(Received for publication, September 1, 1994; and in revised form, November 3, 1994)
From the
The Escherichia coli mutant
nhaA
nhaB (EP432), which lacks the two
specific Na
/H
antiporter genes, is
incapable of efficiently excreting Na
. Accordingly at
low K
(6 mM) medium, its intracellular
Na
concentration is only slightly lower
(1.5-2
) than the extracellular concentration (50
mM), explaining the high sensitivity to Na
(
30 mM) of the mutant. This Na
sensitivity is shown to be a powerful selection for spontaneous
second-site suppressor mutations that allow growth on high
Na
(
0.6 M) with a rate similar to that of
the wild type. One such mutation, MH1, maps at 25.7 min on the E.
coli chromosome. It confers Na
but not
Li
resistance upon
nhaA
nhaB cells and exposes a
Na
-excreting capacity, maintaining a
Na
gradient of about 8-10 (at 50 mM extracellular Na
), which is similar to that of
the wild type. Although lower, Na
excretion capacity
is also observed in the
nhaA
nhaB mutant
when grown in medium containing higher K
(70
mM). This capacity is accompanied with a shift in the
sensitivity of the mutant to higher Na
concentrations
(
300 mM). Whereas Na
excretion by a wild
type carrying
unc is uncoupler sensitive, that of
MH1
unc is dependent on respiration in an
uncoupler-insensitive fashion. It is concluded that under some
conditions (high K
in the medium or in MH1-like
mutants), a primary pump driven by respiration is responsible for
Na
extrusion when the
Na
/H
antiporters are not active.
Na/H
antiporters are essential
for adaptation to Na
during growth since Escherichia coli mutants lacking the
Na
/H
antiporters are sensitive to
Na
(Padan et al., 1989; Pinner et
al., 1993). In accordance, upon transformation of the mutant
strains with multicopy nhaA or nhaB plasmid, the wild
type capacity to grow in the presence of Na
is
restored.
The pattern of regulation of nhaA studied in a
strain carrying nhaA`-`lacZ protein fusion reflects
the importance of nhaA in salt adaptation (Karpel et
al., 1991; Rahav-Manor et al., 1992). Its expression,
which is positively regulated by nhaR, is induced by
Na. The induction is increased with pH similar to the
Na
sensitivity of
nhaA (Padan et
al., 1989).
In view of the Na sensitivity
found in the antiporter mutants (
nhaA or
nhaA
nhaB), it can be anticipated that
unless other Na
-expelling machinery exists and
functions in the absence of the antiporters, Na
will
accumulate in the cells to growth inhibitory levels. Primary
Na
pumps were demonstrated in various bacteria
(Dimroth, 1992a, 1992b; Tokuda and Unemoto, 1985; Heefner and Harold,
1982; Takase et al., 1993). Furthermore, based on studies with
intact cells (Dibrov, 1991; Skulachev, 1984, 1988) and isolated
membrane vesicles (Avetisyan et al., 1989, 1993), it has also
been implied that primary Na
pumps exist in E.
coli. Therefore, the
nhaA
nhaB mutant
affords new approaches to the search for genes and proteins involved in
Na
-excreting machinery other than that of the specific
Na
/H
antiporters. Given that there
exist nonspecific antiporter systems or pumps with low affinity and/or V
to Na
, amplification of the
respective genes by their cloning on multicopy plasmid may confer some
Na
resistance upon transformation into
nhaA
nhaB. Indeed, with this approach, chaA has been cloned (Ivey et al., 1993). On the
basis of its homology to calsequestrin and its promotion of
Ca
/H
exchange activity in membranes,
ChaA appears to be a Ca
/H
antiporter
with low affinity to Na
.
The Na sensitivity of the
nhaA
nhaB strain
provides yet another approach to look for systems conferring
Na
resistance (a powerful selection (growth on
Na
) of second-site revertants resistant to
Na
). The mechanism of resistance of such mutants
should bear upon new Na
-excreting machinery and/or
other mechanisms relevant to Na
resistance and to the
identification of their genes.
