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This work was supported the European Research Council (grant
294678), the Deutsche Forschungsgemeinschaft (grants SFB 665
and Go865/9-1), the NSF (grant 0744979), and the NIH (grants
HL71626 and HL60678). Technical support was provided by
M. Braunschweig, F. Kressin, M. Pippow, and D. J. Visintine. All
relevant data are stored at the Max Delbrück Center for Molecular
Medicine and are available from the authors upon request or are
included in the manuscript and supplementary materials. The
project was conceived and coordinated by G.R.L., T.J.P., J.R., M.G.,
S.K., and N.C.B. Experiments were performed by J.R., B.L.P.,
G.B., D.O., W.H., M.H.R., V.B., R.D.M., B.M.B., J.L., D. T.A., E.St.J.S.,
V.Ga., V.Go., V.G.A., H.L., N.C.B., T.J.P., G.R.L., R.D.M., and E.St.J.S.
Data analysis and bioinformatics were carried out by J.R., P.H.J.L.K.,
C.Z., O.E., T.K., S.K., W.H., M.H.R., and M.G. The paper was written by
G.R.L., T.J.P., and J.R. with input from all authors. Contact S.K. for
correspondence on metabolomics.
Materials and Methods
Figs. S1 to S10
13 June 2016; accepted 1 March 2017
Biased partitioning of the multidrug
efflux pump AcrAB-TolC underlies
long-lived phenotypic heterogeneity
Tobias Bergmiller,1 Anna M. C. Andersson,1 Kathrin Tomasek,1 Enrique Balleza,2
Daniel J. Kiviet,3 Robert Hauschild,1 Gašper Tkačik,1 Călin C. Guet1†
The molecular mechanisms underlying phenotypic variation in isogenic bacterial
populations remain poorly understood. We report that AcrAB-TolC, the main multidrug
efflux pump of Escherichia coli, exhibits a strong partitioning bias for old cell poles by a
segregation mechanism that is mediated by ternary AcrAB-TolC complex formation.
Mother cells inheriting old poles are phenotypically distinct and display increased drug
efflux activity relative to daughters. Consequently, we find systematic and long-lived
growth differences between mother and daughter cells in the presence of subinhibitory
drug concentrations. A simple model for biased partitioning predicts a population
structure of long-lived and highly heterogeneous phenotypes. This straightforward
mechanism of generating sustained growth rate differences at subinhibitory antibiotic
concentrations has implications for understanding the emergence of multidrug resistance
In bacteria, phenotypic heterogeneity arises through stochastic molecular processes in the cell. As a result, isogenic cells can exhibit dis- tinct gene expression levels and phenotypes that fluctuate over time. Contributions of gene
expression noise to phenotypic heterogeneity have
been extensively studied (1), with qualitatively
similar effects expected due to degradation (2)
or random partitioning of cytosolic proteins at
cell division (3). In contrast to cytosolic compo-
nents, outer membrane proteins are diffusion-
restricted due to the formation of supramolecular
islands (4, 5) and thus violate the well-mixedness
assumption. As a consequence, the outer mem-
brane undergoes asymmetric turnover, which
leads to polar localization of at least some outer
membrane proteins (5, 6). In rod-shaped bacteria,
the two sister cells emerging from cell division
can be distinguished by the age of their poles:
One sister cell is characterized by an old cell pole
originating from a division event in the past. Old
poles are considered inert with respect to enve-
lope composition (5, 7), and, as a result, individual
cells—and sister cells in particular—could exhibit
systematic differences in envelope composition.
The bacterial envelope functions as a highly ef-
fective diffusive barrier (8). A key determinant of
envelope permeability to toxic compounds is the
multidrug efflux pump AcrAB-TolC (9, 10), a tri-
partite complex that bridges the entire cell en-
velope and allows transport of harmful chemicals
directly to the outside medium (fig. S1) [see the
supplementary materials (11)]. TolC interacts with
several different excretion and secretion systems (12).
However, AcrAB in complex with TolC is the main
multidrug efflux determinant in Escherichia coli and
enterobacteria (9, 13, 14). Here, we studied pheno-
typic heterogeneity as a result of polar cell enve-
lope localization in E. coli. We monitored the
partitioning dynamics of AcrAB-TolC over many
generations and measured efflux activity and drug
sensitivity of individual cells.
We tracked a chromosomally encoded functional AcrB-GFP (green fluorescent protein) (fig.
S2) as a specific marker of AcrAB-TolC partitioning in single cells growing inside a microfluidic
“mother machine” device (15). Mother cells, which
are the sister cells characterized by an increasingly
old cell pole, remain captured at the closed end of
channels while undergoing cell divisions (Fig. 1A
and fig. S3). Daughter cells are pushed toward the
main trench by cell growth. E. coli cells exhibited
stable and robust growth inside these microfluidic
devices (15) (fig. S4 and table S1).
We found that AcrB-GFP accumulates at the
old pole in mother cells (Fig. 1B, figs. S5 and
S6, and movie S1) in a TolC-dependent manner,
whereas deletion of tolC led to uniformly distributed AcrB-GFP in the inner membrane (Fig. 1C).
As previously reported, deletion of tolC also causes
increased acrAB expression (16). Polar AcrB-GFP
localization was restored by complementing TolC
expression (fig. S7). When comparing the old and
newly formed poles of mothers, we found that
AcrB-GFP localization asymmetry increased slowly with the replicative age of the old cell pole (Fig.
1D). AcrB-GFP accumulated at old cell poles in
general, as is visible in D3 daughters that carry
the next oldest cell pole (Fig. 1D, right panel, and
table S2). AcrB-GFP asymmetry was absent in
DtolC cells (Fig. 1D). Direct comparison of mother
and daughter cells immediately before and after
cell division showed an average of 58% AcrB-GFP
1Institute of Science and Technology Austria, 3400
Klosterneuburg, Austria. 2Faculty of Arts and Sciences,
Center for Systems Biology and Department of Molecular
and Cellular Biology, Harvard University, Cambridge, MA
02138, USA. 3Department of Environmental System Science,
ETH Zurich, Swiss Federal Institute of Technology, 8092
Zurich, Switzerland, and Department of Environmental
Microbiology, Eawag, Swiss Federal Institute of Aquatic
Science and Technology, 8600 Duebendorf, Switzerland.
*These authors contributed equally to this work.
†Corresponding author. Email: email@example.com