An acid-tolerance response system protecting exponentially growing Escherichia coli

Abstract

The ability to grow at moderate acidic conditions (pH 4.0–5.0) is important to Escherichia coli colonization of the host’s intestine. Several regulatory systems are known to control acid resistance in E. coli, enabling the bacteria to survive under acidic conditions without growth. Here, we characterize an acid-tolerance response (ATR) system and its regulatory circuit, required for E. coli exponential growth at pH 4.2. A two-component system CpxRA directly senses acidification through protonation of CpxA periplasmic histidine residues, and upregulates the fabA and fabB genes, leading to increased production of unsaturated fatty acids. Changes in lipid composition decrease membrane fluidity, F0F1-ATPase activity, and improve intracellular pH homeostasis. The ATR system is important for E. coli survival in the mouse intestine and for production of higher level of 3-hydroxypropionate during fermentation. Furthermore, this ATR system appears to be conserved in other Gram-negative bacteria. The ability to grow at acidic pH is crucial for E. coli colonization of the host’s intestine. Here, the authors identify an acid-tolerance response system that is important for E. coli exponential growth at pH 4.2, survival in the mouse intestine, and production of 3-hydroxypropionate during fermentation.

Full text

ARTICLE
An acid-tolerance response system protecting
exponentially growing Escherichia coli
Ying Xu 1,2,8, Zhe Zhao 1,2,8, Wenhua Tong 1,7, Yamei Ding 3, Bin Liu4, Yixin Shi5, Jichao Wang 1,
Shenmei Sun1,2, Min Liu 1, Yuhui Wang4, Qingsheng Qi6, Mo Xian1✉& Guang Zhao 1,6 ✉
The ability to grow at moderate acidic conditions (pH 4.0–5.0) is important to Escherichia coli
colonization of the host’s intestine. Several regulatory systems are known to control acid
resistance in E. coli, enabling the bacteria to survive under acidic conditions without growth.
Here, we characterize an acid-tolerance response (ATR) system and its regulatory circuit,
required for E. coli exponential growth at pH 4.2. A two-component system CpxRA directly
senses acidification through protonation of CpxA periplasmic histidine residues, and upre-
gulates the fabA and fabB genes, leading to increased production of unsaturated fatty acids.
Changes in lipid composition decrease membrane fluidity, F
0
F
1
-ATPase activity, and improve
intracellular pH homeostasis. The ATR system is important for E. coli survival in the mouse
intestine and for production of higher level of 3-hydroxypropionate during fermentation.
Furthermore, this ATR system appears to be conserved in other Gram-negative bacteria.
https://doi.org/10.1038/s41467-020-15350-5 OPEN
1CAS Key Lab of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 266101 Qingdao, China.
2University of Chinese Academy of Sciences, 100049 Beijing, China. 3Institute of Oceanology, Chinese Academy of Sciences, 266071 Qingdao, China.
4TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457 Tianjin, China. 5School of Life Sciences, Arizona State
University, Tempe, AZ 85281, USA. 6State Key Laboratory of Microbial Technology, Shandong University, 266237 Qingdao, China.
7
Present address: Sichuan
University of Science and Engineering, 644000 Yibin, Sichuan, China.
8
These authors contributed equally: Ying Xu, Zhe Zhao. ✉email: xianmo@qibebt.ac.cn;
zhaoguang@qibebt.ac.cn
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Enteric bacteria such as Escherichia coli (E. coli) and Salmo-
nella can colonize and cause disease in the human intestinal
tract. They have to combat acidic environments during the
whole process of invading the host. With pH values as low as
1.5–2.5, the stomach has been recognized as a natural antibiotic
barrier1. With their passage into the small intestine, E. coli cells
will encounter a less acidic environment (pH 4.0–6.0) with the
presence of organic acids produced by the normal intestinal
flora2.
E. coli has developed variable acidic stress response systems,
including the acid resistance (AR) systems response to extreme
acid stress and the acid tolerance response (ATR) system towards
mild and moderate acid stress3,4. Up to now, five AR systems,
AR1−AR5, are reported. The AR1 system is activated by alter-
native σfactor (RpoS) and cAMP receptor protein (CRP)5,6.
Due to the involvement of CRP, the AR1 system is repressed by
glucose. The AR2−AR5 systems are all dependent on a specific
extracellular amino acid, and consist of an antiporter as well as a
decarboxylase enzyme that is usually induced by low pH and
extracellular amino acid3,7, except that AR2 can be induced
at acidic pH in the absence of glutamate8. They confer acid
resistance by consumption of intracellular protons in amino acid
decarboxylation reaction to produce a less acidic internal pH,
using glutamate, arginine, lysine and ornithine as their corre-
sponding substrates, respectively1,5,9–12. All five AR systems can
protect stationary phase cells from the extreme acidity and
prolong survival, while only AR2 and AR3 were reported to
function during the exponential phase5,13.
Among AR systems, AR2 is by far the most effective and the
most complex. The glutamate decarboxylase isoforms, GadA and
GadB, and the glutamate/γ-aminobutyric acid antiporter GadC
are key components of AR2, and their regulation relies on the
action of over 20 proteins and 3 small noncoding RNAs,
including two-component systems EvgAS and PhoPQ; regulatory
proteins RpoS, GadE, RcsB, GadX, GadW and HNS; protease
ClpXP and Lon; and small RNAs DsrA, GadY and GcvB, which
together form a regulatory network with high level of complexity
(for a review, see refs. 3,7). The periplasmic chaperons HdeAB and
their cytoplasmic counterpart Hsp31, which assist the refolding of
denatured proteins during the acid stress7,14,15, are also induced
as part of the AR2 regulon16,17.
The ATR system, though poorly understood, is induced by
exposing E. coli cells to moderate acid stress (pH 4.5–5.8), and
will protect cells from a subsequent challenge of extreme acid pH
(pH 2.0–3.0)4,6. ATR can be activated during adaptation at mild
acidic pH by the regulators Fur and PhoPQ in exponential phase
cells and by RpoS and OmpR in stationary phase cells, but the
stationary phase cells are much more tolerant to acid than the log
phase cells3,4.
Benefited from the complicated AR and ATR systems, E. coli
can survive without growth for several hours at pH 2.01,18–20, and
the acid limit for growth of E. coli is pH 4.0 in rich medium, or
pH 4.5 in minimal medium6,18,20–22. So, E. coli will experience
the transition of pH from no-growth to growth conditions when
passing through the stomach and entering the intestine. It is
exceptionally important to elucidate how E. coli adapts to and
grows at pH 4.0–5.0, because the capability of bacteria to outgrow
hundreds of competing species in gut microbiome in this lower
range of growth pH will determine which strain can colonize the
gut18. Unfortunately, we still barely know that.
In this study, we challenged the exponentially growing cells of
E. coli at pH 4.2, and characterized a regulatory circuit required
for bacterial growth under moderate acidic conditions through
modulation of the membrane lipid composition. The two-
component system CpxRA directly senses acidification through
protonation of the CpxA periplasmic histidine residues, and thus
activates transcription of the essential genes fabA and fabB in
biosynthesis of unsaturated fatty acids (UFAs) to enhance the
UFAs content in membrane lipid. This mechanism enables E. coli
to grow at acidic pH, and also functions in diverse bacterial
species.
Results
UFAs are required for E. coli growth under acidic pH.We
carried out a screening to characterize an E. coli ATR system
required for bacterial survival in exponential growth. At first, the
overnight culture of E. coli BW25113 wild-type strain was directly
inoculated into minimal medium E at pH 4.2 using glucose as sole
carbon source without supplement of any amino acid. However,
the cells were rapidly killed, even preadapted at pH 5.0 (Sup-
plementary Fig. 1). Then the BW25113 strain was grown in
medium E at pH 7.0 to a cell density of ≈3×10
8CFU mL−1, and
the cells were collected, washed and transferred into the same
medium at pH 7.0 and 4.2, respectively. While the cells at pH 7.0
grew normally, the cell density at pH 4.2 decreased continuously,
which led to reduction of the ratio of CFU at pH 4.2 vs. pH 7.0 to
0.21 ± 0.02 after 1 h of exposure at different pH (Fig. 1a). This
result confirmed that exponentially growing E. coli was suscep-
tible to this moderate acidic condition.
