(Received for publication, December 20, 1995)
From the
The ntpJ gene, the tail end in the vacuolar type
Na-ATPase (ntp) operon of Enterococcus
hirae, encodes a putative 49-kDa hydrophobic protein resembling
K
transporter protein in Saccharomyces cerevisiae (Takase, K., Kakinuma, S., Yamato, I., Konishi, K., Igarashi, K.,
and Kakinuma, Y.(1994) J. Biol. Chem. 269, 11037-11044).
Northern blotting experiment revealed that the ntpJ gene was
transcribed as a cistron in the ntp operon. We constructed an Enterococcus strain in which the ntpJ gene was
disrupted by cassette mutagenesis with erythromycin resistance gene.
The growth of this mutant was normal at low pH. However, the mutant did
not grow at high pH in K
-limited medium (less than 1
mM), while the wild type strain grew well; the internal
K
concentration of this mutant was as low as 7% of
that of the wild type strain, suggesting that the K
accumulation at high pH was inactivated by disruption of the ntpJ gene. Potassium uptake activity via the KtrII system,
which had been proposed as the proton potential-independent,
Na
-ATPase-coupled system working at high pH (Kakinuma,
Y., and Harold, F. M.(1985) J. Biol. Chem. 260,
2086-2091), was missing in this mutant strain. However, this
mutant retained as high activities of Na
-ATPase and
Na
pumping as the wild type strain. From these
results, we conclude that the NtpJ is a membraneous component of the
KtrII K
uptake system but not a functional subunit of
vacuolar Na
-ATPase complex; the interplay between the
KtrII system and the Na
-ATPase was discussed.
All living cells show Na circulation across the
cell membrane. This circulation is driven by active transport systems,
which extrude sodium ions and maintain the Na
concentration gradient directed
inward(1, 2, 3) . In animal cells, the
familiar Na
,K
-ATPase expels sodium
ions, in which K
uptake is tightly coupled. Bacteria
have evolved diverse mechanisms for active sodium extrusion. Secondary
Na
/H
antiporters are widely
distributed(4) , and some bacteria have been found to produce
primary sodium pumps coupled with chemical reactions such as
decarboxylation(5) , electron transport(6) , and ATP
hydrolysis(7) . Na
reenters the cells via the
Na
gradient consumer, such as
Na
-coupled secondary co-transport systems, as the
widespread route(8) . Furthermore, Na
-motive
flagellar motor and the Na
potential-driven ATP
synthase are known for their physiological importance in some
bacteria(9, 10) .
The Gram-positive bacterium Enterococcus hirae lacks the respiratory chain; the proton
motive force is generated by proton expulsion via the
FF
, H
-translocating
ATPase(11) . This bacterium has two sodium extrusion systems:
Na
/H
antiporter(12, 13) and an ATP-driven primary pump,
Na
-translocating ATPase (7) . There has been
no clear observation that suggests the Na
gradient-consuming systems in this organism. The physiological
role of sodium extrusion systems may be the elimination of sodium ions
from cytoplasm and making room for K
accumulation(14, 15) .
Our recent biochemical
and molecular biological work on E. hirae Na-ATPase(16, 17, 18, 19, 20, 21, 22, 23) has
suggested that this enzyme is the vacuolar type ATPase distributed in
various eukaryotic endomembrane systems and
archaebacteria(24, 25, 26) . This enzyme is
encoded by a gene cluster (ntp operon) consisting of 11 ntp genes: ntpF, -I, -K, -E, -C, -G, -A, -B, -D, -H, and -J(22) . In addition to the
homologous counterparts of eukaryotic VATPases, (
)A, B, and
K (16-kDa proteolipid) subunits, we found that six other Ntp proteins
(F, I, E, C, G, and D) were similar counterparts of V-ATPase subunits
in eukaryotes(22, 27, 28, 29) .
Thus, the expected molecular structure of E. hirae Na
-ATPase resembles those of the eukaryotic
vacuolar type H
-ATPase complex.
