Cell, Vol. 120, 329–341, February 11, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.cell.2005.01.007
MreB Actin-Mediated Segregation of a
Specific Region of a Bacterial Chromosome
Zemer Gitai,1,3,* Natalie Anne Dye,2Ann Reisenauer,1
Masaaki Wachi,4and Lucy Shapiro1,*
1Department of Developmental Biology
2Department of Biochemistry
Beckman Center, School of Medicine
Stanford, California 94305
3Department of Molecular Biology
Princeton, New Jersey 08544
4Department of Bioengineering
Tokyo Institute of Technology
4259 Nagatsuta, Midori-ku
B. subtilis localize their origins of replication to the mid-
cell and quarter-cell positions at different stages of the
cell cycle (Li et al., 2002; Webb et al., 1997), whereas in
the crescent-shaped Caulobacter crescentus, chromo-
somal origins are at the cell poles (Jensen and Shapiro,
1999). Direct examination of chromosome segregation
in live cells revealed that the origin region (defined as
the site of the initiation of DNA replication) is the first
region of the chromosome to be segregated in all spe-
cies examined (Lau et al., 2003; Li et al., 2002; Viollier
et al., 2004; Webb et al., 1998).
The rapid, directed movement of the origins excludes
the cell growth-related mechanisms initially proposed
for chromosome segregation (Jacob et al., 1963) and is
suggestive of an active mechanism for origin transport.
Several candidates have been put forth as potential
contributors to the force that separates the chromo-
somes. The coincident timing of DNA replication and
segregation suggests that these processes are coupled
and that the act of DNA replication could provide the
motive force for chromosome segregation. The obser-
vation that the DNA replication machinery is stationary
in B. subtilis and E. coli led to the refinement of such
models into the “extrusion-capture” model (Lemon and
Grossman, 2001), wherein the extrusion of DNA from an
immobile replisome forces it poleward, where it is then
captured by as-yet unidentified factors. Coordinate
transcription of origin-proximal reading frames oriented
away from the origin has also been proposed to con-
tribute to the movement of bacterial chromosomes
(Dworkin and Losick, 2002). Though these models are
compelling and elegant, they have yet to be directly val-
In contrast, the mechanisms directing eukaryotic
chromosome segregation are well characterized. Eu-
karyotes use a microtubule-based cytoskeletal system
for chromosome partitioning. After replication, chromo-
some sisters are paired, and microtubules are attached
to specific, centromeric chromosomal loci through a ki-
netochore protein complex. Once attached, the micro-
tubules direct chromosome migration to opposite poles
through dynamic polymerization cycles and the action
of motor proteins (Kline-Smith and Walczak, 2004).
Could a similar mechanism also contribute to bacte-
rial chromosome segregation? The bacterial tubulin ho-
molog, FtsZ (Lowe and Amos, 1998), is an unlikely can-
didate, as ftsZ mutants do not display chromosome
segregation defects, and FtsZ’s midcell subcellular lo-
calization is inconsistent with a role in chromosome
segregation (Lutkenhaus and Addinall, 1997; Quardo-
kus et al., 1996). A more attractive possibility is the re-
cently identified bacterial actin homolog, MreB (Jones
et al., 2001; van den Ent et al., 2001). First, the origin of
replication is found in abnormal numbers and positions
in mreB mutants (Gitai et al., 2004; Kruse et al., 2003;
Soufo and Graumann, 2003); second, MreB localizes to
a spiral that traverses the length of the cell (Figge et
al., 2004; Gitai et al., 2004; Jones et al., 2001; Shih et
al., 2003), such that MreB could potentially be enlisted
to move chromosomes from one end of the cell to an-
Faithful chromosome segregation is an essential com-
ponent of cell division in all organisms. The eukary-
otic mitotic machinery uses the cytoskeleton to move
specific chromosomal regions. To investigate the po-
tential role of the actin-like MreB protein in bacterial
chromosome segregation, we first demonstrate that
MreB is the direct target of the small molecule A22.
We then demonstrate that A22 completely blocks the
movement of newly replicated loci near the origin of
replication but has no qualitative or quantitative ef-
fect on the segregation of other loci if added after ori-
gin segregation. MreB selectively interacts, directly or
indirectly, with origin-proximal regions of the chromo-
some, arguing that the origin-proximal region segre-
gates via an MreB-dependent mechanism not used by
the rest of the chromosome.
To maintain a consistent number of chromosomes,
cells must ensure that during cell division, the chromo-
some is reproducibly replicated and that each of the
new chromosomes is segregated to one of the two
daughter cells. Although the ability of bacteria to faith-
fully replicate and segregate their chromosomes has
been appreciated for over forty years (Jacob et al.,
1963), the molecular mechanism by which bacteria exe-
cute the segregation process has remained elusive.
Observing the placement and movement of chromo-
somal loci revealed that all bacteria examined have
highly reproducible, ordered chromosomal structure,
with loci exhibiting a linear correlation between their
genetic location on the chromosome and their place-
ment along the long axis of the cell (Niki et al., 2000;
Teleman et al., 1998; Viollier et al., 2004). Nonetheless,
the details of chromosomal topology vary among spe-
cies. For example, the rod-shaped bacteria E. coli and
Figure 1. A22 Affects Caulobacter Growth and Morphology
(A) The chemical structure of S-(3,4-dichlorobenzyl)isothiourea, A22.
