Halobacterium salinarum

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1 SUPPLEMENTARY INFORMATION ARTICLE NUMBER: DOI: /NMICROBIOL Direct observation of rotation and steps of the archaellum in the swimming halophilic archaeon Halobacterium salinarum Yoshiaki Kinosita 1, Nariya Uchida 2, Daisuke Nakane 1 & Takayuki Nishizaka 1 1 Department of Physics, Gakushuin University, Mejiro, Toshima-ku, Tokyo , Japan. 2 Department of Physics, Tohoku University, Sendai , Japan This file includes: Supplementary Methods Supplementary Results 1-5 Supplementary Figures S1-S6 Supplementary Table S1 Supplementary References NATURE MICROBIOLOGY 1

2 Supplementary Methods Hydrodynamic model We model the cell body by a cylinder of length L and radius A, which has the center-of-mass velocity V and angular velocity Ω. The flagellum in our model is a helix of radius b, height h and pitch λ and is rotating at frequency ω. The axis of the helix is tilted by an angle β from the axis of the cell body, and is attached to the center of the base plane of the cylinder, whose position is expressed as R L/2 in terms of the center-of-mass position R and length vector L (from the bottom to top) of the cylinder. In the moving coordinate system (x, y, z) that is fixed to the cell body, the configuration of the helical flagellum is specified by the position vector ρ(s) = (b cos θ(s), b sin θ(s), s) = be r + se z, (1) where s is the local height (0 < s < h), θ(s) = ks ωt is the phase of flagellar rotation at time t with k = 2π/λ being the wavenumber, and the orthogonal basis e r = (cos θ, sin θ, 0), e θ = ( sin θ, cos θ, 0), e z = (0, 0, 1) of the cylindrical coordinate system is introduced. The slender-body theory (SBT) gives the linear density of the viscous drag force as f(s) = C ρ(s) [ I 1 2 ρ (s)ρ (s) ], C = 4πη ln(l/a), (2) where ρ = ρ/ t, ρ = dρ/ds, η is the viscosity, and l = h 1 + (2πb/λ) 2 and a are the length and radius of the flagellum, respectively. In the static frame, we need to add R L/2 and its time derivative V Ω L/2 to ρ(s) and ρ(s), respectively. The viscous force F f and torque T f exerted by the flagellum on the fluid is obtained by integrating f(s) and [ρ(s) R] f(s) over the filament as linear functions of V and Ω, in the form and F f = F 0 + M F V V + M F Ω Ω (3) T f = T 0 + M T V V + M T Ω Ω. (4) Here, F 0 and T 0 are the force and torque exerted by the flagellum when the cell body is fixed, and are proportional to ω. For example, F 0 is computed by substituting Eq.(1) into (2) with ρ(s) = bωe θ and ρ (s) = bke θ + e z, as [( ) 1 f 0 (s) = Cbω 2 b2 k 2 1 e θ + 1 ] 2 bke z. (5) Integrating this over the filament, we obtain the total drag force by the flagellum as F 0 = l h h 0 ds f 0 (s) = Clbω h {( ) b2 k 2 1 k [e r] s=h s=0 + 1 } 2 bhke z. (6) The power needed to rotate the flagellum (when the cell body is fixed) reads P = l h h 0 dsf 0 (s) ρ(s) = Clb2 ω 2 h (1 12 b2 k 2 ) h. (7) The viscous drag force and torque exerted by the cell body are given in the form F b = Z F V V and T b = Z T Ω Ω, where the matrices Z F V and Z T Ω are known functions of L and A. Because the cell body is tilted from the flagellar axis, it is convenient to introduce a new coordinate system (X, Y, Z) that is fixed to the cell body with the Z-axis parallel to the axis of cylinder. The basis vectors E X, E Y, E Z in the new coordinate system are related to the basis e x, e y, e z in the original coordinate system via E x = cos βe X sin βe Z, E y = e Y, E z = sin βe X + cos βe Z. (8)

3 The SBT gives the translational drag coefficients in the diagonalized form Z F V = ζ (E X E X + E Y E Y ) + ζ E Z E Z, ζ = 2ζ = 4πηL ln(l/a), (9) The rotational drag coefficients are also calculated by SBT as Z T Ω = L2 12 ζ (I E Z E Z ). (10) Eqs.(9) and (10) give reasonably good approximations for a cylinder of aspect ratio L/A > 4, except for the vanishing ZZ-component of Z T Ω. The vanishing is an artefact of SBT and is remedied by replacing the ZZ-component by the classical formula for a cylinder of finite radius and infinite length [for example, see: L. D. Landau and E. M. Lifshitz, Fluid Mechanics, Pergamon (Oxford, 1959)], Z T Ω,ZZ = 4πηA 2 L. (11) The balance of force and torque due to the flagellum and cell body, F f + F b = 0 and T f + T b = 0, constitute the 6-dimensional matrix equation ( ) ( ) ( ) M F V + Z F V M F Ω V F = 0 (12) M T V M T Ω + Z T Ω Ω T 0 for V and Ω, which is solved numerically.