In previous studies, survival was used to measure the bacterial
acid resistance, which is calculated by the formula, survival (%) =
(CFU after acid challenge/CFU before acid challenge) × 100%.
However, survival was determined under conditions E. coli can
only survive without growth, and is not suitable here as we are
trying to figure out how E. coli grows at moderate acidic pH. So,
the CFU ratio (pH 4.2/pH 7.0) is calculated to represent the acid
tolerance of exponentially growing E. coli because growth of E.
coli at pH 7.0 is steady. A value of CFU ratio close to 1 indicates
that E. coli grows at pH 4.2 to a cell density similar to that at
pH 7.0.
Phospholipids were extracted from exponential phase cells
after exposure of 1 h to pH 7.0 and 4.2, and analyzed as
previously described23. The results showed that exposure to such
acidic condition caused a change of membrane lipid composition
in E. coli cells (Fig. 1b). Specifically, levels of the UFAs, including
palmitoleic acid (C16:1) and oleic acid (C18:1), were elevated by
3.83- and 1.66-fold; meanwhile, the levels of palmitic acid (C16:0)
and stearic acid (C18:0) were reduced. These five fatty acids
shown in Fig. 1b represented more than 96% of total fatty acids in
E. coli cells, consistent with those results reported previously24,25.
As a consequence, the ratio of unsaturated to saturated fatty acids
increased from 0.11 at pH 7.0 to 0.32 at pH 4.2. Comparable to
this result in E. coli, a shift in the unsaturated/saturated ratio in
response to acid stress was also observed in Streptococcus
mutants26,27.
We also found that the expression of two essential genes
required for UFAs biosynthesis, fabA and fabB (Fig. 1c), was
significantly upregulated under the acidic condition since their
protein and mRNA levels were elevated (Fig. 1d, e). A previous
study showed that overexpression of fabA and fabB increased
UFA contents in E. coli28. Therefore, it is plausible that
overexpression of fabA and fabB may allow E. coli to grow under
pH 4.2. To test this hypothesis, we cloned fabA and fabB into
vector pTrcHis2B, and introduced them into BW25113 wild-type
strain. The strain carrying empty vector presented a CFU ratio of
0.26 ± 0.03, similar with that of BW25113 wild-type strain, while
either strain with fabA or fabB showed much higher acid
tolerance at pH 4.2 (Fig. 1f).
In agreement with this result, strains harboring a temperature-
sensitive mutant of fabA or fabB gene became much more
susceptible to acid at 42 °C, whereas the wild-type strain showed
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similar acid tolerance at different temperatures (Fig. 1g). Taken
together, these results demonstrated that elevating the biosynth-
esis of UFAs enhanced the acid tolerance of exponentially
growing E. coli.
Transcription of fabA and fabB is activated by CpxRA system.
To find out the possible regulator of these loci, we compared the
DNA sequences of fabA and fabB promotor regions, and found a
conserved sequence GTAAA-(5 nt)-GCAAA (Fig. 2a, b), which
was similar to the identified CpxR recognition site29. This raised
the hypothesis that the transcription of fabA and fabB is regulated
by two-component system CpxRA, which consists of a sensor
histidine kinase CpxA and a cytoplasmic response regulator
CpxR. Since overexpression of outer membrane protein NlpE was
identified as a Cpx-specific activation signal30,31, we cloned the
nlpE gene and tested its effect on expression of fabA and fabB
loci. As shown in Fig. 2c, d, excessive NlpE protein significantly
enhanced the expression level of fabA and fabB at pH 7.0, and
deletion of cpxR totally abolished this activating effect of NlpE.
Besides those, NlpE overexpression also raised the UFAs content
in E. coli membrane lipid at pH 7.0 (Fig. 2e) and E. coli acid
tolerance at pH 4.2 (Fig. 2f) in a CpxR-dependent manner since
cpxR knockout mutant displayed higher susceptibility to acidic
challenge than wild-type strain even overexpressing NlpE
(Fig. 2f). These observations collectively demonstrate that the
CpxRA system activates transcription of fabA and fabB.
105
106
107
108
109
1010
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Time (h)
CFU mL–1
CFU ratio
pH 7
pH 4
CFU ratio
C14:0 C16:0 C16:1 C18:0 C18:1
0
20
40
60
80
Fatty acid content (%)
pH 7.0
pH 4.2
p = 0.0037
p = 0.0031
p = 0.0009
p = 0.0013
0
1
2
3
4
Relative amount
of proteins
pH 7.0
pH 4.2
p = 0.0022
p = 0.0314
0 2 4 6 8 10 12 14
FabA FabB fabAfabB
0.0
1.0
2.0
3.0
4.0
Relative mRNA level
pH 7.0
pH 4.2
p = 0.0017
p < 0.0001
pfabA
pfabB
0.0
0.5
1.0
1.5
CFU ratio
p < 0.0001
p = 0.0003
Wt fabBTs
fabATs
0.0
0.2
0.4
0.6
0.8
1.0
CFU ratio
30 °C
42 °C
p = 0.0002
p = 0.0001
ab
cde
fg
FabA
FabB
pH 7.0 4.2
S
ACP
OOH
S
ACP
O
S
ACP
O
O
S
ACP
O
Fatty acids
biosynthesis
Unsaturated fatty acids
FabA
FabA
FabB
25 kDa
55 kDa
40 kDa
Vector
Fig. 1 Improved acid tolerance of exponentially growing E. coli caused by increased production of unsaturated fatty acids. a Growth of E. coli
BW25113 strain at different pH. The strain was grown to 3 × 108CFU mL−1in minimal medium at pH 7.0, and transferred into the same medium at pH 7.0
and pH 4.2 (n=3 biologically independent samples). CFU ratio =CFU at pH 4.2/CFU at pH 7.0. bMembrane lipid composition of BW25113 strain after
acidic challenge at pH 4.2 for 1 h. The compositions were determined by GC-MS, and fatty acid content is given as the relative peak area [(peak area of one
fatty acid/total peak area) × 100%] (n=3 biologically independent samples). cBiosynthetic pathway of unsaturated fatty acids, in which FabA and FabB,
3-hydroxyacyl-ACP dehydratase/isomerase and β-ketoacyl-ACP synthase play essential roles. dFabA-His
6
and FabB-His
6
protein level of BW25113 strain
after 1 h of exposure to pH 7.0 and 4.2 determined by western blot. The relative amount of each protein was determined using ImageJ (n=3 biologically
independent samples). eRelative mRNA level of fabA and fabB in BW25113 strain after 1 h of exposure to pH 7.0 and 4.2 determined by qRT-PCR (n=2
biologically independent samples with three technical repeats). fTolerance of BW25113 strain carrying empty vector pTrcHis2B, pfabA or pfabB,
respectively, after acidic challenge at pH 4.2 for 1 h (n=3 biologically independent samples). gTolerance of BW25113 strain, temperature-sensitive FabA
and FabB mutants after acidic challenge at pH 4.2 at 30 or 42 °C for 0.5h (n=3 biologically independent samples). Error bars, mean ± standard error of
mean (SEM). Two-tailed Student’sttests were performed to determine the statistical significance for two group comparisons. The source data are
provided as a Source Data file.
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Transcription start site of fab genes controlled by CpxRA.We
mapped the transcription starts of fabA and fabB using RACE
(rapid-amplification of cDNA ends) experiment. As was pre-
viously reported, for fabA gene, two transcription initiation sites
were detected under growth at pH 7.0: S1 positively regulated by
FadR32 and S2 negatively regulated by FabR33. Interestingly,
when the cell growth was at pH 4.2, our results indicated that the
fabA gene has a third transcription initiation site located at the
203 bp upstream of the start codon, the S3 (summarized in
Fig. 3a). For fabB gene, the transcription was initiated 37 bp
upstream of the start codon in both conditions (summarized in
Fig. 3b), consistent with previous reports34.
qRT-PCR result showed that the mRNA level of fabA and fabB
was upregulated by NlpE overexpression in a fabR fadR double
mutant strain, indicating that they had no obvious effect on fabA
and fabB gene expression under CpxRA-dependent activation
(Supplementary Fig. 2).
CpxR protein directly binds to the fabA and fabB promoters.
