retained in mother cells, corresponding with a
16.0 ± 0.3% mother-daughter difference (Fig. 1E).
In the absence of TolC, AcrB-GFP partitioned
similar to cytosolic mCherry (Fig. 1E, right bar,
and fig. S8). TolC-GFP also accumulated at old
cell poles, with lesser magnitude and higher over-
all variability; its asymmetry was substantially
reduced in the DacrAB strain (fig. S9). Thus, only
complete ternary AcrAB-TolC complexes are par-
titioned unequally at cell division.
To probe the lifetime of AcrB-GFP at the old
cell pole of mothers, we transiently expressed
AcrB-GFP (pulse) (Fig. 1F, fig. S10, and movie
S2). AcrB-GFP asymmetry in mother cells was
constant for several generations during the chase
period and was ultimately followed by a slow
decay in asymmetry. In contrast, soluble GFP
showed no induction-dependent asymmetry. AcrB-
GFP thus segregates with the old mother pole for
an extended period even in the absence of acrB-
gfp expression and is partitioned unevenly at cell
division. Using total internal reflection fluores-
cence microscopy, we found that AcrB-GFP formed
TolC-dependent stationary foci that segregated
312 21 APRIL 2017 • VOL 356 ISSUE 6335 sciencemag.org SCIENCE
Fig. 1. AcrB-GFP accumulates at old cell poles
and undergoes biased partitioning. (A) Schematic representation of a mother machine growth
channel. A mother cell M is captured at the channel
closed end, repeatedly dividing into M and D1
(daughter) cells; a D1 cell divides into daughter
cells D2 and D3. Numbers represent pole ages, and
N represents the increasingly old mother cell pole.
(B and C) Kymographs of wild-type and DtolC strains
expressing AcrB-GFP growing in mother machine.
Snapshots of AcrB-GFP and soluble mCherry expressed within the same cells. (D) (Left) Within-cell
fluorescence asymmetry in M cells expressing AcrB-GFP, quantified as (old cell half – new cell half)/
(whole cell). Data are from 20 mother cells (~34 cell
divisions each), smoothed over three time bins. Dots,
mean generation times; envelope, SD over cells.
(Right) Bar for M shows final asymmetry; bars for
D1/D2/D3 are time averages throughout the entire
experiment. [Left bars, AcrB-GFP; all nonzero at P <
10−4, t test, N = 20 for M (last time point); 691 (D1),
671 (D2), and 655 (D3). Right bars, DtolC; N = 18
for M (last time point); 427 (D1), 383 (D2), and 406
(D3). Error bars, mean ±1 SD (see table S1)]. (E) Partitioning of AcrB-GFP mean cell fluorescence between
M and D1 at cell division [M/D1 different with P <<
10−10 at left and P < 0.002 at right, t tests; data from
(D); N = 691, 425, respectively]. (F) Within-cell asymmetry in M cells during pulse-chase of ParaBAD-acrB-gfp
and soluble ParaBAD-gfp. Gray shading, pulse period.
Dashed line indicates time point when soluble GFP
fluorescence becomes undetectable above background.
Envelope, SD over cells (18 lineages for AcrB-GFP, 17
lineages for GFP).
Fig. 2. Real-time single-cell efflux assays show
differential efflux activity between mother and
daughter cells. (A) Side-by-side comparison of uptake dynamics of dye H33342 in individual M and
D1 cells in strain expressing AcrB-GFP and DtolC
strain (sampled every 90 s). Lines, average dye fluorescence; envelopes, SD over cells. Final saturation
levels, averaged over the last five time points, differ
significantly between M and D1 cells for AcrB-GFP
(P < 10−5, t test, 20 lineages) but not for DtolC (15
lineages). (B) AcrB-GFP and H33342 fluorescence
of individual cells at steady state, averaged over last
five time points, correlate significantly (Pearson correlation, data from two independent experiments;
N = 159 from 66 lineages).