at positions 29, 49, 263, and 269 rapidly induced
lysis, suggesting that MurJ function, and thus
PG synthesis, was inhibited (Fig. 2 and fig. S5).
In contrast, treatment of MurJE273C cells with
MTSES caused cell shape defects and limited
lysis indicative of an incomplete PG synthesis
block due to partial MurJ inhibition. The toxicity
of MTSES labeling was suppressed in all five
strains by the presence of the wild-type murJ
allele (Fig. 2 and fig. S5). Thus, MTSES specifically and rapidly inhibits these single-Cys MurJ
variants. We chose MurJA29C (Fig. 2, and figs. S6
and S7) to assess the effect of MurJ inactivation
on lipid II flipping.
This chemical genetic method for MurJ inactivation was compatible with the in vivo flippase
assay. MTSES treatment of MurJWT cells did not
affect lipid II processing by ColM (Fig. 1, B and
C, and fig. S1). Additionally, in the absence of
MTSES, MurJA29C cells behaved like MurJWT cells
(Fig. 1, B and C, and fig. S1). However, simultaneous addition of MTSES and ColM to MurJA29C
cells failed to produce significant quantities of
the ColM-dependent product PP-Mpep4-G. In fact,
radiolabel in the lipid fraction increased in these
samples (Fig. 1, B and C, and fig. S1). Thus, when
MurJA29C was inactivated with MTSES, lipid II
was protected from ColM cleavage, and label accumulated in the lipid fraction as observed previously for MurJ-depletion strains (4, 6).
The protection of lipid II from ColM cleavage
upon MurJA29C inactivation suggests that either
lipid II is not flipped or that inhibiting MurJA29C
When MurJA29C was inactivated with MTSES,
flippase activity was reduced to a level that was
barely detectable and was incompatible with
life. This observation indicates that the essen-
tial function of MurJ is to translocate lipid II
and that other factors catalyzing lipid II flipping
are unlikely to exist in E. coli. Nevertheless, we
investigated the requirement of SEDS proteins
for flippase activity by depleting Fts W in a DrodA
strain. We found that lipid II flipping remained
robust in this background (figs. S8 and S9). Al-
though it is possible that residual Fts W in these
cells was sufficient for the observed activity, this
result suggests that SEDS proteins are not re-
sponsible for lipid II flippase activity in vivo.
Alternatively, the decrease in levels of PG lipid
intermediates upon Fts W depletion (fig. S9) sug-
gests that either synthesis of PG precursors or
recycling of undecaprenyl-P might be affected by
the loss of SEDS activity. From these data and the
fact that MurJ contains a central solvent-exposed
cavity that is essential for function (8), we conclude
that MurJ is the lipid II flippase in E. coli.
REFERENCES AND NOTES
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We thank D. Mengin-Lecreulx for the generous gift of plasmids
for ColM production and H. Joseph and R. M. Davis for their
technical assistance. Research was supported by funds from the
American Heart Association (L.S.) and the National Institutes
of Health (NIH) under award numbers F32GM103056 (M.D.L.),
R01GM100951 (N.R.), R01AI099144 (T.G.B.), and R01GM76710
(D.K.). The content is solely the responsibility of the authors and
does not necessarily represent the official views of NIH. For
additional data, see the supplementary materials.
Materials and Methods
Figs. S1 to S9
Tables S1 and S2
8 April 2014; accepted 28 May 2014
A Doppler effect in embryonic
Daniele Soroldoni,1,2,3 David J. Jörg,4 Luis G. Morelli,1,5 David L. Richmond,1
Johannes Schindelin,1,6 Frank Jülicher,4 Andrew C. Oates1,2,3†
During embryonic development, temporal and spatial cues are coordinated to generate a segmented
body axis. In sequentially segmenting animals, the rhythm of segmentation is reported to be
controlled by the time scale of genetic oscillations that periodically trigger new segment formation.
However, we present real-time measurements of genetic oscillations in zebrafish embryos
showing that their time scale is not sufficient to explain the temporal period of segmentation.
A second time scale, the rate of tissue shortening, contributes to the period of segmentation through
a Doppler effect. This contribution is modulated by a gradual change in the oscillation profile
across the tissue. We conclude that the rhythm of segmentation is an emergent property controlled
by the time scale of genetic oscillations, the change of oscillation profile, and tissue shortening.
Segmental patterns are common through- out nature. In animals from diverse phyla, segmentation of the body axis occurs dur- ing embryogenesis, and in most cases, seg- ments are added sequentially, with a distinct period as the body axis elongates. Recent findings indicate that a common mechanism involving enetic oscillations underlies this morpholog- ical segmentation in vertebrates and arthro- pods (1). We investigated how the time scale of
222 11 JULY 2014 • VOL 345 ISSUE 6193 sciencemag.org SCIENCE
Fig. 3. MurJ activity is required for ColM-dependent cleavage of lipid II in spheroplasts.
Cells lacking the ColM receptor FhuA and producing
the indicated MurJ variants were grown, labeled, and
treated with MTSES as for Fig. 1. Spheroplasts were
then prepared. In all but one case, spheroplasts were
pelleted and resuspended in ColM reaction buffer
with sucrose, and MTSES (0.8 mM) was added as
indicated. The lysis + sample was resuspended in
buffer lacking sucrose to lyse the spheroplasts. ColM
(100 mg) was added to the prepared spheroplasts as
indicated, and they were incubated for 15 min at
37°C. Lipid intermediates were detected by scintillation
counting after butanol extraction. Statistics are as for
Fig. 1. cpm, counts per minute.