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Applied and Environmental Microbiology, March 2000, p. 943-947, Vol. 66, No. 3
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Expression of Outer Membrane Proteins in
Escherichia coli Growing at Acid pH
Mikiko
Sato,
Kazuhiro
Machida,
Eri
Arikado,
Hiromi
Saito,
Tomohito
Kakegawa, and
Hiroshi
Kobayashi*
Faculty of Pharmaceutical Sciences, Chiba
University, Inage-ku, Chiba 263-8522, Japan
Received 27 September 1999/Accepted 20 December 1999
 |
ABSTRACT |
It is generally accepted for Escherichia coli that (i)
the level of OmpC increases with increased osmolarity when cells are growing in neutral and alkaline media, whereas the level of OmpF decreases at high osmolarity, and that (ii) the two-component system
composed of OmpR (regulator) and EnvZ (sensor) regulates porin
expression. In this study, we found that OmpC was expressed at low
osmolarity in medium of pH below 6 and that the expression was
repressed when medium osmolarity was increased. In contrast, the
expression of ompF at acidic pH was essentially the same as that at alkaline pH. Neither OmpC nor OmpF was detectable in an ompR mutant at both acid and alkaline pH values. However,
OmpC and OmpF were well expressed at acid pH in a mutant
envZ strain, and their expression was regulated by medium
osmolarity. Thus, it appears that E. coli has a
different mechanism for porin expression at acid pH. A mutant deficient
in ompR grew slower than its parent strain in
low-osmolarity medium at acid pH (below 5.5). The same growth
diminution was observed when ompC and ompF were
deleted, suggesting that both OmpF and OmpC are required for optimal
growth under hypoosmosis at acid pH.
| |
INTRODUCTION |
The regulatory mechanism for porin
expression has been well defined in both Escherichia coli
and Salmonella enterica serovar Typhimurium (for reviews,
see references 3, 15, and 18). Gene expression of ompC and ompF is regulated by
medium osmolarity; OmpC and OmpF are synthesized at high and low
osmolarities, respectively. The regulation is mediated via the
OmpR/EnvZ two-component system. OmpR is the regulator and is
phosphorylated by the sensor protein EnvZ.
Expression of outer membrane proteins is also affected by medium pH;
growth at low pH increases OmpC expression and decreases the level of
OmpF (8, 24). Neither OmpC nor OmpF was detectable in an
ompR mutant at acid pH, and mutation of envZ
reduced the expression of ompC- and ompF-lacZ
fusion genes (8). The induction of the ompC-lacZ
fusion gene was greatly stimulated in rich medium but was very low in
minimal medium in S. enterica serovar Typhimurium at acid pH
(6). Thus, the mechanism for pH-dependent porin expression
is still enigmatic.
Two possible mechanisms for the regulation at acid pH can be
postulated: (i) the activity of this two-component regulatory system is
modified by pH, and (ii) E. coli uses different systems at
different pH values for porin expression, as proposed previously for
Na+ extrusion systems (21). In this study, we
found that OmpC was synthesized in medium of low osmolarity at acid pH
and that the level decreased under hyperosmosis. In contrast, the
expression of OmpF was essentially unchanged at both high and low pHs.
Therefore, both OmpF and OmpC are expressed under hypoosmosis at acid
pH. In agreement with these observations, the expression of OmpF and OmpC increased the growth rate in low-osmolarity medium at low pH, but
no difference in growth was observed when medium osmolarity was
increased. Neither OmpF nor OmpC was detectable in an ompR mutant at low pH or at high pH. The expression of OmpF and
OmpC requires envZ at alkaline pH, but both proteins were
expressed at low pH in an envZ mutant, suggesting that porin
expression is regulated in a different manner by medium osmolarity at a
low pH.
| |
MATERIALS AND METHODS |
E. coli strains and growth media.
The E. coli strains used are listed in Table
1. The following three media were used.
Medium A contained 5 mM K2HPO4, 20 mM NH4Cl, and 1% glucose. Medium B contained 1 mM
K2HPO4, 10 mM NH4Cl, and 0.1%
glucose. Medium C contained 1 mM K2HPO4, 1 mM
NH4Cl, and 0.1% glucose. All media contained 1 mM
MgCl2, 0.1 mM CaCl2, 0.1 mM FeSO4,
and 20 µg of thiamine/ml. Fifty millimolar
N-Tris(hydroxymethyl)-methylglycine (Tricine) for
media of pH 8.5 and 2-(N-morpholino)ethanesulfonic acid
monohydrate (MES) for media of pHs 5.5 and 5.0 were used. Medium C
contained 5 mM concentrations of these buffers instead of 50 mM
concentrations. The medium pH was adjusted by the addition of KOH.
