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Bacterial Cells Have Cytoskeletons, Too
Bacterial cells contain cytoskeletal structures that impart
long-range order within the cell
Lawrence Rothfield, Aziz Taghbalout, and Purva Vats
lthough eukaryotic cells contain a
complex internal cytoskeleton, until
recently microbiologists believed
that bacteria contained no comparable elements except for the murein
exoskeleton located outside the cytoplasmic
membrane. Indeed, the absence of a cytoskeleton was one of the hallmarks used to distinguish
bacteria from eukaryotic cells. That view
changed dramatically in 2001 when Laura
Jones, Rut Carballido-López, and Jeff Errington
of Oxford University, Oxford, United Kingdom,
described the cytoskeletal nature of the actinlike MreB and Mbl proteins of Bacillus subtilis.
Within these rod-shaped gram-positive cells, the
two proteins form structures that extend between the two poles and help to regulate cell
shape. This finding prompted a period of rapid
progress that changed our view of bacterial cellular organization.
The eukaryotic cytoplasm has long been
known to contain several types of extended cytoskeletal networks, composed of microtubules,
intermediate filaments, and actin filaments, that
communicate with other intracellular and membrane-associated components. It is now
clear that bacteria contain proteins resembling both the actin and nonactin cytoskeletal proteins of eukaryotic cells as well as
proteins unrelated to the eukaryotic cytoskeletal proteins, organized into extended
membrane-associated structures. These
structures provide long-range order to the
cell—far more than microbiologists once
recognized.
A
Lawrence Rothfield
is a Professor, Aziz
Taghbalout is an
Instructor, and
Purva Vats is a
Postdoctoral fellow
in the Department
of Molecular, Microbial and Structural Biology, University of
Connecticut Health
Center, Farmington.
Homologs of Actin Help
To Shape Bacterial Cells
Cells of B. subtilis contain three actin homologs, designated MreB, Mbl (Fig. 1B),
582 Y ASM News / Volume 71, Number 12, 2005
and MreBH. Further, most other types of rodshaped bacteria, including E. coli, contain actinlike proteins homologous to MreB (Fig. 1C). In
contrast, MreB-related proteins are absent from
those coccal species whose genomic sequences
are known, supporting the idea that a major role
of MreB is to support establishment of a rod
shape. The three-dimensional structures of
MreB and actin are very similar, as shown by F.
van den Ent, Linda Amos, and Jan Löwe of the
MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
Within the cell, MreB is organized into two
helical strands composed of actin-like protofilaments, running along the inner surface of the
cytoplasmic membrane and usually coiling
around the rod-shaped cell along its long axis
(Fig. 1C). The coiled structure changes its appearance and position during the cell cycle, according to work from the laboratories of James
Gober of the University of California, Los Angeles (UCLA) and Lucy Shapiro of Stanford
University, Stanford, Calif. At a specific stage of
the C. crescentus cell cycle, MreB rearranges to
form a ring-like structure near the division site.
• Bacteria, long thought to lack a cytoskeleton,
contain proteins resembling both the actin and
nonactin cytoskeletal proteins of eukaryotic cells
as well as additional proteins that play cytoskeletal roles.
• Actin homologs perform a variety of functions,
helping to determine cell shape, segregate chromosomes, and localize proteins within bacterial cells.
• Nonactin homologs and unique bacterial cytoskeletal proteins are involved in determining cell
shape and in regulation of cell division, chromosome segregation, and probably other cellular
functions.
This resembles the behavior of
FIGURE 1
eukaryotic actin, which assembles into a cytokinetic ring at
the division site early in the division process.
Other bacterial actin-like
structures do not show this degree of plasticity. The overall
coiled structure of Mbl , for
example, appears not to vary
although particular parts of the
structure turn over at a rapid
rate, according to fluorescence
photobleaching analysis. The
different behavior of the MreB
and Mbl structures emphasizes
that bacterial cells contain several cytoskeletal structures,
with different properties.
