Bacterial Gliding Motility: Visualizing
Invisible Machinery
Extracellular filaments covering the surface of the marine gliding
bacterium Saprospira grandis may be its motility organ
Shin-Ichi Aizawa
any flagellated species of bacteria
can swim in liquids and glide
along surfaces, while motile
species without flagella only can
glide. Much is known about flagella, whose rotary mechanism is believed to be
universal. By contrast, gliding motility is a term
used to describe various movements on surfaces
by several different mechanisms, none of which
is fully understood.
Saprospira grandis, meaning a large helical
decomposer, is a gliding bacterium that was first
isolated from the deep sea by J. Gross in 1911.
This gram-negative species, which itself looks
like a multicellular flexible filament, is also
found along seashores in many places, according to R. A. Lewin of the Scripps Institute of
Oceanography at the University of California,
San Diego.
M
A Motility Pattern of the
Cytophaga-Flavobacter-Bacteroides Group
S. grandis is predatory on other bacteria and
plays an important role as a coastal scavenger.
Its manner of catching prey and gliding are
closely related in that both phenomena are
based on its stickiness to surfaces along which it
moves and grazes. First, consider the pattern of
gliding movements of S. grandis. Those movements resemble those of Flavobacterium johnsoniae, Flexibacter polymorphus, and Cytophaga, all of which are members of the same
branch of the eubacterial phylogenetic tree, suggesting that the same mechanism accounts for
the gliding motion of this group. Unlike the
others in this group, each S. grandis cell forms a
long helical filament which can extend to 500
␮m. Consequently, when such cells are suspended in liquid, they easily tangle with one
another to make big aggregates of twisted filaments.
All these species typically glide as fast as 10
␮m/s on glass surfaces. Occasionally, single cells
rotate around one pole while otherwise remaining stationary. Particles or latex beads attached
to the cell surface travel helically along the cell
axis (Fig. 1).
Problems with Extrusion Model
for Gliding Machines
Since this S. grandis behavior and the outlines of
its surface movement were first described during
the 1970s, researchers have been trying to identify its motility organ. Several research groups
have speculated about different ways by which
such cells might move.
For instance, one group of researchers determined that a series of holes aligned across the
cell surface is needed for gliding. They then
speculated that the cells move by extruding lipopolysaccharide (LPS) through those holes. However, this explanation for how cells glide has
several difficulties. First of all, in terms of biophysics, the momentum gained from ejecting
such a low mass of material is negligibly small in
the nearly weightless world of these bacteria.
Another difficulty is that the supposed structures being used for motility is not very well
described. Assuming that the hole along the cell
surface could serve as a rigidly structured exit
for LPS, by what mechanism would cells forcibly eject a mass of LPS?
Shin-Ichi Aizawa is
a team leader with
the “Soft-NanoMachine Project,”
a 5-year national
project at the Science and Technology Agency of
Japan
Volume 71, Number 2, 2005 / ASM News Y 71
physiological condition, some flagella
fully depolymerize; indeed this process begins at pH 5.
UA also has a fixative effect on
proteins, damaging them along their
exposed surfaces. This stain is especially harmful to membrane surfaces, which tend to stain too dark to
observe ultrastructure near their
surfaces. Finally, UA is radioactive,
meaning it can damage our health.
Instead of using UA, our lab conventionally uses phosphotungstic acid
(PTA). When dissolved in aqueous
Schematic patterns of surface movements of the gliding bacterium S. grandis. Beads
solution, its pH can be adjusted
attached to the cell surface travel along the cell axis, often showing a helical movement. A
across a wide range from strongly
cell glides on surface occasionally flip-over at a pole and continue gliding. Sometimes a cell
acidic to alkaline (pH 2–9). Images
stands at a pole and rotates in place like a street dancer. Although the cell shape is drawn
as a rod, the actual shape is a long helical filament.
stained with 2% PTA at pH 7, a
common recipe for negative staining,
look fainter than those stained with
A central tenet of modern biology is that any
UA, which explains why many researchers conmechanical motion should depend on an orgatinue to use UA. However, although images are
nized structure made of protein and perhaps other
fainter with PTA staining, its use provides conmaterials, such as carbohydrates. In other words,
siderable structural detail. Moreover, at pH 4
for this extrusion-propulsion or any other mechPTA provides images with about as much conanism to explain how bacterial cells glide along
trast as those made using UA.
a surface, there must be an accompanying ultrastructure to support that mechanism.