To verify the putative CpxR recognition sites, two mutant strains
were constructed by site-directed mutagenesis, in which the CpxR
binding box on fabA or fabB promoter region was replaced by
CATCT-(5 nt)-CATCT sequence, and expression of fabA and
fabB was determined. Both western blot and qRT-PCR results
FabA
FabB
cpxR pnlpE
Vector
pnlpE
0.0
0.5
1.0
1.5
CFU ratio
Wt
cpxR
p < 0.0001
p < 0.0003
p < 0.0001
0.0
1.0
2.0
3.0
4.0
5.0
Relative mRNA level
Vector
pnlpE
cpxR pnlpE
p < 0.0001
p < 0.0001
p < 0.0001
0
20
40
60
80
Fatty acid content (%)
Vector
pnlpE
cpxR pnlpE
a
cd
ef
1
4631469
2433864
1011408
1011926
1012184
1012198
2435196
2435084
2435182
fabA fabBCB CB
Escherichia coli BW25113
Vector pnlpE
fabA fabB
C14:0 C16:0 C16:1 C18:0 C18:1
FabA FabB
0
1
2
3
Relative amount
of proteins
Vector
pnlpE
cpxR pnlpE
CpxR site
fabA
fabA
–133 –77
–237
–293
fabB
fabB
b
25 kDa
55 kDa
40 kDa
Fig. 2 Transcription of fabA and fabB is activated by two-component system CpxRA. a Schematic diagram showing the genomic locations of fabA and
fabB genes and corresponding CpxR boxes. CB CpxR box. Arrows show the transcription direction of fabA and fabB genes. bAlignment of CpxR binding
sequence and the promoter sequences of fabA and fabB genes. The conserved CpxR binding sequence was highlighted in red. cWestern blot analysis of
FabA-His
6
and FabB-His
6
protein in BW25113 strains carrying empty vector or pnlpE, and cpxR mutant carrying pnlpE grown at pH 7.0. Overexpression of
outer membrane protein NlpE was reported as a specific activating signal of CpxRA system. The relative amount of each protein was determined using
ImageJ from two independent experiments. dRelative mRNA level of fabA and fabB in BW25113 strains carrying empty vector or pnlpE, and cpxR mutant
carrying pnlpE grown at pH 7.0 determined by qRT-PCR (n=2 biologically independent samples with three technical repeats). eMembrane lipid
composition of BW25113 strains carrying empty vector or pnlpE, and cpxR mutant carrying pnlpE grown at pH 7.0 (n=2 biologically independent samples).
The compositions were determined by GC-MS, and fatty acid content is given as the relative peak area [(peak area of one fatty acid/total peak area) ×
100%]. fAcid tolerance of BW25113 strains carrying empty vector or pnlpE, and cpxR mutant carrying vector or pnlpE after acidic challenge at pH 4.2 for 1 h
(n=3 biologically independent samples). Error bars, mean ± SEM. Two-tailed Student’sttests were performed to determine the statistical significance for
two group comparisons. The source data are provided as a Source Data file.
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showed that the activating effect of acid stress on expression of
fabA and fabB was completely eliminated by substitution of CpxR
site (Fig. 3c, d). Moreover, the acid tolerance of these mutants
significantly decreased (Fig. 3e).
The function of CpxR site was further confirmed in vitro
through gel-shift assay and DNase I footprinting analysis using
purified His
6
-CpxR protein and 211- and 219-bp DNA fragments
corresponding to the promoter regions of fabA and fabB,
respectively. The purified His
6
-CpxR protein could shift those
two DNA fragments (Fig. 3f), and protect the fabA promoter at
the −287 to −255 region (numbering from the start codon) and
the fabB promoter at the −119 to −94 region in the noncoding
strand containing the putative CpxR binding site (Fig. 3g). All
these results demonstrate that the CpxR protein enhances
transcription of fabA and fabB by direct binding to their
promoters.
CpxRA is directly activated by acidic environments. As a sensor
histidine kinase in two-component system, CpxA spans the cell
membrane and exposes its sensor domain into the periplasm.
When CpxA detects a specific signal, it autophosphorylates and
then transports the phosphate group to its cognate regulator
CpxR, enabling the regulatory activity of CpxR. Without inducing
signal, CpxA acts as a phosphatase to maintain CpxR in an
inactive state35,36.
To monitor the expression and phosphorylation level of CpxA
protein, we constructed a strain carrying chromosomal His
6
-
tagged CpxA. As shown in Fig. 4a, acid shock significantly
enhanced the amount of CpxA-His
6
protein in exponentially
growing cells. More importantly, increased level of phosphor-
CpxA, the active form of CpxA, was detected at pH 4.2 (Fig. 4a).
To demonstrate direct activation of CpxRA upon exposure to
acidic pH, we used a reconstituted proteoliposome system,
fabA fabB
Free DNA
Bound-DNA
Cold DNA
His6-CpxR
32P-DNA
–322
–232
–142
–52
–173
–83
–– ––++
–+ –+++
++ ++++
fabA fabB
His6-CpxR
AG
His6-CpxR
AG
a
b
c
de
f
g
FabA
FabB
CpxR site
pH 7.0 4.2
Wt CB–Wt CB–
0.0
0.5
1.0
1.5
2.0
Relative amount
of proteins
pH7.0, wt
pH7.0, CB–
pH4.2, wt
pH4.2, CB–
fabA up fabB
0
1
2
3
Relative mRNA level
pH7.0, wt
pH7.0, CB–
pH4.2, wt
pH4.2, CB–
p < 0.0001
p < 0.0001 p = 0.0003
fabB CB
–
fabA CB
–
Wild-type
0.0
0.1
0.2
0.3
0.4
CFU ratio
p = 0.0024
p = 0.0012
–35 –10 CpxR box
FadR box FabR box
–35
–10 S3 CpxR box
–35 –35
–10 S2 –10 S1 FadR box
FabR box
FabA FabB
Start codon
Start codon
25 kDa
55 kDa
40 kDa
200 bp
S
Fig. 3 Identification of CpxR binding sites in the fabA and fabB promoters. a,bDNA sequences of fabA and fabB promoter regions. The FabR box, FadR box
and CpxR box sequences were underlined. Two previously reported transcription start sites of fabA, S1 and S2, and corresponding −35 and −10 regions were
shown in blue and red, respectively. The transcription start site identified in this study and its −35 and −10 regions were shown in green. Numbering is from the
start codon of each gene. cWestern blot analysis of FabA-His
6
and FabB-His
6
in BW25113 wild-type strain at pH 7.0 and 4.2, and strain with CpxR site
substitution at pH 7.0 and 4.2. n=2 biologically independent samples. CB−substituted CpxR box. dRelative mRNA level of fabA and fabB determined by qRT-
PCR in BW25113 wild-type strain at pH 7.0 and 4.2, and strain with CpxR site substitution at pH 7.0 and 4.2 (n=2 biologically independent samples with three
technical repeats). The fabA up primer sequences were shown in purple in (a). eAcid tolerance of BW25113 wild-type strain and mutants with substitution in
fabA or fabB CpxR box after acidic challenge at pH 4.2 for 1 h (n=3 biologically independent samples). fGel-shift assay. 32P-labeled DNA fragments containing
fabA and fabB promoter regions were incubated without and with His
6
-CpxR protein, shown in lanes 1–2. Lane 3 is the same as lane 2 but supplemented with
“cold”DNA fragments. gDNase I footprinting analysis of fabA and fabB promoters with probes for the noncoding strand and increasing amount of His6-CpxR
protein. His
6
-CpxR-DNA mixture were subjected to 5% polyacrylamide electrophoresis, and visualized by autoradiography. The regions protected by CpxR
protein were shown in orange in (a,b). AG, DNA sequence ladder generated with the same primers using a Maxam and Gilbert A +G reaction. fand gare
representative results from two independent experiments. Error bars, mean ± SEM. Two-tailed Student’sttests were performed to determine the statistical
significance for two group comparisons. The source data are provided as a Source Data file.