The actin-like cytoskeletal
structures perform several important cellular functions in
bacteria. One such function is
to regulate the shape of rodshaped bacteria by regulating
the pattern of synthesis of the
cell wall peptidoglycan. Thus,
loss of MreB in all species that
have been examined, and of eiCytoskeletal structures in bacterial cells. (A) Crescentin in Caulobacter crescentus. The crescentin
ther MreB or Mbl in B. subtilis,
filamentous structure (red) is located along the concave edge of the comma-shaped cells, in which
causes cells to lose their rod
DNA (blue) has been co-labeled with DAPI. (Reproduced with permission from Ausmees, Kuhn and
shape. The link between the acJacobs-Wagner, 2003, Cell 115: p. 707.) (B) Mbl protein of B. subtilis- The helical Mbl structure,
visualized by immunofluorescence, is visible along the length of the cell and appears to be
tin-like proteins and the pepticomposed of two intertwined helices. (Reproduced with permission from Jones, Carballido-Lopez
doglycan synthetic machinery
and Errington, 2001, Cell, 104, p. 916.) (C) MreB protein of E. coli- The double helical organization
is exemplified by the observaof the E. coli Yfp-MreB protein is visualized in a three-dimensional reconstruction from a series of
optical sections of fluorescence micrographs. (Reproduced with permission from Shih, Le and
tion that the sites of peptiRothfield, 2003, Proc. Natl. Acad. Sci. U.S. 100: p.7868.) (D) FtsZ protein in sporulating B. subtilisdoglycan synthesis are distribThe FtsZ spiral structure represents FtsZ-Gfp in a fluorescence micrograph of a sporulating cell.
uted in a coiled pattern that
(Reproduced with permission from Ben-Yehuda and Losick, 2002, Cell 109: p. 258.) (E) Representation of panel D. The FtsZ spiral structure is indicated in red. (F) MinD protein in E. coli. The helical
extends along the length of the
structure of Gfp-MinD is visualized in an E. coli cell that is coexpressing Gfp-MinD and MinE. The
cell in a helical arrangement
high level fluorescence in the coils in the upper portion of the cell represents the high concentration
similar to that of Mbl, and this
of MinD within the polar coils (“polar zone”) of the MinD helical array that runs the entire length of
the cell. The MinD in the polar zone oscillates between the two ends of the cell with a periodicity
distribution depends on the
of 1–2 minutes. . (Reproduced with permission from Shih, Le and Rothfield, 2003, Proc. Natl. Acad.
presence of Mbl, as shown by
Sci. U.S. 100: p.7867). (G) Representation of panel F. The high concentration of MinD in the polar
Richard Daniel and Jeff Errzone is indicated in red. The lower concentration of MinD in the remainder of the pole-to-pole helical
array is indicated in blue.
ington. During the process of
cell shape regulation in B. subtilis, Mbl apparently controls
longitudinal growth along the cell cylinder
helical pattern in C. crescentus, according to
whereas MreB controls cell width, according to
Rainer Figge and his collaborators at UCLA.
Daniel and Errington. Similarly, penicillin-bindA second important function of the MreB
ing protein 2 (PBP2), which is involved in pepcytoskeleton is to participate in movement of the
tidoglycan synthesis and is required to maintain
two newly replicated daughter chromosomes to
rod shape, also depends on MreB to form a
the opposite poles of a cell. Depletion of MreB
Volume 71, Number 12, 2005 / ASM News Y 583
Several Forces Helped Steer Rothfield from Clinical onto Research Career Path
When Lawrence Rothfield entered Cornell University before
his 16th birthday, his head was
filled with images of physicians
and scientists from books. “I
wanted to be a doctor from my
early teens because I wanted to
make great discoveries about the
causes and cures of diseases,” he
says. He assumed, as did many
researchers like him, that he
needed to be a physician before he
could make medical discoveries.
After graduating from Cornell,
Rothfield studied medicine at
New York University (NYU) and
soon became a practicing internist
on Park Avenue in Manhattan.
Because he also conducted research, supported by his first research grant for a modest $6,500
from the National Institutes of
Health, “it meant 80-hour weeks,
and eating a sandwich for lunch in
the car, while driving from my
medical office to the medical
school,” he recalls.
“Although I think I was a pretty
good doctor,” Rothfield says, the
lifestyle was too frantic. Thus, after about four years, he stopped
doing clinical work and turned his
full attention to research.
After applying for a more expansive grant, Rothfield experienced what he calls a “life-changing” event—a visit from NIH
reviewers who told him that “my
ideas were good, but that I was
totally unqualified to do the
work.” They advised him to apply
for a fellowship to do biochemical
research with Bernard Horecker,
who was then head of microbiol-
ogy at NYU, where he was assigned to work with Mary Jane
Osborn, then a junior assistant
professor in Horecker’s group,
and still a friend and colleague.
“After two postdoctoral years,
when I was scheduled to return to
medicine, it became clear to
Bernie Horecker that I was doing
so reluctantly, and he offered me a
faculty position in his new department of molecular biology at the
Albert Einstein College of Medicine,” Rothfield says. “I didn’t
hesitate to accept.” He never
again practiced medicine.
Today Rothfield is professor of
molecular, microbial, and structural biology at the University of
Connecticut
Health
Center,
where he served as department
chair from 1968 to 1980. His current research focuses on the mechanism of cell division, and the
long-range organization of the
bacterial cell. “Beyond the obvious biological interest in understanding how cells divide, and the
potential application of this
knowledge in developing antibacterial agents that block bacterial
cell division, I see our work as an
example of a much broader biological question,” he says. “How
do cells determine spatial relationships between different parts
of the cell and use this information to undergo specific differentiation events at predetermined locations?