Growth was monitored by measuring the absorbance of the medium at 600 nm when cells were cultured in media A and B. The viable cells in
medium C were counted on L broth (10) agar plates.
Construction of ompC::Kan.
The
chromosomal ompC of W3110
recD::Tn10 was replaced by the
nonfunctional ompC::Kan with
pBSompC::Kan (1) cut
with KpnI and EcoRI, and the resulting strain was
designated DK3. Plasmid pBSompC::Kan was kindly supplied by
K. Igarashi (Chiba University, Chiba, Japan).
Isolation of outer membrane proteins and analysis.
Outer
membrane proteins were prepared as described previously (12)
with modifications. Cells washed with 10 mM Tris-HCl (pH 8.0) were
suspended in the same buffer containing 20% sucrose, and the
suspension was kept on ice after the addition of 1 mM EDTA and lysozyme
(0.1 mg/ml). Cells were disrupted by sonication. After centrifugation
at 2,500 × g for 5 min, cell envelopes were collected
by centrifugation of the supernatant at 25,000 × g for 10 min, washed twice with 10 mM Tris-HCl (pH 8.0), and then suspended in the same buffer containing 2% Triton X-100. The suspension was
incubated at room temperature for 30 min and centrifuged at 100,000 × g for 30 min. The resulting pellet was
stored at 
20°C before use. The pellet was heated in a 1.25% sodium
dodecyl sulfate (SDS) solution containing 
-mercaptoethanol (1.25%)
at 100°C for 5 min. The proteins (15 µg) were separated by
urea-SDS-polyacrylamide gel electrophoresis as described previously
(26) and stained with Coomassie brilliant blue R250. The
stained gel was scanned with ScanJet IIC (Hewlett-Packard), and
densitometric analysis was performed using NIH Image (version 1.61).
Other methods and chemicals.
Measurement of
-galactosidase activity (13), protein determination
(11), and P1 transduction (10) were carried out as described previously. Reagents used were of analytical grade.
| |
RESULTS |
Effect of medium osmolarity on gene expression of OmpC and OmpF at
low pH.
OmpF was expressed in low-osmolarity medium of initial pH
8.5, and the expression of OmpC was stimulated at high osmolarity under
our growth conditions (Fig. 1). In
contrast to what was observed at alkaline pH, both OmpC and OmpF were
expressed in cells growing in low-osmolarity medium of initial pH 5.5, and expression of both proteins was repressed by the addition of NaCl (Fig. 1). The medium pH decreases rapidly when glucose is used as an
energy source. In our present study, when cells were harvested at an
absorbance of 0.3 to 0.4, the medium pH decreased to 5.1 to 5.3 and 7.9 to 8.1 in media with initial pHs of 5.5 and 8.5, respectively.
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FIG. 1.
Urea-SDS-polyacrylamide gel electrophoresis of outer
membrane proteins. W3110 (wild type) was grown in medium A of initial
pH 5.5 (lanes 1 to 3) and pH 8.5 (lanes 4 to 6) and harvested at an
absorbance of 0.3 to 0.4 as described in Materials and Methods. When
cells were harvested, the medium pH values were 5.1 to 5.3 and 7.9 to
8.1, respectively. Densities of lane 1 were taken as 100, and relative
densities are represented. Lanes: 1 and 4, cells grown without the
addition of NaCl; 2 and 5, cells grown in the presence of 100 mM NaCl;
3 and 6, cells grown in the presence of 200 mM NaCl.
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|
In order to measure the expression more quantitatively, a fusion gene
with
lacZ was used. The expression of OmpF decreased
in
high-osmolarity medium at both pH values (Fig.
2A), whereas
the expression of OmpC
decreased with the increase in medium osmolarity
at acid pH (Fig.
2B).
When 0.3 M NaCl was added, the OmpC expression
decreased to
approximately 50% of the expression without the addition
of NaCl in
medium of initial pH 5.5 (data not shown). Thus, it
appears that the
effect of osmolarity on OmpF expression at acid
pH is the same as that
at alkaline pH but that the effect on OmpC
expression is reversed.
