip and a standard microscope slide. Two pieces of double- For visualization of CheY-GFP, Hfx. volcanii FYK7 cells were infused into a × sided sticky tape, cut to a length of ∼30 mm and aligned on either side of flow chamber and immobilized on a 18 18-mm glass surface (Matsunami an ∼5-mm-wide gap, were used as spacers between coverslips, forming a glass). Unbound cells were washed away with buffer A. GFP signal was tunnel chamber of ∼15-μL volume (38). The glass surface was modified with captured at 5 fps using a fluorescence microscope (Nikon Eclipse Ts2; Nikon) − × a Ca medium containing 5 mg mL 1 bovine serum albumin (BSA) (C1254; equipped with a 40 objective (EC Plan-Neofluar 40 with Ph and 0.75 N.A.; Sigma Aldrich) to avoid cells attaching. Nikon) and a CMOS camera (LRH1540; Digimo). To construct swimming ghosts, 10 μL each of cell culture in Ca medium Evaluation of the Purity of NTP Solutions. NTPs (ATP, CTP, GTP, and UTP) were 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 purchased from abcam, Thermo Fisher Scientific, and Sigma Aldrich. Their Aldrich), and 0.03% sodium cholate hydrate (Sigma Aldrich) were mixed in purity was evaluated using a reverse-phase chromatography column (02485- an Eppendorf tube. Subsequently, the 20-μL mixture was infused into the 81; Cosmosil). The column was connected to a high-performance liquid flow chamber. chromatography (HPLC) (PU-4180; JASCO) and preequilibrated with 20 mM potassium phosphate buffer (KPi), pH 7.0. Nucleotides were dissolved into Phase-contrast images were captured at 20 fps for 15 s. Swimming tra- 20 mM KPi buffer and eluted with a flow rate of 2 mL/min. jectories were determined by the centroid positions of cells and subjected to analysis using Igor pro 6.30. Given the trajectory of cells, r(t) = [x(t), y(t)], the v t v t = r(t+Δt)−r(t) Data Availability. All study data are included in the article and SI Appendix. swimming velocity ( ) was defined as ( ) Δt . ACKNOWLEDGMENTS. We thank Prof. Rikiya Watanabe for technical help in Bead Assay. For the observation of a rotational bead attached to an archaellar revised experiments, especially for an HPLC measurement to check the ATP μ filament, archaellar filaments were sheared by 30 times pipetting with a 200- L contamination in NTP solution; Prof. Achillefs Kapanidis and Dr. Abhishek pipette (F123601; Gilson), infused into a flow chamber, and kept for 10 min. Mazumder for sharing chemicals; Dr. Nariya Uchida for sharing his useful Streptavidin-conjugated fluorescent beads (200 nm [F6774; Molecular Probes], information in the torque calculation; and Dr. Mitsuhiro Sugawa for the

6of7 | www.pnas.org/cgi/doi/10.1073/pnas.2009814117 Kinosita et al. Downloaded by guest on September 28, 2021 technical advice in the microscope measurement. This study was supported Yoshida Scholarship Foundation (to N.M.), the Deutsche Forschungsgemein- in part by the Japan Society for the Promotion of Science (JSPS) Postdoc- schaft with an Emmy Noether grant (to T.E.F.Q.), the Collaborative Research toral Fellowship for Research Abroad, Research Activity Start-up from JSPS Center Grant from the Deutsche Forschungsgemeinschaft (to S.-V.A.), and a (20K22640) and the Uehara Memorial Foundation postdoctoral fellow and grant from the Funding Program for the Biotechnology and Biological Sci- special postdoctoral researchers (SPDR) program from RIKEN (to Y.K.), the ences Research Council (to R.M.B.).

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