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Direct observation of rotation and steps of the archaellum in the swimming halophilic archaeon Halobacterium salinarum

Nature Microbiology volume 1, Article number: 16148 (2016) Cite this article

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Abstract

Motile archaea swim using a rotary filament, the archaellum, a surface appendage that resembles bacterial flagella structurally, but is homologous to bacterial type IV pili. Little is known about the mechanism by which archaella produce motility. To gain insights into this mechanism, we characterized archaellar function in the model organism Halobacterium salinarum. Three-dimensional tracking of quantum dots enabled visualization of the left-handed corkscrewing of archaea in detail. An advanced analysis method combined with total internal reflection fluorescence microscopy, termed cross-kymography, was developed and revealed a right-handed helical structure of archaella with a rotation speed of 23 ± 5 Hz. Using these structural and kinetic parameters, we computationally reproduced the swimming and precession motion with a hydrodynamic model and estimated the archaellar motor torque to be 50 pN nm. Finally, in a tethered-cell assay, we observed intermittent pauses during rotation with ∼36° or 60° intervals, which we speculate may be a unitary step consuming a single adenosine triphosphate molecule, which supplies chemical energy of 80 pN nm when hydrolysed. From an estimate of the energy input as ten or six adenosine triphosphates per revolution, the efficiency of the motor is calculated to be ∼6–10%.

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Figure 1: Swimming of Hbt. salinarum.
Figure 2: Visualization of rotation of the cell body of swimming Hbt. salinarum.
Figure 3: Visualization of archaella rotation of swimming archaea.
Figure 4: Simultaneous determination of rotation direction and helicity of archaella with cross-kymography under TIRFM.
Figure 5: Numerical calculation by a hydrodynamic model based on SBT.
Figure 6: Steps of single cells of Hbt. salinarum detected by tethered-cell assay.

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Acknowledgements

The authors thank S.-V. Albers, M. Beeby, R. Kamiya, H. Noji and I. Sase for discussions that were critical in preparing the manuscript, and T. Minamino and Y.V. Morimoto for supplying Salmonella. This study was supported in part by the Funding Program for Next-Generation World-Leading Researchers Grant LR033 (to T.N.) from the Japan Society for the Promotion of Science (JSPS), by a Grant-in-Aid for Scientific Research on Innovative Areas (‘Fluctuation & Structure’ of JSPS KAKENHI grant nos. JP26103502 and JP16H00792 to N.U. and nos. JP26103527 and JP16H00808 to T.N.; ‘Cilia & Centrosomes’ of grant no. JP87003306 to T.N.; ‘Motility Machinery’ of grant no. JP15H01329 to D.N and no. JP24117002 to T.N.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by JSPS KAKENHI (grant no. JP16H06230 to D.N. and no. JP15H04364 to T.N.). Y.K is a recipient of a JSPS Fellowship for Japan Junior Scientists (no. JP15J12274).

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Authors and Affiliations

  1. Department of Physics, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, 171-8588, Tokyo, Japan

    Yoshiaki Kinosita, Daisuke Nakane & Takayuki Nishizaka

  2. Department of Physics, Tohoku University, 980-8578, Sendai, Japan

    Nariya Uchida

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  1. Yoshiaki Kinosita
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Contributions

Y.K., N.U., D.N. and T.N. designed the research. Y.K. performed experimental work. N.U. developed a framework for computational calculations. Y.K. and T.N. constructed the optical set-up and microscope. Y.K. and T.N. performed data analyses. Y.K., N.U. and T.N. wrote the paper.

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Correspondence to Nariya Uchida or Takayuki Nishizaka.

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Supplementary information

Supplementary information

Supplementary Methods, Supplementary Results, Supplementary Figures 1-6, Supplementary Table 1, Legends for Supplementary Videos 1-11, Supplementary References (PDF 821 kb)

Supplementary Video 1

Swimming of Halobacterium salinarum observed under phase-contrast microscopy. (AVI 17759 kb)

Supplementary Video 2

Fluorescent image of QDs attached to the cell body observed under 3D tracking microscopy, termed as ‘three-dimensional prismatic optical tracking’ (AVI 591 kb)

Supplementary Video 3

Rotation of cell body and archaella simultaneously observed under fluorescent microscopy. (AVI 1300 kb)

Supplementary Video 4

Swimming of fluorescent-labelled cells observed under fluorescent microscopy. (AVI 7504 kb)

Supplementary Video 5

Hbt. salinarum with several flagellar filaments, each one rotating independently. (AVI 3300 kb)

Supplementary Video 6

Archaella rotation observed under TIRFM. (AVI 4389 kb)

Supplementary Video 7

Rotation of bacterial flagellum under TIRFM. Scale bar represents 2 μm. (AVI 2093 kb)

Supplementary Video 8

The reconstructed swimming motion of a single archaeon with observed parameters, based on slender-body theory. (MP4 350 kb)

Supplementary Video 9

Dark-field image of Hbt. salinarum tethered to the glass. (AVI 579 kb)

Supplementary Video 10

Dark-field image of Hbt. salinarum tethered to the glass. (AVI 391 kb)

Supplementary Video 11

Dark-field image of Hbt. salinarum tethered to the glass (AVI 4060 kb)

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Kinosita, Y., Uchida, N., Nakane, D. et al. Direct observation of rotation and steps of the archaellum in the swimming halophilic archaeon Halobacterium salinarum. Nat Microbiol 1, 16148 (2016). https://doi.org/10.1038/nmicrobiol.2016.148

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