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where a different internal and external pH can be stably
maintained (Fig. 4b). The CpxA-His
6
protein was purified from
membranes and reconstituted into vesicles mainly in the inside-
out orientation using the detergent-mediated method as
described37.AsshowninFig.4c, lowering the pH inside
vesicles from 7.0 to 4.2 greatly enhanced the amount of
phospho-CpxA. The phosphoryl group was transferred to
regulator CpxR when the pH inside vesicles was 4.2, while
phosphorylated CpxR could be hardly detected with neutral
lumen pH (Fig. 4c) or without the sensor kinase CpxA. These
CpxA CpxA-Pi
0
5
10
15
Relative amount
of proteins
pH 7.0
pH 5.2
pH 4.2
p = 0.0120
p = 0.0310
p = 0.0036
p = 0.0040
Total CpxA
pH 7.0 4.2
CpxA-Pi
CpxA
5.2
pH 7.0 4.2
CpxA-Pi
CpxA
5.2
CpxR-Pi
CpxR
a c
fg
CpxA-Pi CpxR-Pi
0
5
10
15
20
25
Relative amount
of proteins
pH 7.0
pH 5.2
pH 4.2
FabA
CpxA
Wt
E37A
E42A
E50A
H52A
Wt
E54A
E56A
E91A
E98A
E127A
E101A
E138A
H117A
0.0
0.5
1.0
1.5
Relative amount
of proteins
FabA
CpxA
pH 7.0 4.2
0
1
2
relative amount
ofproteins
Wt
Wt
H52A
H52K
H52R
H117A
H117K
H117R
b
de
119
43 193
216
486
PhoQ
119
43 180
CpxR
ADP
ATP
CpxA
pH 4.2
pH 7.5
P
P
203
473
PhoQ
CpxA sensor domain
PhoP PhoP
PhoP
P
P
Promoter PhoPQ regulated gene
pH 7.0 7.04.2 4.2
pnlpE –+–+
Wild-type PhoQ
PhoQ-CpxA fusion
SlyB
7.0 7.04.2 4.2
–+–+
RstA
0
1
2
Relative amount
of proteins
pH 7.0 vector
pH 4.2 vector
pH 7.0 pnlpE
pH 4.2 pnlpE
SlyB RstA SlyB RstA
Wild-type PhoQ PhoQ-CpxA
25 kDa
25 kDa
25 kDa
25 kDa
15 kDa
15 kDa
55 kDa
70 kDa
55 kDa
70 kDa
55 kDa
35 kDa
25 kDa
Fig. 4 The CpxRA system is activated by protonation of CpxA periplasmic histidine residues. a Western blot of CpxA-His
6
protein in BW25113 strain
after acidic challenge. For total CpxA protein, the same amount of total cellular proteins extracted from cells grown at different pH was applied to SDS-
PAGE. For analysis of phosphorylated CpxA protein, the same amount of CpxA-His
6
protein purified from cells grown at different pH was separated using
SDS-PAGE containing Mn2+and Phos-tag acrylamide, which retard the mobility of phosphoproteins. n=3 biologically independent samples. bSchematic
diagram of the proteoliposome system. cIn vitro analysis of CpxA and CpxR phosphorylation induced by acidic pH, carried out using reconstituted
proteoliposomes, which were preloaded with buffers at different pH and purified CpxA-His
6
protein. To analyze phosphotransfer, purified His
6
-CpxR was
incubated with CpxA-His
6
-containing proteoliposome in phosphorylation buffer. n=2 biologically independent samples. dDesign and construction of a
PhoQ-CpxA fusion protein. PhoPQ is a known two-component system which can sense the acidic pH, and activate transcription of rstA and slyB genes. We
replaced the sensor domain of PhoQ (amino acids 43–193) with the periplasmic domain of CpxA (amino acids 28–164). eWestern blot of RstA and SlyB
proteins in BW25113 with wild-type PhoQ or PhoQ-CpxA fusion at pH 7.0, at pH 4.2, with NlpE overexpression, and at pH 4.2 with NlpE overexpression.
n=2 biologically independent samples. fWestern blot of FabA-His
6
protein in BW25113 strains carrying wild-type CpxA or CpxA mutants in which the
periplasmic histidine/glutamate was replaced with alanine at pH 4.2. n=2 biologically independent samples. gWestern blot of FabA-His
6
protein in
BW25113 strains carrying wild-type CpxA or CpxA mutants in which the histidine at positions 52 and 117 was replaced by alanine, lysine, and arginine,
respectively. n=2 biologically independent samples. Error bars, mean ± SEM. Two-tailed Student’sttests were performed to determine the statistical
significance for two group comparisons. The source data are provided as a Source Data file.
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results suggested that CpxA is capable of activating CpxR upon
direct exposure to acidic environments.
To further verify the sensitivity of CpxA periplasmic domain to
acidification, a chromosomal PhoQ-CpxA fusion strain was
constructed, in which the sensor domain of PhoQ (amino acids
43–193) was replaced by the periplasmic domain of CpxA (amino
acids 28–164) (Fig. 4d). PhoPQ is a known two-component
system that can sense the acidic pH, Mg2+depletion and
antimicrobial peptides, and activate transcription of genes
including rstA and slyB38,39. Western blot results showed that
both low pH and overexpression of NlpE promoted expression of
RstA and SlyB in strain carrying the PhoQ-CpxA fusion protein,
while only acidic environments increased the protein level of
RstA and SlyB in wild-type strain (Fig. 4e). On the other hand,
when the CpxA periplasmic domain was replaced with that from
another kinase AtoS which senses the presence of acetoacetate40,
neither could the protein level of FabA-His
6
and FabB-His
6
be enhanced by acidic conditions in vivo, nor could the CpxR
protein be phosphorylated at pH 4.2 in reconstituted proteolipo-
some (Supplementary Fig. 3), indicating that CpxA periplasmic
domain is sensitive to acidification. All these results collectively
demonstrated that CpxRA system is activated by direct exposure
of CpxA periplasmic domain to acidic environments.
CpxA H52 and H117 are required for sensing of acidic pH.As
reported, the histidine and glutamic acid residues were regarded
as sensors detecting mild acidic pH because they can change their
protonation state upon variation in the surrounding pH3,41–43.
So, we hypothesized that the histidine and glutamate residues in
CpxA sensor domain might be required for pH sensing. To test
this hypothesis, we constructed a series of plasmids encoding
CpxA mutants in which the periplasmic histidine/glutamate
residue was replaced with alanine, respectively. Strains expressing
the mutant CpxA proteins could express fabA in response to
NlpE overexpression normally (Supplementary Fig. 4), indicating
that mutations in residues of the periplasmic domain of CpxA
protein do not impair the kinase activity of CpxA cytoplasmic
domain. As shown in Fig. 4f, only two mutants (H52A and
H117A) resulted in the loss of CpxA capability to upregulate fabA
expression at pH 4.2, while the other mutations in glutamate
residues did not change the expression level of fabA. To confirm
that protonation of H52 and H117 is responsible for pH-
mediated gene regulation, we also mutated these two residues to
other basic amino acids, lysine and arginine, respectively. It was
discovered that the substitution of H52 and H117 by lysine and
arginine had no effect on the response to acidic pH (Fig. 4g).
Moreover, the strain carrying CpxA variant with a basic amino
acid residue at positions 52 or 117 presented a higher level of
FabA protein even at pH 7.0, when compared with strain with
CpxA H52A or H117A mutant (Supplementary Fig. 5), probably
due to the protonation of basic amino acid residue at pH 7.0.
These results demonstrate that protonation of residues at posi-
tions 52 and 117 is required to maintain the response of CpxA to
acidic pH, but the presence of a histidine at those positions is not
a specific requirement for activation.
Physiological effects of changes in lipid composition. To learn
how changed content in membrane lipid affect E. coli cell func-
tions and physiology, several assays were carried out using cells
carrying empty vector as control, pfabA,orpfabB, after acidic
challenge at pH 4.2. The membrane fluidity was determined by
analyzing the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hex-
atriene (DPH), which is negatively correlated to fluidity44.As
shown in Fig. 5a, the strains with overexpression of fabA or fabB
presented much less cell membrane fluidity than strain carrying
empty vector. The membrane permeability was measured by
detecting the leakage of OD
260
materials (predominantly
nucleotides), and there was no significant difference observed
despite the varied UFA contents in membrane (Supplementary
Fig. 6). F
0
F
1
-ATPase spans the cell membrane and transports
periplasmic protons to cytoplasm with production of ATP, and
lipids are required for its optimal functioning45. The activity of
F
0
F
1
-ATPase was measured, and results suggested that the
enhancement of UFAs content repressed its activity (Fig. 5b).