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FIG. 2.
Expression of OmpC and OmpF at low pH. Cells were grown
in medium A of initial pHs 5.5 and 8.5 with and without the addition of
200 mM NaCl. Cells were harvested as described in the legend of Fig. 1.
One Miller unit of -galactosidase activity was defined as described
previously (13). Each bar represents the mean ± standard deviation of four independent experiments. (A) MF8.1
(ompF-lacZ; open bars), MSR6 (ompF-lacZ
ompR::Tn10; solid bars), and MSZ6 (ompF-lacZ
envZ::kan; hatched bars). (B) MKC5.4
(ompC-lacZ; gray bars), MKCR5.4 (ompC-lacZ
ompR::Tn10; dotted bars), and MKCZ5.4
(ompC-lacZ envZ::kan; horizontally striped
bars).
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|
Role of OmpR and EnvZ in porin expression at low pH.
The above
results suggested the possibility that E. coli uses
different mechanisms for porin gene expression at different pH values.
To elucidate this possibility, we constructed mutants containing a
disrupted ompR gene by the insertion of Tn10.
Neither OmpC nor OmpF was detected by electrophoresis of outer membrane proteins from MSR31 (W3110 ompR::Tn10; data
not shown). No significant expression of ompC- and
ompF-lacZ fusion genes in MKCR5.4 (ompC-lacZ ompR::Tn10) and MSR6 (ompF-lacZ
ompR::Tn10) grown in acid and alkaline media was
observed (Fig. 2). EnvZ may not have been expressed when
Tn10 was inserted into ompR, as envZ
and ompR are in an operon with the promoter located upstream
of ompR (23). Next, we used the
ompR101 mutation (7), an in-frame deletion of 57 bp (17). The expression levels of OmpC and OmpF in the outer
membrane fraction from MH1160 (ompR101; Fig.
3) were very low, suggesting that
ompR is essential for gene expression of OmpC and OmpF at
low as well as at alkaline pHs.
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FIG. 3.
Urea-SDS-polyacrylamide gel electrophoresis of outer
membrane proteins. Cells grown in medium A of initial pH 5.5 (lanes 1, 2, 5, and 6) and pH 8.5 (lanes 3, 4, 7, and 8) were harvested as
described in the legend of Fig. 1. Densities of lanes 1 and 5 were
taken as 100, and relative densities are represented. Lanes: 1 and 3, W3110 (wild type); 2 and 4, MH1160 (ompR101); 5 and 7, MSZ31
(W3110 envZ) grown without the addition of NaCl; 6 and 8, MSZ31 (W3110 envZ) grown in the presence of 200 mM NaCl.
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|
Mizuno and Mizushima (
14) showed that an
envZ
deletion mutant produced small amounts of OmpC and OmpF and that their
expression
was regulated by osmolarity. Our data demonstrate a weak
expression
of OmpC and OmpF at alkaline pH (Fig.
3). In contrast to
what
was observed at alkaline pH, both OmpC and OmpF were well
expressed
in
envZ mutants at acid pH (Fig.
3). Expression of
OmpC and OmpF
was repressed by the addition of NaCl to the growth
medium at
acid pH (Fig.
3). Both
ompC- and
ompF-lacZ fusion genes were expressed
at acid pH even if
envZ was deleted, and the expression was again
regulated by
medium osmolarity (Fig.
2). These results suggest
that
E. coli has an EnvZ-independent system for porin expression
functioning at acid
pH.
Effect of OmpF and OmpC on growth at acid pH.
As shown in Fig.
4, growth of MSR31 (W3110
ompR::Tn10) was nearly the same as that of
its parent strain, W3110, in medium A (approximately 0.20 osM) of
initial pH 5.5 but slower than that of W3110 in medium B (approximately
0.12 osM). The difference in growth rate at low pH was enhanced in
medium C, whose osmolarity was approximately 0.02 osM (Fig.
