hydrolysis (19) halted FtsZ movement (Fig. 3E
and movie S8A). Conversely, addition of MciZ, a
Z ring antagonist that, at low levels, increases
FtsZ GTPase activity (20), increased FtsZ velocity
(Fig. 3, F and H, and movie S7B). We next tested
whether FtsZ treadmilling dynamics affected
Pbp2B movement. PC190723 or overexpression
of FtsZ(D213A) caused Pbp2B molecules to become immobile while the remaining colocalized
with FtsZ (Fig. 3, D and E, and movie S8B).
Pbp2B velocity scaled with FtsZ treadmilling
velocity under various perturbations (fig. S5K).
Thus, FtsZ treadmilling is required for the directional motions of both FtsAZ filaments and
septal PG synthases.
Given the mobile nature of septal PG synthe-
sis, we reasoned that the directional movements
of FtsAZ and Pbp2B around the division plane
could be coupled to septal PG synthesis. To test
this, we labeled cells with FDAAs as we altered
FtsZ dynamics. Overexpression of FtsZ(D213A)
created long, slowly growing FtsA-mNeonGreen
spirals, which incorporated FDAAs along their
entire length (Fig. 4A and movie S9). Likewise,
long PC190723 treatments resulted in fragmented
patches of both FtsZ and FDAA incorporation (fig.
S6A), indicating Pbp2B activity is constrained by
FtsAZ location. However, these strong inhibitions
of FtsZ dynamics required much longer pulses
to achieve FDAA labeling, suggesting that FtsZ
treadmilling limits PG synthesis. To test this, we
altered FtsAZ velocity as we pulse-labeled cells
with FDAAs. This revealed that both the total
amount and total area of PG synthesis within the
ring are modulated by FtsAZ velocity: Conditions
that slowed dynamics decreased both the total
amount and area of FDAA labeling (Fig. 4B and
fig. S6, B and C). Conversely, increasing FtsAZ
velocity (with MciZ) increased both the total
amount and area of labeling. Thus, in B. subtilis,
both the amount and spatial distribution of
septal PG synthesis are directly coupled to, and
limited by, the rate of FtsZ filament treadmilling.
Because FtsAZ dynamics control the rate of
septal synthesis, we asked whether the rate of
Fig. 2. FtsAZ and Pbp2B move directionally around the division site.
(A) mNeonGreen-FtsZ (bAB185) shows directional motion inside (left) and outside
(right) the Z ring; montage at 8-s intervals. (B) FtsA-mNeonGreen (bAB167) shows
directional motion inside (left) and outside (right) the ring. (C) mNeonGreen-
FtsZ and FtsA-Halo Tag-JF549 (bAB229) colocalize and move together. Yellow
is the overlap of green and red. (D) FtsAZ overexpression [100 mM isopropyl
b-D-1-thiogalactopyranoside (IPTG) in bAB221] creates increased FtsZ filaments
showing directional motion outside the Z ring. (E) Vertically immobilized cells
(SH41) show multiple, independent mNeonGreen-FtsZ filaments moving in both
directions around the division site. Cropped rings and radial kymographs in
early (left), middle (center), and late (right) divisional stages. Blue arrow-
heads indicate directional FtsZ tracks. (F) Single molecules of Pbp2B (bGS31,
15-min incubation of 50 pM JF549) move directionally around the division site.
Blue to yellow indicates trajectory time. Kymographs drawn at yellow arrow-
heads. Scale bars, 0.5 mm. (G) Velocity distributions of FtsA filaments, FtsZ
filaments, and single Pbp2B molecules.