meso-diaminopimelic acid (Dap-Dap) cross-links
(Fig. 4F), indicating that the nature of cross-linking
reactions was also perturbed. We observed that the
differences in PG composition between minicells
(round DNA-less cells produced by polar divisions)
isolated from FtsZD212G and BW25113 DminC cells
were similar to the differences seen with intact
cells, indicating that the shorter glycans and greater
cross-linking are not attributable to aberrant placement of the division site (fig. S17B). A Caulobacter
crescentus FtsZ mutant showed opposite changes
(less cross-linking and longer glycan strands),
along with bulging of the septal wall (34). Thus,
the balance between glycan strand polymerization
and cross-linking activities is likely an important
factor in defining the shape of the septum, with
FtsZ coordinating the two enzymatic activities.
Our results show that FtsZ engages in tread-
milling powered by GTP hydrolysis to spatially or-
ganize the septal PG synthesis machinery (Fig. 4G)
without limiting the rate of septum closure. In
Bacillus subtilis, FtsZ treadmilling appears to dis-
tribute septal PG synthesis and also to dictate the
synthesis rate (35). The differences between the two
organisms may reflect differential requirements for
PG synthesis between Gram-negative and Gram-
positive bacteria. Furthermore, whereas the actin
homolog MreB relies on wall synthesis for its
movement, FtsZ exploits its innate treadmilling
capacity to control the movement of septal syn-
thesis enzymes. The broad conceptual similarities
among FtsZ, MreB, and the movement of cellulose
synthase complexes along cortical microtubules in
plants (36) suggest that coupling cytoskeletal mo-
tion to wall synthesis may be a general strategy
across the kingdoms of life to ensure evenly dis-
tributed, robust septal wall synthesis and morpho-
genesis through time-averaging.
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We thank members of the Xiao and Huang labs for helpful discussions
and technical assistance; E. Goley, P. Levin, C. Coltharp, J. Buss, G. Lan,
J. Lutkenhaus, E. Garner, and G. Squyres for critical discussions;
E. Kuru, Y. Brun, and M. VanNieuwenhze for the HADA dye and
assistance in its use; J. Lutkenhaus for the MC123 strain; H. Erickson for
the FtsZ antibody and internal FtsZ-FP fusion; P. de Boer for the TB43
strain; E. Goley, P. Levin, and their lab members for helpful advice on
protein purification and GTPase assay; R. Tsien for the TagRFP-t
construct; M. Delannoy for assistance with SEM; K. Esther, D. Jesse, and
Nikon for assistance with N-SIM imaging; and J. Franklin for assistance
with whole-genome sequencing. Supported by NIH New Innovator
award DP2OD006466 and NSF career award MCB-1149328 (K.C.H.); an
NSF graduate research fellowship and an ARCS Scholar Awards
fellowship (A. M.); NIH grant R01 GM086447, NSF EAGER award MCB-
1019000, and a Hamilton Smith Innovative Research award (J. X.); and
NSF grant PHYS-1066293. We are grateful for the hospitality of the
Aspen Center for Physics. HADAs were received under a material
transfer agreement from M. VanNieuwenhze at Indiana University.
Materials and Methods
Figs. S1 to S17
Tables S1 to S6
Movies S1 to S19
26 September 2016; accepted 20 January 2017
Fig. 4. Altered directional movement of FtsI and septal PG composition in FtsZmut cells. (A) Directional
movement of multiple (left) or single (right) TagRFP-t-FtsI molecules along the septum in FtsZ cells. Images with
yellow arrowheads are maximum intensity projections; kymograph images are from positions denoted by the
arrowheads. (B to D) Examples of TagRFP-t-FtsI directional movement in E250A (B), D158A (C), and D212G (D)
cells. (E) Mean TagRFP-t-FtsI movement speed is highly correlated with FtsZ treadmilling speed; error bars
denote SD (see table S1). (F) UPLC analysis reveals altered PG composition in FtsZD212G cells. The relative
percentage of each component of D212G was normalized to that of wild-type cells. Error bars denote SD, n = 3;
*P < 0.05, **P < 0.01 (unpaired t test). (G) A schematic model depicting FtsZ treadmilling (gray circles) that
drives directional movement (wavy arrows) of the septal PG synthesis machinery (brown rectangles), leading to
processive synthesis of new septal PG (yellow). IM and OM, inner and outer membranes; scale bars, 0.5 mm.