4 Supplementary Results 1. Angle of archaella bundle against cell axis In the main text, we showed that one polar curved archaella-bundle was observed per cell (Fig. 1c), and estimated the angle in inset of Fig. 1d as (Fig. 1e). Note that the value is based on the assumption that cells adhered to the substrate during fixation keeping the plane containing both archaella and the cell parallel to the substrate. Therefore, the value 24 could be underestimated counting the possible variation of the orientation of cells at the moment of the fixation. 2. Heterogeneity in rotation rate As shown in Fig. 2f in the main text, the histogram of rotation rate of the swimming cell body suggested two peaks, being partially attributable to the heterogeneity among cells. Because the hydrodynamic model indicates that the angle of archaella bundle against the long cell axis, in Fig. 5a, couples to rotation rate (Fig. 5b), the existence of the heterogeneity possibly suggests the difference of among cells. This possibility coincides with the observation under the electron microscope (Fig. 1e). 3. Quantification of archaella configuration under TIRFM From a still image of archaella under TIRF illumination, multiple parameters of archaellar structure were determined (Supplementary Figure 5a). 1) Pitch. The intensity profile along the straight line represented multiple peaks (Supplementary Figure 5b) corresponding to the helical pitch of the archaella bundle, which allowed us to estimate the pitch as in Fig. 5 in the main text. The pitch sizes in CW- and CCW rotation were and 2.09±0.32 m (Supplementary Figure 5c, n = 33 in CW and n=31 in CCW), respectively, which were similar to the observation by TEM ( m, n = 43). Although the main peak was identical, the other peak appeared in CCW rotation, which was 1.41±0.07 m (Supplementary Figure 5c lower). The distribution and apparent multiple peaks in the histogram of the pitch may suggest the possibility of the change in quaternary structure, which has been reported only in purified archaella 1. 2) Pitch angle. This value was directly determined from the still image as the shallow angle of archaella, and summarized as in Supplementary Figure 5d. In addition to, the radius of the helix structure b was calculated 2 (Supplementary Figure 5e). 4. Discrepancy between model and observation The deviations of the theoretical results from the experimental data may be explained by two factors. First, we neglected the non-local nature of the hydrodynamic interaction and calculated the total hydrodynamic force by integrating the local force density (see hydrodynamic model in Supplementary Methods). This way of approximation is called the resistive force theory and tends to produce larger force than the original and non-local version of slender-body theory. According to a recent study 3, the resistive force theory may result in over-estimation of the force by a factor of around two for a helix of pitch-to-radius ratio λ/b = 6. Intuitionally, this over-estimation arises by the fact that we neglect the effect of the tip of the helix in calculating the local force density. Therefore, the estimated swimming speed shown above sets an upper limit to the actual swimming speed. Second, in our calculation of the rotation rate, we adopted the rotational drag coefficient for a uniform and infinitely long cylinder, which is converted to the resistance per length. Thus we neglected the effect of the tips of the cell body, which leads to over-estimation of the rotational drag and thus under-estimation of the rotation rate. Since the cell body has a smaller aspect ratio than the archaellum, this approximation could cause a larger deviation than the approximation for the archaellum, which explains the tendency of deviations from the observed values. 5. Details in unitary steps In tethered cell assay, tethered cells changed their rotational direction only a few times during observation period up to 1 min. (120,000 frames), the size of steps were not similar before and after the switching of the direction and changed in most cases; more than 20% deviation was observed in 8 out of 10 cells. As shown in Fig. 6e in the main text, there is no clear bias for small ( 30 ) and large ( 60 ) steps in CCW and CW rotation. Exceptional large steps ( 120 and 240 ) were observed in 10%. We also found that some traces did not show smooth orbit, but accompanied the thorn-like outward slip in every unitary step (Fig. 6c, inset). This feature is quite unusual and has never been reported in steps of the bacterial flagella motor 4. Because we tracked the cell body as a marker for rotation detection, the outward shift of the center of the mass should correspond to the tilting of the whole cell (the black arrow in Fig. 6f) as easily recognized in Supplementary Video 11. Presumably, rotation of archaellum is not limited within a plane perpendicular to the rotation axis, but accompanied by a piston-like, push-pull motion and synchronous tilting during rotation, as FlaI, the archaellum motor ATPase 5, should share common conformational changes with PilB that is exquisitely specialized to the elongation of pilus.