“For example, in the case of cell
division proteins, the site is usually at the midpoint of the cell,
whereas other cellular proteins
or certain mutational changes in MreB impairs
chromosome segregation, according to work by
Herve Soufo and Peter Graumann at the University of Marburg, Marburg, Germany, and from
584 Y ASM News / Volume 71, Number 12, 2005
will be located at one or both cell
poles,” he continues. “How does
the cell distinguish between these
locations? This problem of topological recognition is a general biological problem that is encountered in all types of cells.”
Rothfield grew up in New York
City, where his father—a “man of
total honesty and integrity” —
worked as a pharmacist. His
mother, who became a full-time
housewife, dropped out of law
school to support her husband
during pharmacy school.
Rothfield’s wife Naomi, a physician and rheumatology expert,
is a professor of medicine at Connecticut. He credits her—along
with the NIH grant review
group—with propelling him
along a research career path. “She
encouraged me despite the significant blow to our not-too-robust
financial situation,” he says. They
have four grown children—not a
scientist in the group—two involved with music professionally,
the third a computer programmer, and the fourth a faculty
member in a university English
department.
Rothfield vividly recalls Ludwig Eichna, professor of medicine
at NYU, who influenced him
greatly. “Eichna never accepted
sloppy thinking and always demanded of students ‘Show me the
evidence!’” Rothfield adds, “I
never forgot that lesson.”
Marlene Cimons
Marlene Cimons is a freelance writer
in Bethesda, Md.
the laboratories of Lucy Shapiro at Stanford
University and of Kenn Gerdes at the University
of Southern Denmark, Odense, Denmark. Zemer Gitai and his collaborators at Princeton
University and Stanford University also showed
that MreB molecules associate, directly or indirectly, with the Caulobacter chromosome near
its origin of replication. Thus, the MreB cytoskeleton is likely to be associated with the
bacterial equivalent of a centromere, working
cooperatively with other bacterial proteins at
this site to move the oriC region of each daughter chromosome to a cell pole, perhaps by moving it along the MreB filament toward a polar
binding site.
Yet a third MreB function is to deliver or
maintain certain proteins at the cell poles, according to work from the laboratories of Shapiro, Marcia Goldberg at Harvard University,
and Yu-Ling Shih and her collaborators at the
University of Connecticut (UC). This process
also could involve movement of the proteins
along the MreB cytoskeletal track.
Bacteria Encode Homologs to Other
Eukaryotic Cytoskeletal Proteins
In addition to cytoskeletal elements composed
of actin homologs, some bacteria contain cytoskeletal structures formed by homologs of the
two other main groups of eukaryotic cytoskeletal proteins—intermediate filament proteins
and tubulins.
For instance, the comma-shaped bacterium C.
crescentus contains a homolog of intermediate
filament proteins, called crescentin. In C. crescentus cells, crescentin forms a curved filamentous structure adjacent to the inner surface of
the cytoplasmic membrane (Fig. 1A), according
to Nora Ausmees, Jeffrey Kuhn, and Christine
Jacobs-Wagner at Yale University. Crescentin is
required for the cells to maintain their crescent
shape and, in its absence, the cells grow as rods.
Moreover, when MreB is removed, the cells also
lose their normal crescent shape, but become
spherical. Therefore, this species contains two
shape-determining cytoskeletal proteins, one
(the actin homolog MreB) that is responsible for
the underlying rod shape of the cell and the
other (the intermediate filament protein homolog crescentin) responsible for imparting curvature to the rod.
Additionally, almost all bacterial species encode FtsZ, a close structural homolog of the
tubulin proteins that are the major components
of eukaryotic microtubules. FtsZ is required for
cell division and forms a ring structure at the
division site during the first step of assembling
the bacterial cell division machinery. The FtsZ
ring acts as a scaffold for assembly of the large
number of other proteins of the division apparatus. However, FtsZ can also assume a coiled
structure that winds around the length of rodshaped cells, and it is likely that what appears to
be a FtsZ-containing ring at the division site is a
tightly compressed coiled structure. Thus, in E.
coli cells, a portion of FtsZ is present in a dynamic helical structure that resembles the structures formed by the cytoskeletal MreB and Min
proteins, according to Swapna Thanedar and
William Margolin at the University of Texas
Medical School in Houston. Furthermore, in
sporulating B. subtilis cells, a coiled FtsZ structure extending from midcell toward the cell
poles appears to represent an intermediate stage
in redistributing FtsZ from the ring structure at
midcell to a new FtsZ ring that is used to form
the spore septum near the end of the cell (Fig.
