predicted to also be repressed by CsrA in EPEC,
and pgaA is a K12-exclusive gene (table S7). We
found that expression of Ces T in E. coli K12, or
EPEC, is sufficient to activate the expression of
these genes (figs. S11 to S13). We further determined that, in EPEC, expression of glgC and ydeH
is specifically activated upon contact with host
cells (fig. S14). Taken together, our analyses show
that, in attached EPEC, the CsrA-Ces T-T3SS regulatory circuit influences the expression patterns
of nleA, glgC, ydeH, BFP, LEE4, and probably
other genes as well.
Here we show that the T3SS serves to sense
attachment to the host cell and, as a result activates, the CsrA-Ces T-T3SS regulatory circuit. Integration of our results with the broad knowledge
of CsrA regulation in E. coli K12 points to possible
massive remodeling of gene expression upon EPEC
attachment (fig. S15). In this study, we used EPEC
to analyze the link between the T3SS and gene
expression and provide data supporting the
premise that the same regulation occurs in EHEC
and CR. We suggest that this remodeling of
gene expression allows AE pathogens to colonize the intestinal epithelium surface and successfully survive and flourish in this niche, which is
essentially free of microbiota (28). We predict
that a similar link between T3SS activity and
expression of metabolic and signaling genes exists
in other pathogens.
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We thank J. Kaper, S. Gruenheid, M. Donnenberg, and G. Frankel
for providing antibodies and strains; R. Kulkarni for providing and
running the CsrA predicting script; A. Winer, M. Elgradly-Weiss,
T. Hershko-Shalev, and L. Argaman for technical help; and S. Ben
Yehuda, G. Segal, and J. Sivaraman for reading the manuscript.
The work was funded by a grant from the Israel Academy of
Sciences and Humanities. N.K. is a recipient of a fellowship from
the Carol and Leonard Berall Endowment. I.R. is an Etta Rosensohn
Professor of Bacteriology. Plasmids and bacterial strains described
in this paper are available from I.R. under a material transfer
agreement with the Hebrew University of Jerusalem.
Materials and Methods
Figs. S1 to S15
Tables S1 to S7
5 July 2016; accepted 19 January 2017
Treadmilling by FtsZ filaments drives
peptidoglycan synthesis and
bacterial cell division
Alexandre W. Bisson-Filho,1‡ Yen-Pang Hsu,2‡ Georgia R. Squyres,1‡ Erkin Kuru,2*‡
Fabai Wu,3† Calum Jukes,4 Yingjie Sun,1 Cees Dekker,3§ Seamus Holden,4§
Michael S. VanNieuwenhze,2,5§ Yves V. Brun,6§ Ethan C. Garner1§
The mechanism by which bacteria divide is not well understood. Cell division is mediated by
filaments of FtsZ and FtsA (FtsAZ) that recruit septal peptidoglycan-synthesizing enzymes to
the division site. To understand how these components coordinate to divide cells, we visualized
their movements relative to the dynamics of cell wall synthesis during cytokinesis. We found
that the division septum was built at discrete sites that moved around the division plane. FtsAZ
filaments treadmilled circumferentially around the division ring and drove the motions of the
peptidoglycan-synthesizing enzymes. The FtsZ treadmilling rate controlled both the rate of
peptidoglycan synthesis and cell division. Thus, FtsZ treadmilling guides the progressive insertion
of new cell wall by building increasingly smaller concentric rings of peptidoglycan to divide the cell.
In most bacteria, cell division involves the in- ward synthesis of peptidoglycan (PG), creat- ing a septum that cleaves the cell in two. The location of the septal PG synthases is regu- lated by filaments of the tubulin homolog
FtsZ, which associate with the cytoplasmic side
of the membrane via the actin-like FtsA and other
factors. FtsZ forms membrane-associated filaments with FtsA (FtsAZ) (1, 2). Together, they
form a dynamic structure, the Z ring, which encircles the cell at the future division site (3) and
recruits PG synthases and other proteins involved
in cytokinesis (4). Once the division machinery is
mature, the Z ring constricts while the associated
synthases build the septum that partitions the
cell in two.
We do not have a clear understanding of how
the components of cell division interact in space
and time to carry out cytokinesis, as we have
been unable to observe the dynamics of each
component relative to each other or to the structure they build: The organization and dynamics
of FtsZ filaments within the Z ring remain ill-defined; it is not known how FtsAZ filaments
control the activity of PG synthases; and the dynamics of septal PG synthesis have never been
directly observed. To gain insight into how these
components work together to divide bacteria, we
visualized the dynamics of septal PG synthesis in
relation to the movements of FtsAZ filaments
and the septal PG synthase Pbp2B in the Gram-positive Bacillus subtilis.
To assess the dynamics of septal PG synthesis,
we sequentially pulse-labeled growing cells with
different colors of fluorescent D-amino acids (FDAAs)
(table S1), which are incorporated into PG (5) by
the D,D-transpeptidation activity of penicillin-binding proteins (PBPs) (6). Three-dimensional
(3D)–structured illumination microscopy (3D-
SIM) showed that sequential three-color FDAA
pulse-labeling resulted in bull’s-eye patterns at
the division plane (Fig. 1A), demonstrating that
the septum is progressively synthesized inward
from the cell surface. Short, sequential pulses of
two FDAA colors resulted in discrete spots or
arcs distributed around the septum, with the
sciencemag.org 17 FEBRUARY 2017 • VOL 355 ISSUE 6326 739
1Molecular and Cellular Biology, Faculty of Arts and
Sciences Center for Systems Biology, Harvard University,
Cambridge, MA 02138, USA. 2Department of Molecular and
Cellular Biochemistry, Indiana University, Bloomington, IN
47405, USA. 3Department of Bionanoscience, Kavli Institute of
Nanoscience Delft, Delft University of Technology,
Netherlands. 4Centre for Bacterial Cell Biology, Institute for
Cell and Molecular Biosciences, Newcastle University,
Newcastle upon Tyne NE2 4AX, UK. 5Department of
Chemistry, Indiana University, Bloomington, IN 47405, USA.
6Department of Biology, Indiana University, Bloomington,
IN 47405, USA.
*Present address: Department of Genetics, Harvard Medical
School, Boston, MA 02115, USA. †Present address: Division of
Geology and Planetary Sciences, California Institute of Technology,
Pasadena, CA 91125, USA. ‡These authors contributed equally to
this work. §Corresponding author. Email: firstname.lastname@example.org
(E.C.G.); email@example.com (M.S.V.); firstname.lastname@example.org
(Y.V.B.); email@example.com (S.H.); c.dekker@
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