5B). Since medium C contained a low
concentration of MES buffer, the medium pH dropped rapidly at a high
cell density. Therefore, we started the cell culture at a low density
of 0.5 × 106 to 1.1 × 106 cells/ml,
and growth was monitored by determining the viable cell count. When
growth in medium A was measured in terms of viable cell count, it was
found that the cell number increased in proportion to the absorbance of
the culture medium in both strains. MSR31 (W3110
ompR::Tn10) grew in medium C containing 200 mM NaCl at the same rate as W3110 (Fig. 5B), suggesting that the low
growth rate of the ompR mutant is due to a low osmolarity
but not to a low concentration of some nutrients of this medium. In
contrast to the results for growth at acid pH, no significant
difference in the growth rates of the two strains was observed at
alkaline pH in medium C (Fig. 5A) and medium A containing 200 mM NaCl
(data not shown).
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FIG. 4.
Growth of W3110 and MSR31. W3110 (wild type; solid
symbols) and MSR31 (W3110 ompR::Tn10; open
symbols) were grown in media A (A) and B (B) of initial pH 5.5. Growth
was monitored by measuring the absorbance of the medium.
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FIG. 5.
Growth of W3110 and MSR in low-osmolarity medium. W3110
(wild type; solid symbols) and MSR31 (W3110
ompR::Tn10; open symbols) were grown in
medium C of initial pHs 8.5 (A) and 5.5 (B). Growth was monitored by
determining the viable cell count. Symbols: circles, no addition of
NaCl; triangles, 200 mM NaCl added.
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|
The difference in growth rates between W3110 (wild type) and MSR31
(W3110
ompR::Tn
10) was enhanced in medium of
initial pH
5.0 (Fig.
6A). It is quite
likely that growth diminution is due
the absence of porin proteins. To
confirm this idea, we constructed
a mutant deficient in
ompC
and
ompF. MKCF36 (
ompC ompF) grew slower
than
MKW505 (
ompC+ ompF+) in medium B of
initial pH 5.0, and no difference in the growth
rate was observed when
200 mM NaCl was added to the medium (Fig.
6B). The same results were
obtained in strains deficient in
ompC and
ompF
constructed separately by the same procedure. MKW505
(
ompC+ ompF+) grew faster than
W3110, probably due to a different genetic
background.
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FIG. 6.
Growth of mutants deficient in OmpC and OmpF in medium
of initial pH 5.0. Cells were grown in medium B of initial pH 5.0 (open
symbols) and the same medium containing 200 mM NaCl (solid symbols).
Growth was monitored by measuring the absorbance of the medium. (A)  and  , W3110 (ompR+);  and  , MSR31
(ompR). (B) and , MKW505 (ompC+
ompF+); and , MKCF36 (ompC ompF);
 , MKC505 (ompC ompF+);  , MH621
(ompC+ ompF).
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|
The growth rates of MKC505 (
ompC) and MH621
(
ompF) were the same as that of MKW505
(
ompC+ ompF+) in medium B of initial
pH 5.0 (Fig.
6B) as well as in the same
medium containing 200 mM NaCl
(data not shown). MKFC36 (
ompC ompF)
grew at the same rate
as MKW505 (
ompC+ ompF+), MKC505
(
ompC), and MH621 (
ompF) in medium B of initial
pH 8.5
with and without the addition of 200 mM NaCl (data not shown).
The growth yield was low in medium B of initial pH 5.0 (Fig.
6B).
Since
the buffering capacity of MES decreases rapidly at a pH
below 5, the
low yield is probably due to the decrease in medium
pH. In fact, the
medium pH dropped to approximately 4 when growth
stopped.
| |
DISCUSSION |
In agreement with previous reports (8, 24), our data
showed that the level of OmpC was high at low pH when cells were cultured without the addition of NaCl. However, the OmpC level was low
in cells grown in high-osmolarity medium at acid pH, as the OmpC
expression is down-regulated by medium osmolarity at low pH. Thomas and
Booth (24) have reported that OmpF expression is repressed
in medium of pH 6.0 when glucose is used. However, OmpF was expressed
in cells growing in glucose medium of initial pH 5.5 under our
experimental conditions (Fig. 1 and 2). As described in Results, the
medium pH decreases rapidly when glucose is used as an energy source.
Therefore, the discrepancy might be due to a different pH value when
the cells were harvested.
Our present observations lead us to conclude that E. coli
has two kinds of mechanisms for porin expression. One is working at
neutral and alkaline pH values, and the other is for acid pH. OmpR
participates in both mechanisms, but EnvZ is involved only in the
former system. OmpC was expressed at pH 5.5 in an S. enterica serovar Typhimurium mutant deficient in ompR
(6). Thus, the regulation at low pH might be different
between the two strains.