1D, E), as shown by Sigal Ben-Yehuda and
Richard Losick at Harvard University.
Cytoskeletal-Like Organization
of Other Bacterial Proteins
Extended helical cytoskeletal-like structures are
also formed in bacterial cells by proteins that are
unrelated to eukaryotic cytoskeletal proteins.
For instance, the E. coli MinCDE proteins that
determine placement of the division site wind
around the cell between the two poles in an
arrangement similar to that of MreB (Fig. 1F,
G), according to Shih and her collaborators at
UC. This structure is distinct from the MreB
cytoskeleton. The basic MinD coiled array may
act as a lattice for the assembly and disassembly
of the MinE and MinC site selection proteins
and possibly other structures involved in division site placement.
The E. coli SetB protein that affects chromosome segregation is also organized into a helical
structure, according to Olivier Espeli and Kenneth Marian of Sloan-Kettering Institute, New
York, N.Y. In this case, the SetB coiled array
likely involves interactions with the MreB cytoskeleton. Additional bacterial proteins that
form extended intracellular helical structures
include the SecY protein, which is involved in
exporting proteins across the cytoplasmic membrane; the ParA protein of plasmid pB171 that is
involved in segregating plasmid DNA; and the
Volume 71, Number 12, 2005 / ASM News Y 585
LamB protein of the E. coli outer membrane.
The large and increasing number of proteins
that adopt this long-range coiled organization is
intriguing and implies that some common mechanism may be involved in their assembly.
Future Challenges in Studying the
Bacterial Cytoskeletons
The discovery in 2001 of an actin-like cytoskeleton in B. subtilis quickly led to a new view of
bacterial cells in which the cell is highly organized, with internal protein structures that show
extensive long-range order.
Based on this new knowledge, researchers
studying the bacterial cytoskeleton face several
major questions: Are there a limited number of
cytoskeletal structures, with several functional
elements associated with each one, or are there a
large number of independent structures, each
specialized to carry out a single function? Is
there an underlying membrane-associated scaffold that is responsible for organizing all these
coiled cytoskeletal structures? Are there additional structures that traverse the bacterial cytosol—perhaps facilitating the intracellular
movement of specific proteins and other molecules—in addition to those thus far identified,
which extend along the undersurface of the cytoplasmic membrane? Are there novel bacterial
cytoskeleton-associated proteins within the
structures that have thus far been identified? For
example, in eukaryotic cells, several low-abundance proteins play key roles in the dynamic
behavior of the tubulin-based microtubules, and
the submembranous cytoskeleton of eukaryotic
cells often contains proteins that communicate
with cytosolic structures or with integral membrane proteins that in turn communicate with
the outside world. Are there comparable proteins in bacterial cytoskeletal networks?
One thing is certain. We are in the early stages of
defining the nature of the bacterial cytoskeleton
and are likely to encounter many surprises over the
next few years that will further change our views
of the bacterial cell.
ACKNOWLEDGMENT
Our research is supported by USPHS grant GM37– 60632.
SUGGESTED READING
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Ben-Yehuda, S., and R. Losick. 2002. Asymmetric cell division in B. subtilis involves a spiral-like intermediate of the
cytokinetic protein FtsZ. Cell 109:257–266.
Daniel, R.A., and J. Errington. 2003. Control of cell morphogenesis in bacteria: two distinct ways to make a rod-shaped cell.
Cell 113:767–776.
Figge, R. M., A. V. Divakaruni, and J. W. Gober. 2004. MreB, the cell shape-determining bacterial actin homologue,
coordinates cell wall morphogenesis in Caulobacter crescentus. Mol. Microbiol. 51:1321–1332.
Gitai, Z., N. Dye, and L. Shapiro. 2004. An actin-like gene can determine cell polarity in bacteria. Proc. Natl. Acad. Sci. USA
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Jones, L., R. Carballido-Lopez, and J. Errington. 2001. Control of cell shape in bacteria: helical actin-like filaments in Bacillus
subtilis. Cell 104:913–922.
Kruse, T., J. Moller-Jensen, A. Lobner-Olesen, and K. Gerdes. 2003. Dysfunctional MreB inhibits chromosome segregation in
Escherichia coli. EMBO J 22:5283–5292.
Shih, Y.-L., T. Le, and L. Rothfield. 2003. Division site selection in Escherichia coli involves dynamic redistribution of Min
proteins within coiled structures that extend between the two cell poles. Proc. Natl. Acad. Sci. USA 100:7865–7870.
Soufo, H. J., and P. L. Graumann. 2003. Actin-like proteins MreB and Mbl from Bacillus subtilis are required for bipolar
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Thanedar, S., and W. Margolin. 2004. FtsZ exhibits rapid movement and oscillation waves in helix-like patterns in
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