FIGURE 1
Fragile Surface Structures
Practical Problems Reviewing
Observations by Electron Microscopy
Seeing is believing. However, the opposite also
appears to be true: if you don’t see, you don’t
believe. Obtaining clear and unambiguous images of nano-sized structures along the surfaces
of micrometer-sized bacterial cells totally depends
on which techniques are being used. In general,
the electron microscope is the best tool to use in
observing nano-sized structures, while negative
staining with heavy metal ions is the simplest
procedure for highlighting those structures.
However, although negative staining is
straightforward to use, its use can prove problematic. For example, because uranium acetate
(UA) gives very high contrast in negatively
stained images, it is widely used in electron
microscopy labs. However, this high-contrast
stain tends to damage samples. First of all, to use
UA, the pH of the solution needs to be adjusted
to 4 because this salt precipitates at neutral or
alkaline pH. Because pH 4 is far from a normal
72 Y ASM News / Volume 71, Number 2, 2005
Diluting cell suspensions of S. grandis with water instantly abolishes gliding motility, suggesting that the motility organ is so fragile that it
disassembles when salts are absent. When we
sought factors that would stabilize these structures, we found that magnesium works the best
in maintaining the structures needed for motility. At higher than 40 mM magnesium, cells can
glide in the presence of as low as 10 mM sodium
chloride, whereas at lower than 40 mM magnesium, the motility is lost even in the presence of
100 mM sodium chloride. Seawater contains
about 50 mM magnesium salts.
Electron microscopy reveals bundles of filaments covering the cell surface in the presence of
40 mM magnesium (Fig. 2A). The filament bundles are surrounded by a membrane. As cell
density increases, the filament bundles in membrane envelopes also increase. When the cells
glide on surfaces, some of those filament bundles
are left behind (Fig. 2B-C).
When we analyzed the protein components of
the filament by SDS-PAGE, we found three ma-
Aizawa Is a Long-time Student of Flagellar Motors—and Haiku
When Shin-Ichi Aizawa was a professor of biosciences at Japan’s Teikyo University, in Utsunomiya, he
developed two key precepts for keeping undergraduates who did research with him happy: recognize
their achievements and feed them.
“I published papers every year in
good journals [and] I always put
their names on the papers,” he says.
“Also, I cooked rice at lunch for
them every day.” Despite his departure from the university to pursue
other ventures, he adds, “Even today I cook rice for the young scientists.”
Aizawa is a team leader with the
“Soft-Nano-Machine Project,” a
5-year national project at the Science and Technology Agency of Japan. “Many others work on hard
materials, such as carbon nanotubes
or semiconductors,” he says. “Our
project is to focus on biological materials. Many of the teams on our
project work on something moving,
such as muscle and flagella.”
Aizawa, 55, has been studying
flagellar motors all his adult life,
repeatedly asking: “How does such
a tiny, nano-sized motor run as fast
as 100 revolutions per second? How
does the flow of protons power the
motor? What is the physical role of
this energy conversion?
“This is one of the most fundamental science questions, a matter of basic physics. . .as yet unsolved,” he says. “The answers may
not change our immediate future,
but it reminds me of the discovery
of gravity by Newton. The only
difference is that you cannot see the
rotation of flagella by your naked
eyes in daily life— but they are there
around you, everywhere, at this moment.”
Much earlier, while an undergraduate student, Aizawa studied
physics, but “soon found that it
was not for me,” he says. He found
himself worrying that a focus on
physics would require him “to be a
genius— or part of a gigantic project,” he recalls. Thus, he switched
to studying biology, which he found
“a vast world filled with lots of
mysteries and interesting phenomena, where I might be able to stand
by myself—even though I was not
smart— but patient enough to think
about one problem for a long time.”
