advances.sciencemag.org/cgi/content/full/4/5/eaar7975/DC1

Supplementary Materials for

The mechanism of two-phase motility in the spirochete Leptospira:

Swimming and crawling

Hajime Tahara, Kyosuke Takabe, Yuya Sasaki, Kie Kasuga, Akihiro Kawamoto, Nobuo Koizumi,

Shuichi Nakamura

Published 30 May 2018, Sci. Adv. 4, eaar7975 (2018)

DOI: 10.1126/sciadv.aar7975

The PDF file includes:

fig. S1. Swimming motility of Leptospira.

fig. S2. Example kymographs of Leptospira cells crawling without slip.

fig. S3. WT and “unbent” mutant (ΔfcpA) strains of Leptospira.

fig. S4. Example kymographs of ΔfcpA mutant cells crawling without slip.

fig. S5. Movement of a microbead attached to the cell surface.

fig. S6. Movement of a microbead attached to the cell surface.

fig. S7. Kymographs of Leptospira cells labeled with Ab-LPS–coated beads or

noncoated beads.

fig. S8. Relationship between swimming speed and bead movement.

fig. S9. Crawling motility of the pathogenic Leptospira.

Other Supplementary Material for this manuscript includes the following:

(available at advances.sciencemag.org/cgi/content/full/4/5/eaar7975/DC1)

movie S1 (.avi format). A L. biflexa cell swimming in a 10% Ficoll solution.

movie S2 (.avi format). WT L. biflexa cells crawling on a glass surface.

movie S3 (.avi format). Effect of an anti-LPS antibody on crawling.

movie S4 (.avi format). ΔfcpA mutant cells crawling on a glass surface.

movie S5 (.avi format). Effect of CCCP on Leptospira crawling.

movie S6 (.avi format). Fluorescent observation of the Leptospira outer

membrane using Cy3-NHS.

movie S7 (.avi format). Movement of a small bead aggregate on the Leptospira

cell body.

movie S8 (.avi format). Movement of a single 200-nm bead on the Leptospira cell

body.

movie S9 (.avi format). Movement of a large bead aggregate on the Leptospira

cell body.

Supplementary Materials

fig. S1. Swimming motility of Leptospira. Pairwise representation of swimming speeds and rotation rates in

media with and without Ficoll are shown. Black, red, and blue circles indicate data obtained in media without

Ficoll, with 5% Ficoll, and with 10% Ficoll, respectively. Regression lines are fitted to data points in each

condition. The viscosity was measured with a tuning-fork-type viscometer (SV-1A; A&D): 0.9 mPa × s for

motility medium without Ficoll, 6 mPa × s for 5% Ficoll, and 26 mPa × s for 10% Ficoll.

fig. S2. Example kymographs of Leptospira cells crawling without slip. Cells crawling on surfaces without

any coating were observed. Yellow lines indicate cell displacements. PC appears to be fixed (as shown in Cell 1

by red dotted lines), indicating a movement without slip.

fig. S3. WT and “unbent” mutant (ΔfcpA) strains of Leptospira. The top panels show 3D-CG of the WT and

ΔfcpA strains (see also dark-field micrographs, as shown in Fig. 2C). The middle panels show the WT and

ΔfcpA cells, as observed by cryo-electron microscopy. The lower panels are transmission electron micrographs

of negative-stained PFs that were isolated from the WT and ΔfcpA cells.

fig. S4. Example kymographs of ΔfcpA mutant cells crawling without slip. Cells crawling on surfaces

without any coating were observed. Yellow lines indicate cell displacements. PC appears to be fixed (as shown

in Cell 1 by red dotted lines), indicating a movement without slip.

fig. S5. Movement of a microbead attached to the cell surface. (A) Kymograph of the movements of a cell

and bead; analysis was performed at the sections indicated by vertical (blue) and horizontal (green) dashed

lines, and each result is surrounded by the same color. The movie was recorded at a frame rate of 250 Hz. The

cell swims in the direction indicated by a white arrow; the bead does not move in the X direction, but a time

trace of the Y position shows periodic undulation with a frequency of 20–30 Hz. X and Y positions of the bead

were measured and plotted against time (right panel). Analysis of the time period indicated by yellow and black

lines is shown in B. (B) Rotation analysis of the bead shown in A. The focal plane recognized from the helix

angle of PC (shown by an enlarged image in A, surrounded by a red rectangle) and a change in area of the bead

image (see main text) indicate the clockwise rotation of the bead. Numbers of images correspond to 0.20 s (1),

0.22 s (2), 0.26 s (3), 0.28 s (4), 0.29 s (5), 0.32 s (6), 0.37 s (7), 0.41 s (8), and 0.45 s (9). The schematics show

the bead rotation viewed from the direction indicated by a black arrow.

fig. S6. Movement of a microbead attached to the cell surface. Example cell 2. (A) Kymograph of the

movements of a cell and bead. Analysis was performed at the sections indicated by vertical (blue) and

horizontal (green) dashed lines, and each result is surrounded by the same color. The movie was recorded at a

frame rate of 30 Hz. The time trace of the bead position shows a vertical undulation of the bead, with a

frequency of <1 Hz. Images contained in the period indicated by a black line are shown in B. (B) Rotation of

the bead. The numbers indicated in each image correspond to 1.0 s (1), 1.3 s (2), 2.2 s (3), 2.5 s (4), 2.7 s (5),

3.1 s (6), 3.6 s (7), 3.8 s (8), 4.2 s (9), and 4.7 s (10). The yellow arrows in the third panel indicate the helix

angle of the PC, allowing us to determine the rotational direction (see main text). The schematics show the bead

rotation viewed from the direction indicated by a black arrow; the bead rotated in CW. The bottom panel

indicates a change in the area of the bead image.

fig. S7. Kymographs of Leptospira cells labeled with Ab-LPS–coated beads or noncoated beads. Kymographs of Leptospira cells labeled with Ab-LPS coated beads (A) or non-coated beads (B). White arrows

in stills displayed above each kymograph indicate beads, and yellow lines indicate displacements of cells.

fig. S8. Relationship between swimming speed and bead movement. (A) Changes in the

relative positions of the Ab-LPS coated beads to the cell bodies and swimming speeds were

analyzed in individual cells (n = 12 cells); the swimming direction was defined as positive. The

red line is a regression line fitted to all data points; the black line has a slope of −1. An extended

view is shown on the right. (B) The relative bead speed is equal to the swimming speed of the

cell, indicating that the bead migration is impeded by a drag force generated by cell translation, so

that the bead position on the video monitor is fixed. (C) When the relative bead speed is faster

than the cell swimming speed, as shown in A, additional forces other than drag force from

swimming would be exerted on the bead.

fig. S9. Crawling motility of the pathogenic Leptospira. Crawling of Leptospira interrogans

serovar manilae strain. UP-MMC-NIID was analyzed; kymographs of two different cells are

shown. Yellow dashed lines indicate that the cells moved on a glass surface without slipping.