We show that
nhaA
nhaB is Na
sensitive
and defective in Na
excretion. This Na
sensitivity has provided a strong selection for the isolation of
a spontaneous second-site revertant, which bears a mutation at locus
25.7 min on the E. coli chromosome. The mutation confers
Na
but not Li
resistance and exposes
a Na
excretion capacity, which is driven by
respiration but not by
t;ex2html_html_special_mark_amp;mgr;
.
In several
experiments, cells were grown in modified L broth (pH 7.5) from which
concentrations of K and/or Na
were
modified and indicated, respectively, in mM by a number (n, m) following the atomic symbol
(LBKnNam). Thus, LBK70Na7 contains 70 mM KCl, and although Na
is not added, it contains 7
mM Na
(the endogenous contaminating
Na
as measured by atomic absorption); LBK6Na300
contains 6 mM endogenous contaminating K
(as
determined by atomic absorption) and 300 mM NaCl, etc. In
other experiments, the L broth medium was further modified by the
addition of 50 mM 1,3-bis(tris[hydroxymethyl]methylamino)-proprane (pH
7.5) and 50 mM glucose and the K
or
Na
concentration indicated as above
(LBBGKnNam).
Cells were also grown in two minimal
media. Medium A (Davies and Mingioli, 1950) was used without sodium
citrate but supplemented with thiamine (2.5 µg/ml) and the carbon
source 10 mM melibiose or 10 mM glucose as indicated.
When required, threonine (0.1 mg/ml) and/or thiamine (0.1 mg/ml) were
added. The other minimal medium was GMM containing 30 mM KHPO
, 50 mM NaCl, 50 mM 1,3-bis(tris[hydroxymethyl]methylamino)-proprane (pH
7.5), 7.5 mM (NH
)
SO
, 1
mM MgSO
, and 50 mM glucose. For plates,
1.6% agar was used. Concentrations of antibiotics were 100 µg/ml
ampicillin, 50 µg/ml kanamycin, and 12.5 µg/ml tetracycline.
Resistance of cells to Na
was tested by determining
colony-forming units on unmodified L broth plates containing various
concentrations of Na
. Conjugation and P1 transduction
were conducted as described (Miller, 1992).
Protein concentration in membrane vesicles was determined as described (Bradford, 1976).
Figure 1:
High
K improves the growth rate and decreases the
Na
sensitivity of the
nhaA
nhaB mutant. Wild type (TA15) (A),
nhaA
nhaB (EP432) (B), and MH1 (C) cells were grown in LBBGK6-based
medium (
) or in LBBGK70-based medium (
) in the presence of
various concentrations of Na
. Note that the lowest
[Na
]
= 7 mM,
the contaminating Na
.
Figure 2:
MH1 mutation confers Na
but not Li
resistance. A, cells EP432/pGM36
(
) or EP432 (
), as indicated, were grown in LBK70Na7 (pH
7.5) to the logarithmic phase and at zero time diluted (1:100) in the
medium containing 0.3 M NaCl (LBK6Na300). B-D,
cells (TA15,
; EP432,
; and MH1,
) were grown in
LBK70Na7 (pH 7.5) as above and at zero time diluted (1:100) at the same
pH in LBK70Na7 (B), LBK6Na300 (C), or (LBK6Li100) (D).
The salt-resistant phenotype (0.6 M NaCl) of these mutants is stable even after several transfers on
medium devoid of added Na (LBK70Na7). As expected for
a genotype derived from EP432, these cells are
NhaA
NhaB
Thr
Lac
Kan
CM
.
The frequency of spontaneous appearance of such mutants is about
1/10
cells. One mutant designated MH1 was isolated for
further study.
In marked contrast to the significant Na resistance
conferred by the MH1 mutation, this mutant remains as sensitive to
Li
as EP432 (Fig. 2D). Thus, MH1 is a
mutation that specifically affects Na
resistance.