A native of Hiroshima, he received a B.S. in physics from Tohoku University in 1974 and a Ph.D.
in biophysics from Nagoya University in 1979. He did postdoctoral
training at the department of biophysics and biochemistry at Yale
University, supervised by the late
Robert M. Macnab, who served
both as a mentor and friend. He
returned to Japan and joined a
project similar to the work he is
conducting now. “As group leader,
I worked hard on flagellar structure
and Bob was always helpful in writing papers,” he recalls.
“Our efforts publishing many
papers from such unpopular projects—at that time the projects were
not highly regarded and university
people thought we were wasting
money— got a high profile on television, newspapers, and magazines,”
he says. “Since then, university people paid attention, and now the projects are very popular and competitive.”
In 1990, he began teaching and
doing research at Teikyo University, where he remained for a dozen
years until joining his current project. “I was happy as a scientist, but
the teaching loads were getting
heavier every year, and the undergrads per lab were getting to be
more than 30,” he says. “I quit the
university on the occasion that I got
this project. I opened up my own
lab in a huge space of a warehouse,
and about eight people are working
with me.”
He and his wife have two grown
daughters, the older one a biology
major at the University of Hawaii
and the younger a law student at
Kyoto University. Because they both
were born in New Haven, “they are
technically Americans—and the elder one stays in Hawaii as an American,” he says.
Aizawa writes haiku, which are
17-syllable Japanese poems, and
has organized an e-mail haiku club.
Every month, each of the 17 members sends him a poem. He erases
their names, shuffles the poems,
and distributes them to club
members for votes and commentary. “One difference of our club
from many other traditional haiku
clubs is that I, as a leader, don’t
choose the best haiku,” he says.
“All the members do. So I can learn
new feelings from the younger
members, so that I can stay young.”
He has been writing Haiku for 20
years. While it is difficult to translate
poems from one language to another, he nevertheless gave it a try:
Hot sake
No more ambitions
Do I need?
(Atsu-kan ya/ nande imasara/
yashin nado)
“As you can see, I love hot sake
and often tell my feelings when I am
drunk,” he says, laughing. While
there may be no direct connection
between haiku and his science,
“Haiku definitely helps me to write
papers,” Aizawa says. “Choosing
the right words and cutting sentences as short as possible to meet
the requirements of cruel editors
are the everyday efforts we do.”
Marlene Cimons
Marlene Cimons is a freelance writer
in Bethesda, Md.
Volume 71, Number 2, 2005 / ASM News Y 73
FIGURE 2
Electron micrographs of S. grandis cell surface. (A) A bundle of filaments covers the cell surface of S. grandis. (B) When S. grandis cells
were incubated on an EM grid, filaments wrapped by membrane remained on the trails. (C) An enlarged image of filaments wrapped
with membrane. (D) Wild-type Salmonella typhimurium SJW1103 cells were trapped along the filamentous cell surface of S. grandis.
They were caught through their flagella. Samples were negatively stained with 2% PTA (pH7) and observed by JEOL 1200EX.
jor bands, corresponding to proteins of 53, 40,
and 38 kDa. Because our efforts to determine
the amino-terminal amino acid sequences of the
three proteins did not succeed, we suspect that
they are chemically modified.
Because bundles of filaments cover the surface
of S. grandis, and because they appear to be left
along the path as such cells slowly glide along
surfaces, we think that the filaments are a part of
the motility mechanism. Whether true or not,
we would like to find whether motor proteins
74 Y ASM News / Volume 71, Number 2, 2005
are involved in motility of these cells. In searching for such proteins, it will be helpful to know
the genomic sequence of this organism as one
way of looking for matches to genes specifying
other motor proteins.
Prey-Predator Relationship
S. grandis preys not only on marine bacteria but
also on enteric bacteria such as Salmonella and
Escherichia coli, both of which can surFIGURE 3
vive in seawater. S. grandis traps prey
cells passively, doing little until actively
motile prey cells bump against its long
filaments and then adhere to its cell
surface.