Then the intracellular pH was monitored using ratiometric pH-
sensitive GFP pHluorin246. When growing under pH 5.0–9.0, E.
coli can maintain the intracellular pH between pH 7.4 and
7.947,48. Upon exposure to pH 4.2, the internal pH of control
strain decreased to 6.82 ± 0.06, and the homeostasis of pH in E.
coli cells with overexpression of fabA or fabB was good (Fig. 5c),
probably due to the lowered membrane fluidity and F
0
F
1
-ATPase
activity.
As E. coli has to combat a moderate acidic environment
(pH 4.0–6.0) and proliferate in host’s small intestine, we carried
out mouse gastrointestinal passage experiment to test the
in vivo effect of the UFAs-CpxRA system. BALB/c mice were
administered BW25113 wild-type strain or mutant with
substituted fabA CpxR binding site, and three independent
trials with six mice in each trial were performed for each strain.
After 24 h, fecal samples were collected to test the presence of
each strain. Out of 18 inoculated mice, the wild-type
BW25113 strain was recovered from a total of 8 mice, while
the fabA CpxR box mutant was detected in only 2 fecal samples
(Fig. 5d). The results between the parental and mutant strains
were statistically different (p< 0.05), demonstrating that this
ATR system significantly promotes E. coli survival in mouse
intestinal lumens.
In bio-production of organic acids, product accumulation acidifies
the fermentation broth and inhibits the growth of producing strain.
Previously, we had constructed a 3-hydroxypropionate (3HP)-
producing E. coli recombinant strain. In shaking flask cultivation,
the pH of culture decreased to pH 5.2–5.5 along with the production
of 3HP, repressing the further production and cell growth (Fig. 5e).
So, the pH of fermentation broth had to be adjusted to 7.0
periodically. Then fabA gene was overexpressed to enhance E. coli
tolerance to acidic environments, leading to similar 3HP production
andcellgrowthwithandwithoutpHadjustment(Fig.5e). This
result demonstrates the great prospect of UFAs-CpxRA-dependent
ATR system in organic acids bio-production.
The UFAs-CpxRA system functions in diverse bacteria species.
Salmonella Typhimurium LT2 and Shigella flexneri 2a str. 2457T
are both common enteric pathogens, and their FabA proteins
share more than 98% identity with E. coli FabA (Supplementary
Fig. 7). To test whether UFAs are required for growth under
acidic pH in these bacteria, the empty vector and recombinant
plasmid carrying E. coli fabA gene were transformed into Sal-
monella Typhimurium LT2 and S. flexneri 2a str. 2457T. In acidic
challenge tested at pH 5.0, the transformation of empty vector did
not affect the acid tolerance of those two strains, but the over-
expression of fabA gene elevated those CFU ratios by 2.32- and
1.99-fold, respectively (Fig. 6a). Moreover, mRNA level of fabA
and fabB was upregulated by acid challenge in Salmonella,
Klebsiella and Pseudomonas strains (Fig. 6b), and the consensus
sequence of CpxR site was also found upstream of the fabA and
fabB genes in additional bacteria species (Table 1). Additionally,
Cronobacter sakazakii clone carrying transposon insertion in
cpxR gene was identified as acid-sensitive mutant49. All these facts
suggest this ATR system functioning in exponential phase is
highly conserved across bacteria species.
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Discussion
According to the results shown above, we present a previously
uncharacterized ATR system as well as its regulatory pathway
in exponentially growing E. coli. We demonstrated that the
two-component system CpxRA can directly sense the acidic
environments, and activate transcription of UFAs synthetic genes,
resulting in increased UFAs contents in cell membrane lipid and
normal growth of E. coli at pH 4.2. Our findings greatly enriched
our understanding of the networks contributing to bacterial acid
resistance, and the mechanisms for stress response governed by
the CpxRA system.
As reported, the acid limit for E. coli growth is pH 4.0–4.5, and
all known AR and ATR systems only prolong survival of E. coli
cells under acidic conditions, but cannot support growth at pH
4.0–4.518,21,22. In our study, overexpression of fabA and fabB
genes for UFAs biosynthesis, or activation of the two-component
regulatory system CpxRA both restore the growth capability of
E. coli at pH 4.2. Compared with those previously known AR
systems, this growth-conferring ATR system is expected to have
more important physiological significance. Firstly, bacteria
can grow normally under acidic pH with activation of the
CpxRA- and UFAs-dependent system, achieving higher biomass,
which may be required for successful pathogenesis and efficient
bio-production. Secondly, de novo mutations during DNA
duplication play a critical role in bacterial stress resistance
development50, and the rapid proliferation at low pH provides an
opportunity for the evolution of novel AR and ATR systems.
Thirdly, functioning of previously known AR systems is subject to
more external limitations, such as AR1 is repressed by glucose
and AR2−AR5 are dependent on exogenous amino acids,
whereas the UFAs-CpxRA system can be activated by acidic pH
alone because CpxA is capable of phosphorylating CpxR upon
exposure to pH 4.2 in a reconstituted proteoliposome system.
ShigellaSalmonella
0.0
0.1
0.2
0.3
0.4
0.5
CFU ratio
Wild-type
Vector
pfabA
p = 0.0004
p = 0.0008
0.0
Klebsiella
Salmonella
Pseudomonas
2.0
4.0
6.0
Relative mRNA level
pH 5.0/pH 7.0
fabA
fabB
ab
Fig. 6 The exponential phase ATR system based on UFAs and CpxRA is
conserved in bacteria. a Acid tolerance of Salmonella Typhimurium LT2 and
Shigella flexneri 2a str. 2457T wild-type strain and strains carrying empty
vector or pfabA after acidic challenge at pH 5.0 for 1 h (n=3 biologically
independent samples). bRelative mRNA level of fabA and fabB in Salmonella
Typhimurium LT2, Klebsiella pneumoniae ATCC25955 and Pseudomonas
aeruginosa PAO1 grown at pH 7.0 and pH 5.0 (n=2 biologically
independent samples with three technical repeats). Error bars, mean ± SEM.
Two-tailed Student’sttests were performed to determine the statistical
significance for two group comparisons. The source data are provided as a
Source Data file.
Wild-type
fabA CB–
0
1
2
3
4
No. of mice with BW25113
in fecal samples
p = 0.0132
0.0
0.1
0.2
0.3
DPH polarization
p =
0.0048
p =
0.0213
0.0
2.0
4.0
6.0
8.0
Pi (nmol)
p = 0.0016
p = 0.0005
Vector
pfabB
6.5
7.0
7.5
8.0
Intracellular pH
p = 0.0082
p = 0.0012
3HP (g L
–1
)OD
600
pH
0.0
1.0
2.0
5.0
6.0
7.0
8.0
9.0
3HP (g L–1), OD600, pH
Vector w/o pH adjustment
pfabA w pH adjustment
Vector w pH adjustment
pfabA w/o pH adjustment
p = 0.0006
p = 0.0099 p = 0.0015 p = 0.0013
ab c
de
pfabA
Vector
pfabB
pfabA
Vector
pfabB
pfabA
Fig. 5 The UFAs content affects membrane properties, E. coli survival in host’s intestine and production of organic acid. The membrane fluidity (a),
F
0
F
1
-ATPase activity (b), and intracellular pH (c) of BW25113 strains carrying empty vector pTrcHis2B, pfabA or pfabB, after acidic challenge at pH 4.2 for
1 h. The membrane fluidity is determined by the anisotropy of fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). F
0
F
1
-ATPase activity is assayed in
terms of the release of inorganic phosphate using permeabilized cells in ATP-containing buffer. The intracellular pH was monitored using ratiometric pH-
sensitive GFP pHluorin2. dRecovery of E. coli BW25113 wild-type strain and mutant with substitution in fabA CpxR site from feces following oral
administration to BALB/c mice. Three independent trials with six mice in each trail were performed for each strain. e3-Hydroxypropionate production,
OD
600
, and final pH of fermentation broth of recombinant strains with and without fabA overexpression. For experiments with pH adjustment, the medium
was adjusted to pH 7.0 every 12 h. Data are presented as mean ± SEM of three independent experiments. Two-tailed Student’sttests were performed to
determine the statistical significance for two group comparisons. The source data are provided as a Source Data file.