Supplementary Figures Supplementary Figure 1 Histograms of swimming speed of Halobacterium salinarum (Hbt. salinarum) at room temperature R.T. (Left, n= 97) and 45 C (Right, n= 62), respectively.

5 Supplementary Figures Supplementary Figure 1 Histograms of swimming speed of Halobacterium salinarum (Hbt. salinarum) at room temperature R.T. (Left, n= 97) and 45 C (Right, n= 62), respectively. Solid line represents the Gaussian distribution with 3.3±0.9 and 4.4±1.2 μm s -1 at R.T. and 45 C, respectively (P = < 0.05 by t-test). In the observation at 45 C, the flow chamber was set on the thermoplate (MATS-55RAF20; Tokai-hit). The experiments at R.T and 45 C were reproducibly performed five and three times, respectively.

6 Supplementary Figure 2 Analysis of a swimming of Hbt. salinarum that was sparsely labeled with multiple quantum dots (QDs). (a) Experimental setup (left) and the micrograph of a typical sample (right). (b) The kymograph of a right. (c) The intensity changes along the blue in b. The oscillation correlates the rotation rate of the cell body. (d) The frequency analysis of c. (e) The histogram of the rotation rate. Solid line represents the Gaussian distribution with 3.4±1.2 Hz (n = 28 cells). These experiments were reproducibly performed at least 20 times for 4 days.

7 Supplementary Figure 3 Upper: Sequential images of a swimming cell at 60-ms intervals. Scale bar represents 2 μm. Lower: Kymograph of upper images for 1 sec.

8 Supplementary Figure 4 Schematics showing the orientation of the image under an inverted microscope. The image of a specimen becomes inverted because the light coming from the objective is reflected once in order to change the direction of the light towards the camera port located at the left side. Therefore, images captured under inverted microscopes always become mirror images for the viewer looked from the bottom side. Likewise, right-handed helix appears as a left-handed helix on the camera, although helicity does not depend on the view direction from which the observer watches the helical specimen, i.e., from the top or the bottom.

Supplementary Figure 5 Quantification of structural parameters of archaella under total internal reflection fluorescence microscope (TIRFM).

9 Supplementary Figure 5 Quantification of structural parameters of archaella under total internal reflection fluorescence microscope (TIRFM). (a) Schematics of archaella bundle to explain how to measure the pitch and the helix angle. (b) Upper: Fluorescent image of the cell. Scale bar, 2 μm. Lower: An example of intensity profile of labeled archaella along the green line in upper. Green solid line represents the sum of four Gaussians. (c) Histograms of pitches in archaella structures during CW rotation (upper, n = 33) and CCW rotation (lower, n = 31). Solid line represents the Gaussian fitting. Peaks and SDs were 2.11±0.24 and 2.09±0.32 μm in CW and CCW rotation, respectively. Additionally, the other peak in CCW rotation was 1.41±0.07 μm. (d) Histograms of pitch angles (upper, n = 20 lower, n=16). (e) Histograms of radius of helix (upper, n = 20 lower, n=16). Peaks and SDs were 0.24±0.04 and 0.23±0.04 μm in CW and CCW rotation, respectively. These experiments were done at least 50 times for 10 days.

Supplementary Figure 6 Additional examples of rotation time courses (left) and histograms of pairwise-distance-function (PDF) analysis with a moving window (right).

10 Supplementary Figure 6 Additional examples of rotation time courses (left) and histograms of pairwise-distance-function (PDF) analysis with a moving window (right). The rotational direction & the angle of unitary step are (a) CCW & 60.8, (b) CW & 56.5, (c) CCW & 34.6 and (d) CW & These are traces obtained in different four cells, and also different from the cell shown in Fig.5a-d in the manuscript. These data were taken from 19 independent experiments for 3 days.

11 Supplementary Table 1 Structural parameters of the cell and kinetics in Hbt. salinarum. Values were directly measured by either the electron microscope or optical microscope techniques as denoted in the bottom line except the radius of archaella helix. Length of cell body L Radius of cell body A Archaella angle Archaellum radius a Archaellum length h Archaellum helix pitch Archaellum rotation speed Archaellum pitch angle Archaellum helix radius b ( m) ( m) (nm) ( m) ( m) 24 3 (Hz) ( m) EM reference 6 OM calculation a EM, estimation from data taken by electron microscope; OM, estimation from sequential images or the still image acquired under optical microscopes; equation for calculation a, b = 1/2 tan.