This entrapment process is far from
perfect, and seemingly trapped cells
can escape from the predator. Lewin
calls this kind of approach to gathering nutrients “ixotrophy,” meaning
that the cells feed on prey caught on
sticky substances, resembling bird-lime
or flypaper. Ixotrophy is a complicated
process, and can be divided into several
simpler steps, namely (i) adhesion, or
trapping of prey cells on the cell surface of the predator; (ii) translocation,
or collecting trapped cells at one place,
usually the poles of the predator cells;
Electron micrographs of rhapidosome, a bacteriocin of S. grandis. Rhapidosome resem(iii) aggregation, or bringing both prey
bles TMV or the tail of labda phage. A thin core rod penetrating a thick tubular rod often
and predator cells together and within
slip out (A), and thick tubular rod gradually disassemble into a helical filament (B).
an envelope, probably consisting of
LPS; and (iv) digestion, or assimilating
sticky surface, S. grandis shows no particular
the nutrients that prey cells provide.
preference for prey so long as they come into
The adhesion process is the first step of
close contact.
ixotrophy. Flagellated bacteria are trapped more
often than are nonflagellated ones. We examined
Bacteriocin for Killing
roles of flagella of prey cells using various flagellar shape mutants of Salmonella. Because S.
The final step in S. grandis ixotrophy, digestion,
grandis trap prey cells with different kinds of
is poorly understood. One possible mechanism
flagella, the antigenicity of the flagellar surface is
is that S. grandis uses bacteriocins in killing prey
not important for the trapping mechanism. Neicells. Whether S. grandis produces bacteriocins
ther is the helical handedness of the flagella
is not known. However, it does produce phagebeing trapped, because cells with right-handed
like particles called rhapidosomes (Fig. 3) that
helical flagella are trapped as well as are those
strikingly resemble the R-type pyocin of Pseuwith left-handed helices (Fig. 2D). S. grandis
domonas aeruginosa that is a phage-tail-type
also traps nonmotile cells whose flagella are
bacteriocin. Myxococcus xanthus, another glideither straight or paralyzed, albeit with less effiing, predatory bacterial species, also produces
ciency.
phage-type bacteriocins. Although the bacteriAll in all, S. grandis finds more opportunities
ocidal mechanism of colicin-type bacteriocins
for contact with prey having helical flagella
are well understood, those of the phage-tailalong their surfaces. However, because of its
type bacteriocins are only poorly understood.
ACKNOWLEDGMENTS
I am grateful to R. A. Lewin for strains and advice, to S. Ishii for illustration, and to N. Ishikawa, M. Kanbe, T. Mori, and K.
Komoriya for their work on S. grandis.
SUGGESTED READING
Lapidus, I. R., and H. C. Berg. 1982. Gliding motility of Cytophaga sp. Strain U67. J. Bacteriol. 151:384 –398.
Lewin, R. A. 1997. Saprospira grandis: a flexibacterium that can catch bacterial prey by “ixotrophy.” Microb. Ecol.
34:232–236.
McBride, M. 2004. Cytophaga-Flavobacterium gliding motility. J. Mol. Microbiol. Biotechnol. 7:63–71.
Volume 71, Number 2, 2005 / ASM News Y 75
Michel-Briand, Y., and C. Baysse. 2002. Pyocins of Peudomonas aeruginosa. Biochimie 84:499 –510.
Ohsumi, M., Shinomiya, T., and Kageyama, M. 1980. Comparative study on R-type pyocins of Pseudomonas aeruginosa.
J. Biochem. 87:1119 –1126.
Ridgeway, H. F., and R. A. Lewin. 1988. Characterization of gliding motility in Flexibacter polymorphus. Cell Motility
Cytoskeleton 11:46 – 63.
Sangkhobol, V., and B. D. Skerman. 1981. Saprospira species–natural predators. Curr. Microbiol. 5:169 –174.
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