Since all of these
exconjugants were Cam (in the recipient chromosome, 25.6
min) (Pinner et al., 1992), it is suggested that the mutation
is localized between 25.6 and 27.7 min. It should be noted that 10 out
of 11 other MH1-like mutations, isolated in the same way and shown to
have similar growth phenotype, were found by conjugation to map in the
same region.
To define the position of MH1 more closely, we used P1
transduction (Table 2). When the donor strain was the exconjugant
(obtained in one of the crosses described above) Kan Cam
Tet
Na
(
nhaA::kan
nhaB::cat
trp::Tn10
MH1) and the recipient strain NM81
(
nhaA1::kan
nhaB
MH
),
Cam
or Tet
transductants were selected, and
each scored for Na
resistance. Out of 253 Cam
transductants, 199 were Na
resistant, and out of
410 Tet
colonies, 54 were Na
resistant.
None of the transductants were both Cam
and
Tet
. The cotransduction to each marker suggests map
location between 25.8 and 26.5 min.
When tre::Tn10 was the
donor strain and the recipient was MH1, Tet transductants
were selected, and each scored for Cam
and Na
resistance. Out of 266 Tet
transductants, 193
became Cam
as expected from the distance between nhaB::cat (25.6 min) and tre::Tn10 marker (26 min).
Since not all Tet
transductants are Na
, it is
apparent that the MH1 mutation does not reside in tre.
Furthermore, out of the 266 Tet
transductants, 173 became
Na
(Table 2), suggesting that MH1 is located in close
proximity to tre (within 0.3 min).
To determine on which
side of tre MH1 is located, an extransductant of the latter
transduction, nhaA1::kan
nhaB1::cat
MH1tre::Tn10
(Kan
Cam
Na
Tet
), was
used as a donor strain and NM81 as an acceptor. All tet
cat
transductants were
also Na
as expected from the location of MH1 between tre and nhaB. Hence, MH1 maps at 25.7 min on the E. coli chromosome.
Figure 3:
Membrane vesicles isolated from MH1 do not
exhibit Na/H
antiporter activity.
Everted membrane vesicles were isolated from cells grown in LBK70Na7;
EP432, MH1, or MH1 transformed with pGM36, a multicopy plasmid bearing nhaA (Karpel et al., 1988).
pH was monitored
with acridine orange in a medium containing 140 mM potassium
chloride, 10 mM tricine (pH 8), 5 mM MgCl
, acridine orange (0.5 µM), and
membrane vesicles (50 µg of protein). At the onset of the
experiment, Tris-D-lactate (5 mM) was added, and the
fluorescence quenching was recorded. NaCl (10 mM) was then
added, and the new steady state of fluorescence obtained (dequenching)
was monitored. When indicated, monensin (1 µM) was
added.
Figure 4:
MH1
but not EP432 maintains a low concentration of intracellular
Na at low K
medium. TA15 (
),
EP432 (
), or MH1 (
) cells were grown to the logarithmic
phase (A
= 0.25) in LBBGK6Na50,
Na (0.5 µCi, carrier free) was added at zero time, and
measurement of intracellular
Na commenced at 1 h, after a
steady state level of the label had been established. The calculated
intracellular Na
quantity and concentrations are
shown. Opensymbols, no additions; closedsymbols, anaerobic conditions in the presence of 50
µM CCCP.
Since
the presence of 70 mM K in the rich medium
increased the growth rate of
nhaA
nhaB with
no effect on either the wild type or MH1 strain (Fig. 1), we
also measured the Na
gradient maintained by the
different strains in the high K
medium (LBBGK70) at
various [Na
]
(Fig. 5).
It is apparent that, up to 400 mM Na
, TA15
maintains a relatively constant gradient of 15-20-fold, which
results in a low [Na
]
. MH1, up
to 350 mM [Na
]
, also
maintains a gradient of 10-11-fold, which is only slightly lower
than that of the wild type. Beyond this concentration,
[Na
]
of MH1 increases much more
drastically than that of the wild type. At 400 mM [Na
]
, the
[Na
]
of these strains are 20
and 80 mM, respectively. It was impossible to measure
accurately [Na
]
at still higher
extracellular Na
concentrations.