Why does E. coli have multiple regulatory systems for
porin expression?
Expression of OmpF in envZ mutants is
slightly lower than that in the wild type at pH 5.2, suggesting that
the activity of the mechanism mediated by EnvZ is low at acid pH. Thus,
the most likely explanation would be that EnvZ has a low activity at
acid pH. It is also possible that the expression of EnvZ decreases at
low pH. In both cases, a mechanism independent from EnvZ may be
required in acid environments. An unknown factor might function as a
sensor instead of EnvZ at acid pH. In agreement with the previous
report by Mizuno and Mizushima (14), weak expression of OmpF
and OmpC was observed at alkaline pH in our envZ mutant (Fig. 2 and 3). These results suggest that the activity of the system
independent from EnvZ proposed here is very low at pH around 8.0 but detectable.
How does the environmental pH affect the activity of the OmpR/EnvZ
system?
Since EnvZ is a membrane protein, a domain located outside
of the cytoplasmic membranes might function as a pH sensor.
Alternatively, the regulatory system may be affected by cytoplasmic pH.
Mugikura et al. (16) demonstrated that the cytoplasmic pH is
kept constant within the range of medium pH from 6 to 8 and that the
cytoplasm is acidified in acid medium below pH 6. Since porin
expression at pH 6.5 was similar to that at pH 8.0 (data not shown),
the latter possibility is more likely. Heyde and Portalier
(8) have reported that the porin expression was mediated via
EnvZ in Luria-Bertani medium of initial pH 5.6 without glucose. The medium they used is alkalinized rapidly at a high cell density. Therefore, if the medium pH was above 6 when cells were harvested (absorbance of 0.6 to 0.8), expression of OmpC could be mediated via EnvZ.
To the best of our knowledge, this is the first report to demonstrate
the effect of OmpC or OmpF on growth of
E. coli. It
has been
shown in this study that the expression of either OmpC
or OmpF is
required for growth in low-osmolarity medium at acid
pH (Fig.
6). The
decrease in the OmpC expression due to NaCl was
only about 50% (Fig.
2). However, the above results suggest that
the higher expression of
OmpC and OmpF is essential for growth
under hypoosmosis at acid pH. It
is an open question why these
porins are required under such
conditions. It can be argued that
the active accumulation of nutrients
decreases at acid pH because
of the low activity of energy metabolism.
Therefore, an acceleration
of the movement of nutrients across the
outer membranes through
porins would be especially important at low pH
when the surroundings
contain a low concentration of
nutrients.
When
E. coli cells were transferred from acid medium of pH
5.5 to alkaline medium, cells became sensitive to alkalinity (
19,
20). The sensitivity to alkalinity was reduced by
ompC
lesion
(
20), and our results show that OmpC is expressed at
low pH.
These observations suggest that the presence of OmpC reduces
the
ability to survive at alkaline pH. The pore size of OmpC is
regulated
by pH; an increase in pH induced the structural change from a
small-sized channel conformation to a large-sized one (
25).
Kennedy (
9) reported that membrane-derived oligosaccharides
(MDO) were accumulated in the periplasmic space at low osmolarity.
The
deletion of MDO synthesis enhanced OmpC expression (
5).
Thus, porins might participate in the maintenance of osmolytes
in the
periplasmic space under
hypoosmosis.
| |
ACKNOWLEDGMENTS |
We thank K. Igarashi (Chiba University) R. G. Matthews
(University of Michigan, Ann Arbor, Mich.), T. Mizuno (Nagoya
University, Nagoya, Japan), K. Nishimura (National Institute of
Genetics, Mishima, Japan), and T. J. Silhavy (Princeton
University, Princeton, N.J.) for gifts of strains and plasmids. We are
grateful to R. L. Kaspar for his help with the preparation of the
manuscript and valuable suggestions.
This work was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, Sports and Culture of Japan.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Faculty of
Pharmaceutical Sciences, Chiba University, 1-33, Yayoi-cho, Inage-ku,
Chiba 263-8522, Japan. Phone: 81-43-290-2916. Fax: 81-43-290-2918. E-mail: hiroshi{at}p.chiba-u.ac.jp.
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Applied and Environmental Microbiology, March 2000, p. 943-947, Vol. 66, No. 3
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