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We proved that the transcription of fabA stimulated by CpxRA
is required for multiplication of E. coli in mouse intestine, indi-
cating that UFAs-CpxRA system is also related to pathogenesis of
E. coli pathovars, as well as those previously known AR systems.
While various pathotypes of E. coli colonize and infect different
organs, they all have to combat acidic environments during
invading the host’s digestive tract. With pH values as low as
1.5–2.5, the stomach has been recognized as a natural antibiotic
barrier1. Benefited from AR1−AR5 systems, E. coli can survive in
the gastric acid for hours22,51. With their passage into the small
intestine, E. coli cells will encounter a less acidic environment (pH
4.0–6.0) with the presence of organic acids produced by the
normal intestinal flora2. As pathogenic E. coli strains must
reproduce rapidly to cause disease ultimately52, the UFAs-CpxRA
system is likely to play a key role. In summary, the successful
enteric pathogen must possess two abilities, survival in extreme
acidic condition and quick growth in moderate acidic environ-
ment. Consequently, this ATR system may be a new target for the
development of antimicrobials.
UFAs-CpxRA-dependent ATR system also has potential
application in bio-production of organic acids, which are valuable
platform chemicals and have been successfully produced by
recombinant E. coli strains53,54. However organic acids cause
acidification of fermentation broth and inhibit E. coli growth at
concentrations far below what is required for economical pro-
duction. Now large quantity of base titrant are required to raise
pH of the media in organic acids production process, and large
amounts of acid must be consumed to recover the organic acids
in the protonated form after production. If we could construct
acid-tolerant strains growing at a pH less than the pKa of the
produced acid, the additional consumption of acid and base
titrants will be circumvented and the overall production cost will
be lowered remarkably. As the UFAs-CpxRA system functions in
exponential phase, is not repressed by glucose (the carbon source
in most fermentation), and does not need exogenous amino acids,
it is believed that this ATR system could be effectively applied in
the field of organic acids bio-production.
Our data suggested that UFAs played an important role in
protection of exponential phase E. coli from acid shock. The
increase of UFAs content in membrane lipid not only affected
the fluidity of lipid bilayer but also changed the activity of the
F
0
F
1
-ATPase, conducing to reduced membrane proton perme-
ability and improved internal pH homeostasis. This phenomenon
is consistent with that S. mutants cells grown at pH 5.0 had
higher UFAs composition and lower proton permeability than
those grown at pH 7.027,55. Moreover, changes in fatty acid
composition probably also affect the PTS system and enzyme
secretion56,57. Overall, changing membrane fatty acid composi-
tion may improve the bacterial ability to adapt to acidic envir-
onment and be an important factor in bacterial acid response. In
this study, the cyclopropane fatty acid (CFA) could not be
detected in exponential phase E. coli cells, although it was
regarded as a major factor in acid resistance of stationary phase E.
coli24,58. However, UFA can be converted into CFA by CFA
synthase59, and increased UFAs content will potentially enable
the synthesis of CFA in stationary phase.
We demonstrate that the E. coli kinase CpxA is a direct sensor
for acidic pH. Our data provide strong evidence that a decrease in
pH protonates the histidine residues at positions 52 and 117 in
CpxA periplasmic domain, leads to events catalyzed by its cyto-
plasmic domain, including phosphorylation of CpxA, transfer of
phosphoryl group to regulator CpxR, as well as activation of
CpxR-dependent gene transcription. As titratable by pH, histidine
has been regarded as sensor detecting mild acidic pH, and also
plays an essential role in the activation of sensor kinase PhoQ by
acidic pH60. But the molecular details of how histidine senses the
low pH signal in PhoQ and CpxA are different. Protonation of
residues at positions 52 and 117 is effective to activate CpxA at
acidic pH, whereas the imidazole ring of histidine is important in
maintaining the response of PhoQ to acidity60.
Our results evidenced that CpxRA is a key system in the acid
stress response of exponentially growing E. coli (Fig. 7), consistent
with a previous proteomic analysis highlighting the importance of
CpxRA in acid stress61. Upon exposure to acidic environments,
CpxRA system stimulates the transcription of UFAs synthetic
genes, resulting in improved intracellular pH homeostasis. Addi-
tionally, CpxRA upregulates some genes involved in cell wall
modification, including peptidoglycan (PG) cross-linking proteins
YcfS, YcbB and DacC; PG cleaving proteins AmiA, AmiC and
Slt61–63. The induction of those genes led to an increase of cross-
linking between PG and outer membrane proteins, and an increase
of cell wall stability, which may help protecting E. coli cells from
acidic challenge. That proteomic study also indicated the repression
of AR2 system by CpxRA61. Because AR2 system is responsible for
survival below pH 3.0 and log phase cells with overexpression of
AR2 genes are not more acid resistant4, the CpxRA-mediated
repression of AR2 in exponentially growing cells above pH 3.0 will
guarantee that AR2 system is not induced in an inappropriate
situation to avoid the metabolic burden. Furthermore, the sensor
kinase CpxA was proved to cross-talk with noncognate response
regulator OmpR64, which itself is involved in the acid stress
response of E. coli65,66.AllofthesedataindicatethatE. coli has
Table 1 Putative CpxR box of fabA and fabB genes from Gram−bacteria (The genome sequences of various bacteria used in this
table are under the accession numbers CP009273,NC_003197,NC_002516,NC_011283,NC_013716,CP001235,NC_004741,
CP001589, and NC_009778, respectively).
Bacteria species fabA gene fabB gene
SequenceaPositionbSequenceaPositionb
E. coli BW25113 GTAGAagaagGCAAA (8) −272 to −258 GTAAGgctgcGCAAA (8) −112 to −98
Salmonella typhimurium LT2 GTAAAcagggcGTGAT (8) −343 to −328 GCAAAtttccGTACT (8) −103 to −89
Pseudomonas aeruginosa PAO1 GGAATtgcaacGCATA (6) −279 to −264 GTCGAtggccGCGAA (6) −83 to −69
Klebsiella pneumoniae 342 GTAAAcagggcGTCAT (8) −341 to −326 GCAAAtttatACACA (6) −102 to −88
Citrobacter rodentium ICC168 GTAAGcaagggGTAAT (8) −343 to −328 GCAAAtttcgGCAGA (7) −102 to −88
Vibrio cholerae O395 ATAAAtcagtaGTAGA (8) −282 to −267 GAAATcaggctGAATA (6) −110 to −95
Shigella flexneri 2a str. 2457 T GTAGAagaagGCAAA (8) −272 to −258 GTAAGgctgcGCAAA (8) −112 to −98
Yersinia pestis D182038 GTACAgaaagGTCAA (8) −269 to −255 GTAAAattagtGGAAG (8) −25 to −10
Cronobacter sakazakii ATCC BAA-894 GCAAAcagggcGTGAT (8) −340 to −325 GCAAAtttcttGCCAA (7) −101 to −86
aNumber of bases same with the CpxR site consensus was presented in the parenthesis.
bNumbering is from the start codon of fabA or fabB gene.
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evolved multiple acid response mechanisms as well as precise reg-
ulatory circuits, to target specific stress conditions.
In many Gram-negative pathogens, the CpxRA system plays an
important role in the regulation of virulence factors, including
pilus, secreted virulence effectors and type III secretion system36.
For CpxRA to upregulate expression of these virulence genes,
there must be an activating signal for CpxA under in vivo con-
ditions. The identity of this signal still remains a topic of much
debate. According to the results in this study, we propose that
moderate acidic pH (such as the intestine and macrophage) is the
activating signal of CpxA in vivo. As the previous study showed
that CpxRA also contributed to bacterial resistance to anti-
microbial peptides, a component of the host antimicrobial
response62, acidic pH and antimicrobial peptide may have
synergistic effect on CpxRA activation. Further study is ongoing
to address this hypothesis.
Methods
Bacterial strains and growth conditions. All strains and plasmids used in this
study are listed in Supplementary Data 1, and all primers used are listed in Sup-
plementary Data 2. Phage P1 was used for generalized transductions in E. coli.