Figure 5:
The capacity of the wild type,
nhaA
nhaB, and MH1 to maintain a
Na
gradient at various extracellular Na
concentrations. TA15 (
), EP432 (
), or MH1 (
)
cells were grown in LBBGK70 (pH 7.5) medium. NaCl was added to the
indicated concentrations 1 h before measurement together with
Na
. Internal Na
was
determined as described under ``Materials and Methods.'' Dashedline, [Na
]
=
[Na
]
.
In contrast to MH1
and TA15, intracellular concentration of
nhaA
nhaB mutant increases linearly with a
higher slope; at 50 mM [Na
]
, the gradient was
3-5-fold (Fig. 5) and at 300 mM [Na
]
, whereas
[Na
]
of MH1 or TA15 is
10-20 mM, that of EP432 is above 100 mM (Fig. 5).
To
compare the pattern of energization of Na transport in
MH1 with that of the wild type (TA15), we have deleted most of the
H
/ATPase genes of these strains (
unc derivatives). Both MH1
unc and TA15
unc (Fig. 6) maintained a similar Na
gradient
of about 5-6. Similar to previous results obtained by Borbolla
and Rosen(1984) in another wild type-derived strain, addition of CCCP
to TA15
unc collapsed the Na
gradient (Fig. 6A). Note that in the high K
medium dissipation of the gradient by CCCP was faster than in the
low K
medium, suggesting that K
serves as a counter-ion to the movement of Na
across the membrane. This result agrees with the notion that
via the
Na
/H
antiporters maintains the
Na
gradient in E. coli cells. Furthermore,
this result shows that under these conditions, any
t;ex2html_html_special_mark_amp;mgr;
-independent
mechanism of Na
excretion, if present, has a very weak
activity.
Figure 6:
Respiration but not
t;ex2html_html_special_mark_amp;mgr;
drives
Na
excretion in MH1
unc cells. A, TA15
unc cells were grown in medium LBBGK6Na50
(
) or LBBK70Na50 (
) and manipulated as described in Fig. 4with minor modifications needed for rapid inter-conversion
between anaerobic and aerobic conditions. At zero time, 15-ml aliquots
of mid-logarithmic phase cells were placed into 125-ml Erlenmeyer
flasks, and
Na was added; after 1 h of aerobic growth
(with shaking) in the presence of the isotope, CCCP (final
concentration of 50 µM) was added (
) (fullsymbols). B, MH1
unc cells grown in
LBBK6Na50 and manipulated as above.
, no addition
, 50
µM CCCP added at 60 min (
). For anaerobic cells
(
), immediately after addition of CCCP, cell suspension was
transferred into a 50 ml-test tube and covered with 1 ml of the light
mineral oil. 1 h later, the cell suspension was exposed to oxygen
(indicated by an arrow) by rapid transfer into the original
flask, and aerobic incubation continued with shaking
(
).
In marked contrast to TA15unc, addition of
CCCP to MH1
unc had no effect on the Na
gradient (Fig. 6B). This result is consistent
with the lack of the Na
/H
antiporters
in the latter strain and reveals that MH1 possesses a Na
excretion machinery, which is independent of
t;ex2html_html_special_mark_amp;mgr;
.
Total
collapse of the Na gradient was obtained in MH1 by
applying anaerobiosis in the presence of CCCP (Fig. 6B). Similar but somewhat slower decay of the
gradient occurred under anaerobic conditions (not shown). The
Na
gradient was reestablished upon reaeration of the
anaerobic experimental systems (Fig. 6B). We conclude
that respiration drives Na
excretion in MH1.
In this work, we show that MH1, a spontaneous point mutation
in the mutant nhaA
nhaB (EP432) which lacks
the two specific Na
/H
antiporters,
renders Na
resistance upon the cells and exposes a
Na
extrusion capacity that is independent of
t;ex2html_html_special_mark_amp;mgr;
but
driven by respiration.
