3E and table S5), as found previously (11); septum
closure rate measurements in D212A and D212G
were unreliable because of the frequent failure
of cell division (movie S15). These data are most
consistent with FtsZ GTPase activity affecting
the spatiotemporal distribution of synthesis, rather
than the septal PG synthesis rate.
Next, we reasoned that because FtsZ recruits
many proteins involved in septal synthesis, the dynamics of the essential septal transpeptidase
FtsI would likely follow that of FtsZ. Using single-molecule tracking in wide-field epifluorescence
microscopy, we tracked the movement of a complementing (fig. S15), N-terminal fluorescent protein
fusion of FtsI (TagRFP-t-FtsI) at visible constriction
sites in FtsZWT and three FtsZmut backgrounds. Kymographs of TagRFP-t-FtsI fluorescence in FtsZWT
cells showed diagonal tracks similar to those of
FtsZ (Fig. 4A and fig. S16), indicating that individual TagRFP-t-FtsI molecules moved directionally
along the septum, in contrast to stationary FtsZ
molecules in treadmilling FtsZ polymers (Fig. 2 and
fig. S11) (22). The TagRFP-t-FtsI movement was
not unidirectional and exhibited large variations in
time and in different cells (Fig. 4A and movies S16
to S18). In the FtsZmut strains, TagRFP-t-FtsI moved
more slowly (Fig. 4, B to D), with mean speed comparable to the corresponding FtsZ treadmilling
speed (Fig. 4E). Thus, FtsZ treadmilling guides the
directional movement of FtsI and thereby directs
the distribution of new septal PG.
Finally, we used ultraperformance liquid chromatography (UPLC) to investigate whether the
biochemical composition of the PG itself was altered in D212G cells (12). In both LB and minimal
media, D212G cells had shorter glycan strands and
greater cross-linking than did wild-type cells (Fig.
4F and fig. S17A), indicating imbalances in the
relative levels of septal glycan strand polymerization and cross-linking in FtsZD212G cells. We
also observed a large increase in alternative
746 17 FEBRUARY 2017 • VOL 355 ISSUE 6326
Fig. 2. FtsZ polymers exhibit treadmilling dynamics in live E. coli cells. (A and B) Maximum
intensity projection (left panels) and montages from
time-lapse imaging (movies S8 and S9) of a cell in
which a midcell Z-ring was not assembled (A) and a
cell with a clearly visible midcell Z-ring (B). (C) Kymographs of the cells in (A) and (B) computed from the
intensity along the line between the two yellow arrows.
(D) Distributions of polymerization and depolymerization
speeds as measured from the leading and trailing edges
of individual cells’ kymographs [blue and red lines in
(C)]. (E) Structured illumination microscopy maximum-intensity projection (left panel) and montage from
time-lapse imaging of counterclockwise Z-ring treadmilling. (F) Kymograph of fluorescence along circumference of cell in (E). (G and H) Treadmilling speeds
correlated with kcat (G) and Z-ring dynamics (H). Error
bars denote SD. Scale bars, 0.5 mm.
Fig. 3. FtsZ GTPase mutants change the spatial distribution pattern but not the rate of septal
PG synthesis. (A) Representative scanning electron microscopy images of FtsZ wild-type, E250A,
D158A, and D212G cells. Red arrows denote deformed, asymmetric septa. (B) Representative images of
HADA-labeled septa for short (<10 s), intermediate (90 s), and long (810 s) labeling pulses. Red arrows
and yellow arrowheads denote incomplete and complete septa, respectively. (C) Severe GTPase mutants
had large percentages of cells with incompletely labeled septa even for long pulses. (D) Integrated septal
HADA fluorescence increased similarly with labeling pulse duration in all strains. (E) The ratio of septum
closure rate (vc) to elongation rate during constriction (vec) was similar among tested GTPase mutants.
Error bars denote SEM. Scale bars, 1 mm.