12 Captions for Supplementary Videos Supplementary Video 1 Swimming of Halobacterium salinarum observed under phase-contrast microscopy. The yield of swimming cells was 85% in the optimum growth condition. Area: μm. Supplementary Video 2 Fluorescent image of quantum dots (QDs) attached to the cell body observed under 3-D tracking microscopy, termed as three-dimensional prismatic optical tracking. Area: μm. Supplementary Video 3 Rotation of cell body and archaella simultaneously observed under fluorescent microscopy Cell was sparsely labeled with multiple quantum dots (QDs). Area: μm. Supplementary Video 4 Swimming of fluorescent-labeled cells observed under fluorescent microscopy. Area: μm. Supplementary Video 5 Hbt. salinarum with several archaellar filaments, each one rotating independently. Area: μm. Supplementary Video 6 Archaella rotation observed under total internal fluorescence microscope (TIRFM). The cell body was attached to the glass, and so archaella rotation was quantified by the unidirectional propagation of spots originated from curved peaks of archaella coming closer to the surface. Area: μm. Supplementary Video 7 Rotation of bacterial flagellum under total internal fluorescence microscope (TIRFM). Scale bar represents 2 μm. [Preparation of bacteria] Salmonella enterica serovar Typhimurium SJW 1103 (ref. 7 ) was inoculated on 1.5 % agar containing T-broth 8 (1% Tryptone, 0.5 % NaCl) and grown at 37 ºC. A single colony was scratched by a loop, and subsequently inoculated into 10 ml of T-broth medium in a 40-ml tube. Cells were grown for 4-5 hours to mid-exponential phase with a shaker (200 r.p.m) at 37 ºC. Culture with 1 ml of volume were collected by centrifugation at 8,000 g for 4 min at 25 ºC, resuspended in buffer (10 mm HEPES-NaOH ph 7.8, 70 mm KCl and 30 mm NaCl) containing Cy3-NHSester 8 (GE health care) for 30 min at room temperature. Cells were washed two times to remove extra dye above a condition, and resuspended into a buffer containing 1.5 mg/ml bovine-serum albumin (Sigma-Aldrich). For observation, 1 μl volume of labeled S. typhimurium was dropped on a cleaned glass (24 36 mm) with a hydrophilic treatment device, subsequently tapped by a small glass (18 18 mm), and sealed with nail polish to prevent an evaporation. Supplementary Video 8 The reconstructed swimming motion of a single archaeon with observed parameters, which were described in Supplementary Table 1, based on slender-body theory. Supplementary Video 9 Dark-field image of Hbt. salinarum tethered to the glass. 1/10 playback speed. Scale bar represents 1 μm. Supplementary Video 10 Dark-field image of Hbt. salinarum tethered to the glass. 1/10 playback speed. Scale bar represents 1 μm. Supplementary Video 11 Dark-field image of Hbt. salinarum tethered to the glass. 1/10 playback speed. Scale bar represents 1 μm.

13 Supplementary References 1. Alam, M. & Oesterhelt, D. Purification, reconstitution and polymorphic transition of halobacterial flagella. J. Mol. Biol. 194, (1987). 2. Goldstein, S.F. & Charon, N.W. Multiple-exposure photographic analysis of a motile spirochete. Proc. Natl. Acad. Sci. USA 87, (1990). 3. Rodenborn, B., Chen, C.H., Swinney, H.L., Liu, B. & Zhang, H.P. Propulsion of microorganisms by a helical flagellum. Proc. Natl. Acad. Sci. USA 110, E (2013). 4. Sowa, Y. et al. Direct observation of steps in rotation of the bacterial flagellar motor. Nature 437, (2005). 5. Reindl, S. et al. Insights into FlaI functions in archaeal motor assembly and motility from structures, conformations, and genetics. Mol. Cell 49, (2013). 6. Cohen-Krausz, S. & Trachtenberg, S. The structure of the archeabacterial flagellar filament of the extreme halophile Halobacterium salinarum R1M1 and its relation to eubacterial flagellar filaments and type IV pili. J. Mol. Biol. 321, (2002). 7. Yamaguchi, S., Fujita, H., Sugata, K., Taira, T. & Iino, T. Genetic analysis of H2, the structural gene for phase- 2 flagellin in Salmonella. J. Gen. Microbiol. 130, (1984). 8. Turner, L., Ryu, W.S. & Berg, H.C. Real-time imaging of fluorescent flagellar filaments. J. Bacteriol. 182, (2000).

The flagellar motor of Caulobacter crescentus generates more torque when a cell swims backwards

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