The MH1 mutation was selected, without
mutagenesis, by growth of the Na-sensitive EP432 cells
on Na
(0.3 M, pH 7.5) under conditions in
which it is highly sensitive to Na
(medium of low
K
(6 mM)). Similar selection previously
allowed us to clone chaA, a putative
Ca
/H
antiporter gene, from a DNA
library prepared from and transformed into EP432 (Ivey et al.,
1993). A recent study of a
chaA mutation in the genetic
background of the wild type or the Na
/H
antiporter mutants (
nhaA,
nhaB,
nhaA
nhaB) has shown that chaA may
have a role in Na
circulation at alkaline pH (pH 8.5)
but not at neutral pH (Ohyama et al., 1994). Although both MH1
and chaA (Ivey et al., 1993) confer Na
resistance but not Li
resistance they are
different loci and confer different phenotypes. MH1 maps at 25.7 min,
whereas chaA maps at 27 min (Ivey et al., 1993) on
the E. coli chromosome. In contrast to chaA, which
apparently in multicopy increases the Na
/H
and the Ca
/H
antiporter
activity of isolated membrane vesicles of both EP432 as well as MH1
transformants, we could not find any Na
/H
antiporter activity in MH1, and its
Ca
/H
antiporter activity was
identical to that of the wild type. MH1 was isolated at neutral pH and,
in contrast to chaA, exerts its effect both at neutral and
alkaline pH.
The spontaneous frequency of mutants, similar to MH1 in
growth phenotype, is high (1:10cell). Interestingly, out of
the 11 independently selected mutations analyzed, 10 were localized to
the MH1 region. The ease in selection of the MH1-like mutations
reflects the very strong selection pressure imposed on EP432 mutant by
Na
and the efficient capacity of MH1 to confer
Na
resistance.
Determination of Na
distribution across the cytoplasmic membrane of the wild type cells
(TA15) under growth conditions in low K
medium
(LBBGK6Na50, pH 7.5) shows a Na
gradient of about 10 (Fig. 4A). Similar results were previously obtained
with endogenously respiring resting cells of another wild type strain
resuspended in buffer containing 80 mM Na
and
5 mM K
using NMR technology to quantitate
intracellular Na
( Fig. 4in Castle et
al., 1986; Pan and Macnab, 1990).
In marked contrast to the
wild type cells, EP432 cells have very low capacity to maintain a
Na gradient (Fig. 4B and 5). This
result is consistent with the suggestion (West and Mitchell, 1974;
Padan and Schuldiner, 1992, 1994) that the
Na
/H
antiporters are the main
Na
extrusion machinery of wild type E. coli cells. As a result of the lack of the antiporters, the
[Na
]
of EP432 is around 30
mM (at 50 mM [Na
]
in the low K
medium), whereas that of the wild
type is around 5 mM (compare Fig. 4, A and B). We therefore suggest that as opposed to the wild type that
grows up to 0.8 M NaCl in this medium (Padan et al.,
1989), this increase in intracellular Na
is the cause
of the dramatic growth inhibition of EP432 by extracellular
Na
(>30 mM) (Pinner et al.,
1993).
Addition of K (to 70 mM), which
does not affect the wild type growth (Fig. 1A),
sensitivity to Na
(Fig. 1A), or
Na
gradient (compare Fig. 4A with Fig. 5), increases the growth rate of EP432 even without added
Na
, and this effect seems to account for the shift in
the Na
sensitivity of EP432 toward higher
concentrations observed in the high K
medium (Fig. 1B). Most interestingly, concomitantly the
addition of K
improves the capacity of EP432 to
maintain a Na
gradient, and at 50 mM [Na
]
, a gradient of
3-5 is maintained (Fig. 5). However, the capacity of EP432
to maintain a Na
gradient is much lower than that of
the wild type at increasing
[Na
]
. At
[Na
]
of 300 mM,
[Na
]
of EP432 is 125
mM, as opposed to 15 mM of TA15. Therefore, it
appears that by allowing certain capacity of Na
excretion, the sensitivity of EP432 is shifted toward higher
[Na
]
in the high K
medium. Although as yet the mechanism of the effect of
K
is unknown, it may be related to a protective effect
of the ion against the inhibitory effect of Na
on many
enzymatic reactions (Walderhaug et al., 1987).