(B) Growth curve of cells treated for up to 6 hr with 0, 0.1, 1, 10, and 100 ?g/ml of A22 or an equivalent volume of methanol (MeOH).
(C) Colony formation assay curve for cells treated with 0, 0.1, 1, 10, and 100 ?g/ml of A22 or an equivalent volume of methanol (MeOH).
(D) DIC images of Caulobacter grown for 6 hr in 0, 1, 10, or 100 ?g/ml of A22.
(E) Timecourse of cells grown in 10 ?g/ml of A22 for 0, 2, 4, 6, and 8 hr.
(F) Timecourse of cells depleted of mreB for 0, 6, 12, 18, and 24 hr. Scalebars represent 1 ?m.
other; finally, there is precedent for actin’s involvement
in bacterial DNA segregation, as another bacterial actin
homolog, the R1 plasmid-specific ParM protein, has
been directly implicated in R1 plasmid partitioning
(Moller-Jensen et al., 2002, 2003).
Though these results are tantalizing, demonstrating a
direct role for MreB in chromosome segregation has
proven difficult. MreB loss of function is lethal and pleio-
tropic (Figge et al., 2004; Gitai et al., 2004; Jones et
al., 2001), disrupting multiple cellular processes distinct
from chromosome dynamics, including cell shape de-
termination, polar protein localization, and cell division.
Thus, it remains unclear whether MreB plays a primary
role in chromosome segregation, or if MreB’s effect on
chromosome dynamics is a secondary consequence of
Here we present the first evidence that supports a
direct role for MreB in the segregation of a specific re-
gion of the chromosome. We reached these conclu-
sions by first characterizing a small molecule, A22, that
specifically, rapidly, and reversibly perturbs MreB func-
tion. Our studies focus on the differentiating bacterium,
Caulobacter crescentus, whose ability to be synchro-
nized renders it particularly powerful for the analysis
of chromosome segregation (Jensen et al., 2002). By
administering the MreB-perturbing compound at dif-
ferent stages of the Caulobacter cell cycle we show
that origin-proximal loci segregate through an MreB-
dependent mechanism, and that the rest of the chro-
mosome follows the origin using an MreB-independent
mechanism. The ability of A22 to block DNA segrega-
tion without affecting DNA replication also demon-
strates that the process of replication is not sufficient to
separate chromosomes, as was previously proposed.
Microscopy and Timelapse Imaging
All experiments other than the A22 flow-cell analysis were per-
formed by mounting the samples on 1% agarose pads as de-
scribed (Ryan et al., 2002), with the appropriate concentration of
A22 added to the agarose. The rates of origin-distal locus segrega-
tion were determined by inducing Pxyl:lacI:cfp expression with
0.03% xylose for 75 min, isolating swarmer cells, and growing the
swarmer cells in M2G liquid medium for 50 min. After 50 min, 10
?g/ml of A22 was added to these liquid cultures, they were
mounted onto 1% M2G agarose pads containing 10 ?g/ml A22,
and a phase and CFP fluorescence image was captured every 2
min for 60 min. The images were analyzed by previously described
software (Viollier et al., 2004) to determine the average position and
rate of movement of the observed foci.
The A22 flow-cell treatment timelapse images were performed
on Pxyl:gfp-mreB cells induced for 2 hr with 0.03% xylose. These
cells were adhered to polylysine-coated cover slips. The cover
slips were then adhered to microscope slides with two parallel
strips of double-sided tape (3M), leaving a central channel. An ex-
cess of medium containing 0 or 10 ?g/ml A22 was pipetted onto
one opening of this channel and wicked through by placing a tissue
(VWR) on the other end. Images were collected every 30 s.
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Analysis of DNA Replication
DNA replication was assessed by incorporation of
dGTP (Marczynski et al., 1990) or chromomycin-stained DNA-content
FACS analysis (Winzeler and Shapiro, 1995) as described pre-
Formaldehyde-crosslinked chromatin immunoprecipitation was
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raised by cloning the mreB coding sequence into the pET28a vec-
tor (Novagen). The resulting His6-tagged MreB was overexpressed
in BL21(DE3)/pLysS E. coli and purified using Qiagen Ni2+-NTA
agarose as described previously (Viollier et al., 2002). Two milli-
grams of purified His6-MreB was used to immunize two rabbits
(Josman Laboratories, Napa, California). Oligonucleotide primer
sequences for the regions PCR amplified are available upon re-
We would like to thank Tom Wandless (Stanford University) for syn-
thesizing additional A22 compound based on its published struc-
ture (Iwai et al., 2002), Patrick McGrath for assistance with the
quantitative analysis of locus movement and positioning, and
members of the Shapiro and McAdams labs and Coleen Murphy
for critical discussions. Z.G. is supported by a Ruth Kirschstein NIH
postdoctoral fellowship. This work was supported by NIH grant
Received: November 5, 2004
Revised: December 22, 2004
Accepted: January 6, 2005
Published: February 10, 2005
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