Bacteria were grown at 37 °C in Luria-Bertani broth (Oxoid) or in E minimal
medium (0.8 mM MgSO
4
, 10 mM citric acid, 57.5 mM K
2
HPO
4
, 16.7 mM
NaNH
3
HPO
4
, 0.5% glucose). When necessary, antibiotics were added at final
concentrations of 100 μgmL
−1for ampicillin, 20 μgmL
−1for chloramphenicol or
50 μgmL
−1for kanamycin. E. coli DH5αwas used as host for the preparation of
plasmid DNA, and E. coli χ7213 was used for preparation of suicide vectors.
Diaminopimelic acid (50 μgmL
−1) was used for the growth of χ7213 strain. LB
agar containing 10% sucrose was used for sacB gene-based counter selection in
allelic exchange experiments. The software ImageJ (version 1.52a) was used to
analyze western blot results, and the online version of Clustal Omega (https://www.
ebi.ac.uk/Tools/msa/clustalo) was used for sequence alignment.
Plasmids were constructed by digesting PCR fragments containing target gene
and cloning into corresponding vectors as normal. Derivatives of pTrc-cpxA with
nucleotide substitutions were constructed using Q5 Site-Directed Mutagenesis Kit
(New England Biolabs) according to the manufacturer’s specifications. All plasmids
were confirmed by DNA sequencing. Strains harboring chromosomal epitope-
tagged proteins were generated using λRed recombinase system67,68. DNA
fragments encoding PhoQ-CpxA fusion or carrying substituted CpxR site were
generated by joint PCR using primers shown in Supplementary Data 2, and cloned
into the suicide vector pRE11269. The resulting plasmids were used to mediate the
allelic exchange to generate strains with chromosomal PhoQ-CpxA fusion and
CpxR site mutation.
In acidic challenge experiments, the strains were grown in E medium (pH 7.0)
to an OD
600
of 0.6. For strains with plasmid, IPTG was added to final
concentration of 0.5 mM at an OD
600
of 0.4, and the strain was further grown to an
OD
600
of 0.6. Then, the cells were harvested and washed twice with fresh E medium
(pH 7.0), and inoculated into E medium with various pH as indicated, and strains
were grown for another 30–60 min before the cells were collected to determine the
CFU, membrane lipid composition, mRNA and protein levels.
Determination of fatty acid compositions. Phospholipids were extracted as
described by Wang and Cronan23. Briefly, E. coli cells were harvested and resus-
pended in 1 mL sterile water, and 5 mL of chloroform-methanol (2:1 vol/vol) was
added and vortexed for 3 min then stewing overnight. The solution was centrifuged
and the upper phase was removed, an equal volume of 2 M KCl was added, fol-
lowed by mixing and centrifugation. The top KCl phase was removed, an equal
volume of water was added, followed by mixing and centrifugation. The resulting
bottom organic phase was dried under a stream of nitrogen. Two milliliters of
methyesterification reagent (BF
3
:CH
3
OH =1:4) was added and incubated at 60 °C
for 30 min. The resulting solution was extracted with n-hexane for three times and
then detected with Agilent GC-MS (7890A–5975C; Column: Agilent-HP-INNO-
Wax). The fatty acids were identified by comparing the retention times and mass
fragmentation patterns with authentic standards. Content of each fatty acid is given
as the relative peak area [(peak area of one fatty acid/total peak area) × 100%].
Immunoblot analysis of His
6
-tagged proteins. The E. coli cells were disrupted by
sonication, the cell lysates were centrifuged, and the supernatants were used for
western blot. Protein concentration was determined using BCA protein assay kit
(Pierce), and the same amount of protein sample was separated in 12% SDS-PAGE,
transferred to nitrocellulose membranes (Bio-Rad), and incubated with mono-
clonal HRP-conjugated anti-6×His antibodies (1:10,000 diluted) (Abcam). Protein
signals were detected using Immobilon Western HRP substrate (Millipore) and X-
ray film. To separate the phosphorylated proteins, 20–50 μM Phos-tag Acrylamide
(WAKO) and 0.1 mM Mn2+were added into the SDS-PAGE. Quantification was
conducted using ImageJ software (NIH).
Quantitative RT-PCR and RACE. Total RNA was isolated from bacterial culture
using EASYSpin Plus bacterial RNA quick extract kit (Aidlab Biotechnologies,
China) according to the manufacturer’s instructions. RNA concentration was
determined by spectrophotometry at 260 nm. Removal of genomic DNA and
synthesis of cDNA were carried out using PrimeScript RT reagent Kit with gDNA
Eraser (Takara). qRT-PCR was conducted using TB Green Premix Ex Taq (Takara)
with the QuantStudio 1 system (Applied Biosystems). Constitutively transcribed
gene rpoD was used as a reference control to normalize total RNA quantity of
different samples. The relative difference of mRNA level was calculated using the
ΔΔCt method70. Two independent biological samples with three technical repeats
for each sample were performed for each qRT-PCR analysis.
The RACE experiment was performed using SMARTer RACE cDNA
Amplification Kit (Clontech), according to the manufacturer’s instructions. The
primers 1128 +1129 and 1130 +1131 were used to determine the transcription
start sites of fabA and fabB under the NlpE overexpression conditions, respectively.
Purification of His
6
-CpxR and CpxA-His
6
. Purification of His
6
-CpxR and CpxA-
His
6
was conducted according to Fleischer et al. 37. Briefly, E. coli strain BW25113
with pTrc-cpxR or pTrc-cpxA was grown at 37 °C with aeration in LB medium.
Gene expression was induced with 0.5 mM IPTG for 3–4 h. Membrane fractions
and cytosolic fraction were separated by ultracentrifugation37. His
6
-CpxR was
solubilized in cytosolic fraction and purified by Ni-affinity chromatography.
Membrane proteins were solubilized with 1% dodecyl-β-D-maltoside (DM), and
then CpxA-His
6
was also purified by Ni-affinity chromatography.
Preparation of proteoliposomes. Proteoliposome was reconstructed as previously
described with small modification37. Briefly, E. coli phospholipids (Avanti) were
dried under a stream of nitrogen, and slowly dissolved in sodium citrate-
hydrochloric acid buffer (pH 7.0, 5.0, 4.2) with 10% glycerol (vol/vol) and 0.47%
Triton X-100 (vol/vol), respectively. Purified CpxA-His
6
was added to the mixture
and stirred at room temperature for 20 min. Bio-Beads SM-2 (Bio-Rad) were added
in a bead/detergent ratio of 10:1(w/w), and the mixture was gently stirred at 4 °C
overnight. After 16 h, fresh Bio-Bead s were added, and the mixture was stirred for
another 6 h. The proteoliposomes were collected by ultracentrifugation. To test
autophosphorylation, proteoliposomes were incubated with 300 μmol ATP in
phosphorylation buffer (50 mM Tris-HCl, pH 7.5, 10% glycerol (vol/vol), 2 mM
dithiothreitol (DTT), 50 mM KCl, 5 mM MgCl
2
) at room temperature for 30 min.
5× SDS sample buffer was loaded to termination reaction. To analyze
CpxA
PADP
ATP
PCpxR
pH 4.2
52 117
+
+
ycfS, ycbB, dacC
amiA, amiC, slt
gadA, gadC
hdeABD
Repression of
AR2 system
Increased cell
wall stability
fabA, fabB
Increased internal
pH homeostasis
OmpR
P
Fig. 7 Model illustrating the CpxRA-dependent gene regulation involved
in exponential phase ATR system. In exponentially growing E. coli, CpxRA
system is activated by moderate acidic pH through protonation of
periplasmic histidine residues, and upregulates the transcription of UFAs
biosynthesis genes fabA and fabB, and cell wall modification genes including
ycfS,ycbB,dacC,slt,amiA and amiC, and represses the expression of
AR2 system genes. The sensor kinase CpxA also interacts with noncognate
response regulator OmpR, which itself plays an important role in E. coli acid
resistance.
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phosphortransfer, purified His
6
-CpxR was added to this mixture and incubated at
room temperature for 20 min. Then samples were ultracentrifugated and the upper
phase was collected. 5× SDS sample buffer was loaded to stop the reaction. To detect
the phosphorylation level of CpxA and CpxR, all the samples were subjected to 8%
SDS-PAGE with 20–50 μM Phos-tag Acrylamide (WAKO) and 0.1 mM Mn2+,
which can retard the mobility of phosphoproteins to show the phosphorylated and
nonphosphorylated forms in two separated bands.