Remarkably,
MH1, even though it lacks both nhaA and nhaB
Na/H
antiporters, maintains a
Na
gradient of 5-8 (directed inward) in the
presence of 50 mM [Na
]
, as does the wild
type ( Fig. 4and Fig. 5). Furthermore, up to 350 mM [Na
]
, the gradient of MH1
is only slightly lower than that of the wild type and, only beyond it,
decreases (Fig. 5). At 400 mM [Na
]
,
[Na
]
of MH1 and the wild type
are 90 and 15 mM, respectively. Most interestingly, at 400
mM [Na
]
, when
[Na
]
concentration of MH1
reaches 90 mM, a difference in growth rate between MH1 and
wild type becomes apparent (Fig. 1C). These results
imply that the mutation MH1 exposes a Na
export
machinery, which at least up to 350 mM [Na
]
is similar in its
capacity to that of the wild type.
unc strains cannot
interconvert phosphate bond energy with the electrochemical proton
gradient, allowing conditions to be established in which the sources of
energy available for transport are both phosphate bond energy and an
electrochemical gradient (during metabolism of glucose), only phosphate
bond energy (glucose metabolism in the presence of uncouplers or
respiratory inhibitors), or only an electrochemical gradient of protons
or redox (during respiration of substrates of the electron transport
chain). As previously shown by Borbolla and Rosen(1984), the driving
force for Na
extrusion in wild type E. coli (in our case TA15) is
t;ex2html_html_special_mark_amp;mgr;
. Thus,
TA15
unc maintains a Na
gradient like
TA15, and the uncoupler CCCP collapses the gradient in
TA15
unc. In contrast to TA15
unc, MH1
unc maintains the Na
gradient
even in the presence of uncoupler. It could be argued that
MH1
unc is insensitive to uncouplers. This is highly
unlikely since, as described above, the isogenic strain
TA15
unc is uncoupler sensitive. Furthermore, the collapse
of the Na
gradient in MH1 by anaerobiosis is
accelerated in the presence of uncouplers. Hence, Na
extrusion in MH1
unc is not driven by
t;ex2html_html_special_mark_amp;mgr;
. Since
only anaerobiosis in the presence or absence of uncouplers collapsed
the gradient, we suggest that the Na
extrusion in MH1
can be directly coupled to electron transport.
This report
demonstrates the presence of a respiration-dependent,
t;ex2html_html_special_mark_amp;mgr;
-independent,
Na
-extrusion mechanism in E. coli. This
mechanism is evident in MH1, a mutant devoid of both antiporters and
carrying a mutation. We suggest that a similar, although weaker device
is responsible for the Na
extrusion detected in EP432,
a mutant devoid of both antiporters but still capable of generating a
small Na
gradient in the presence of high
K
. The two above mutants allow us to describe a
mechanism that in the wild type, if present, is masked by the activity
of two antiporters: NhaA and NhaB.
MH1 can be a mutation in a
Na pump itself, increasing its activity and/or
expression. On the other hand, it is also possible that MH1 affects
another system that is needed for expression or that limits the
activity of the pump. In this respect, as described above, EP432 shows
a limited Na
extrusion capacity. Hence, it is possible
that this low activity can be increased by induction, under conditions
unfavorable to the H
cycle (in the presence of
protonophores or at alkaline pH) as suggested before (Avetisyan et
al., 1989, 1992, 1993; Skulachev, 1984, 1988; Dibrov, 1991) or by
MH1-like mutations as shown here.