Electrophoretic mobility shift assay (EMSA). Primers 683 and 685 were labeled
using T4 polynucleotide kinase (New England Biolabs) and γ-32P ATP (Perki-
nElmer Life Sciences). The promoter regions of fabA and fabB genes were
amplified with primers 682 +683 and 684 +685, respectively. Ten nmol of 32P-
labeled DNA was incubated at room temperature for 30 min with 0 or 50 pmol of
His
6
-CpxR protein in 20 μL of an EMSA buffer consisting of 10 mM Tris-HCl, pH
7.5, 1 mM ethylene diamine tetraacetic acid, 5 mM DTT, 10 mM NaCl, 1 mM
MgCl
2
, and 5% glycerol. The mixture was subjected directly to 4% TAE-PAGE.
Signals were detected by autoradiography.
DNase I footprinting assay. DNase I footprinting assays were carried out using
the fabA promoter region amplified from BW25113 chromosome with primers 682
and 32P-683 and using the fabB promoter region amplified with primers 684 and
32P-685 for the noncoding strand. Approximately 25 pmol of 32P-labeled DNA and
0, 25, 50, or 100 pmol of His
6
-CpxR protein were mixed in a 100-μL reaction
containing 20 mM 2-[4-(2-hydroxyethyl)pi perazin-1-yl]ethanesulfonic acid pH 8.0,
10 mM KCl, 1 mM DTT, and 0.1 mg mL−1bovine serum albumin. The reaction
mixture was incubated at room temperature for 20 min. Then 1 μL of 100 mM
CaCl
2
,1μL of 100 mM MgCl
2
, and 0.005 units of DNase I (Fermentas) were added,
and the mixture was incubated at room temperature for 2 min. The DNase I
digestion was stopped by phenol treatment, and the DNA was precipitated. Sam-
ples were analyzed by 6% polyacrylamide electrophoresis by comparison with a
DNA sequence ladder generated with the same primers using a Maxam and Gilbert
A+G reaction.
Membrane property assays. The membrane fluidity was measured by applying
DPH as a fluorescence probe44.Briefly, the washed cells were incubated in DPH
(Sigma) at a final concentration of 2 μM, then shaken in the dark at 30 °C for
40 min. After incubation, the unincorporated DPH was removed by washing with
phosphate-buffered saline (PBS) twice. The cells were resuspended in PBS to get a
final density of OD
600
=0.2 for the measurement of fluorescence anisotropy on a
FluoroMax-4 spectrofluorometer (HORIBA Jobin Yvon). The wavelengths were
342 and 432 nm and slit widths were 5 and 10 nm for excitation and emission light,
respectively. The fluorescence anisotropy, which was negatively correlated with
membrane fluidity, was calculated44. For the permeability assay, the washed cells
were resuspended in PBS for 30 min. The release of nucleotides was then measured
at an optical density of 260 nm.
Intracellular pH measurement. The gene encoding ratiometric pH-sensitive green
florescent protein pHluorin2 was synthesized and cloned into vector pBAD-18kan.
Excitation assays were performed at wavelengths 395 and 475 nm with the emis-
sion at 510 nm using FluoroMax-4 Spectrofluorometer (HORIBA Jobin Yvon), and
the ratio of fluorescence at 395 to 475 nm was used to calculate the intracellular pH
according to a standard curve46.
F
0
F
1
-ATPase activity assay. The cells were washed once and resuspended in 1.8
mL membrane buffer (75 mM Tris pH 7.0, 10 mM MgSO
4
). Toluene was added to
afinal concentration of 10% (vol/vol), and the suspension was vortexed for 30 s and
subjected to two rounds of freeze-thawing. Cells were collected, resuspended in
membrane buffer. ATPase activity was assayed in terms of the release of inorganic
phosphate in 50 mM Tris-maleate buffer pH 7.0, containing 10 mM MgSO
4
and
50 μM ATP. Phosphate was assayed by the malachite green method, using Mala-
chite Green Phosphate Assay Kit (Cayman Chemical).
Mouse survival passage experiments. Female BALB/c mice were housed indi-
vidually in pathogen-free facility and given food and water ad libitum until 6-week-
old. BW25113 strain and mutant with substitution in fabA CpxR site were grown
in LB at 37 °C to log phase, and then 104CFUs of bacteria were orally admini-
strated to mice. To allow for inoculum clearance through the stomach, the mice
were not provided feed for 4 h after oral administration. Fecal samples were col-
lected 24 h after inoculation and suspended in PBS (0.5 g feces/4.5 mL PBS) and
subsequently diluted. The diluted samples were then plated on LB agar containing
nalidixic acid (20 μgmL
−1) to test for the presence or absence of corresponding
strains. Three independent trials were performed for each strain, and in each trial
six mice were inoculated. This experiment was performed following the Guide for
the Care and Use of Laboratory Animals (National Institutes of Health, 1985).
3-Hydroxypropionate production. The strains were grown overnight in LB broth
and 1:100 diluted into 250 mL Erlenmeyer flasks with 50 mL of minimal medium
containing 14 g L−1K
2
HPO
4
•3H
2
O, 5.2 g L−1KH
2
PO
4
,1gL
−1NaCl, 1 g L−1
NH
4
Cl, 0.5 g L−1MgSO
4
, 0.2 g L−1yeast extract, and 20 g L−1glucose. All
shake flask experiments were carried out in triplicates. After incubation at 37 °C,
0.05 mM IPTG was added for induction at OD
600
0.8, and 3 h later, biotin
(40 mg L−1) and NaHCO
3
(20 mM) were added. The antibiotics were supplied
periodically after induction of IPTG every 12 h until 48 h. 10 g L−1glucose was
added once again after 24 h induction. 3HP concentration in medium was deter-
mined using Agilent 1200 Infinity HPLC system with an Aminex HPX-87H
column (300 × 7.8 mm, Bio-Rad)54.
Ethics declarations. The animal experiments were performed according to the
standards set forth in the Guide for the Care and Use of Laboratory Animals
(National Institutes of Health, 1985). Experimental protocols were approved by the
Institutional Animal Care Committee at Qingdao Institute of Bioenergy and Bio-
process Technology.
Reporting summary. Further information on research design is available in
the Nature Research Reporting Summary linked to this article.
Data availability
The source data underlying Figs. 1a, b, d−g, 2c−f, 3c−g, 4a, c, e−g, 5a−e, and 6a, b and
Supplementary Figs. 1, 2, 3b, c, 4, 5, and 6 are provided as a Source Data file. The FabA
sequences used in this study are under the accession numbers AIN31422, NP_460041,
and EFS15200. The genome sequences used in this study are under the accession
numbers CP009273,NC_003197,NC_002516,NC_011283,NC_013716,CP001235,
NC_004741,CP001589, and NC_009778. Other data supporting the findings of this
study are available from the corresponding authors upon request.
Received: 18 June 2019; Accepted: 5 March 2020;
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Acknowledgements
We thank Dr. Roy Curtiss III (University of Florida) for pRE112 and E. coli χ7213,
Dr. Lixue Zhang (Qingdao University) for help on proteoliposome reconstruction,
Dr. Qinggang Wang (Qingdao Institute of Bioenergy and Bioprocess Technology) for
helpful discussion, and NBRP-E.coli at NIG for BW25113 strain. This study was
ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15350-5
12 NATURE COMMUNICATIONS | (2020) 11:1496 | https://doi.org/10.1038/s41467-020-15350-5 |www.nature.com/naturecommunications
Content courtesy of Springer Nature, terms of use apply. Rights reserved

financially supported by the NSFC (31722001 and 31670089), and Natural Science
Foundation of Shandong Province (JQ201707).
Author contributions
G.Z. designed the experiments. Y.X., Z.Z., W.T., Y.S., Y.D., B.L., J.W., M.L., Y.W. and S.S.
performed the experiments. G.Z., M.X., Y.X. and Q.Q. analyzed the results. G.Z., M.X.,
Y.X. and Q.Q. wrote the manuscript. All authors edited the manuscript before submission.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41467-
020-15350-5.
Correspondence and requests for materials should be addressed to M.X. or G.Z.
Peer review information Nature Communications thanks Daniela De Biase, Peter Lund
and the other, anonymous, reviewer for their contribution to the peer review of this
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