On the other hand,
one decade ago, Kakinuma and Harold (30) reported a peculiar
feature of E. hirae Na-ATPase. They examined
the proton potential-independent K
transport activity
(KtrII system) in this bacterium (31) and found that this
activity depended on the activity of Na
-ATPase. An
apparent equimolar exchange of the internal Na
for the
external K
was observed in the absence of the proton
potential. They proposed the mechanism of the KtrII system as the
direct Na
/K
exchange by the
Na
-ATPase. Since the molecular mechanism of this
enzyme had not been elucidated, it was the simplest
explanation(32) . In this connection, we paid attention to the
function of the ntpJ gene of the ntp gene cluster.
This gene encoded a putative 49-kDa hydrophobic protein, which
resembles those of K
transport systems of Saccharomyces cerevisiae (Trk1 and Trk2) and of Escherichia coli (Trk) (22) and has not been assigned
so far to other V-ATPase subunits. Thus, we thought that the NtpJ
protein was the K
transporting component for the KtrII
activity in the Na
-ATPase.
In this work we
disrupted the ntpJ gene by cassette mutagenesis and examined
the properties of this mutant. Although the Na-ATPase
was alive in this mutant, the KtrII K
uptake activity
was deficient, suggesting that the NtpJ protein is a membraneous
component of this K
uptake system but not the
essential one of the Na
-ATPase complex. The KtrII
K
transport system is important for this organism in
K
-limited medium at high pH.
Figure 1: Structure of the ntp operon and its neighboring genes. The ntp operon is composed of 11 genes: ntpF, -I, -K, -E, -C, -G, -A, -B, -D, -H, and -J. The arrows indicate the direction of the genes. There are two genes (ntpR and ORFX) with the opposite direction of the ntp operon at the flanking regions. The DNA segments (shaded boxes) labeled as I, II, and III represent the probes used for Northern blotting as shown in Fig. 3.
Figure 3:
Northern blotting. Cells of strain 9790
were grown in KTY medium at pH 7.5 (lane 1), NaTY medium at pH
7.5 (lane 2), or NaTY medium at pH 10.0 (lane 3), and
total RNA was extracted from the cells as described under
``Experimental Procedures.'' Northern blotting was carried
out using three different DNA fragments (Fig. 1) (probe I,
2.5-kb HindIII-HindIII fragment; probe II, 0.8-kb PvuII-PvuII fragment; probe III, 1.2-kb PvuII-PvuII fragment) as the probes labeled with a
random primer labeling kit using
[-
P]dCTP.
Figure 2: Disruption of the ntpJ gene. A, schematic representation of disruption of the ntpJ gene. The 2.4-kb HindIII-HindIII fragment from ntpD to ORFX in the ntp gene cluster was subcloned into pUC119 (pKAZ132). The genes are represented by open boxes with italic type, and the portion of the vector is represented with thick lines. The shaded box represents the erythromycin resistance gene that was introduced into the SphI site of the ntpJ gene of pKAZ132 (pJEM2). The details of the plasmid manipulation and the disruption of the chromosomal gene are described under ``Experimental Procedures.'' Restriction enzyme sites were as follows: HindIII (H) and SphI (S). B, Southern hybridization. The pJEM2 (lane 3), genome DNAs isolated from 9790 (lane 2), and mutant JEM2 (lane 1) were digested with HindIII, and hybridization was performed with pJEM2 as the probe as described under ``Experimental Procedures.''
Figure 4:
The growth of strain 9790 and the ntpJ-disrupted mutant JEM2 at different pHs. Cells were
cultured in NaTY medium (pH 7.5) (A and B) or NaTY
medium (pH 7.5) supplemented with 10 mM KCl (C) (open circles), and the cell growth was monitored by the
optical density at 600 nm. At the arrows, the medium pH was
shifted to 10.0 by the addition of NaCO
(closed circles). A, strain 9790; B and C, mutant JEM2.
Fig. 4C shows the growth of
mutant JEM2 in NaTY medium where 10 mM KCl was also added. It
is noteworthy that the mutant JEM2 grew well in this medium even after
shifting the medium pH to 10.0 (Fig. 4C, closed
circles). The internal concentrations of K and
Na
of JEM2 growing at pH 10.0 in this medium were 120
and 80 mM, respectively (Table 1). It is important to
point out here that the Na
-ATPase mutant, Nak1, did
not grow in the same medium: NaTY medium supplemented with additional
10 mM KCl(33) . These results suggest that E.
hirae grows at high pH even where the internal K
concentration was relatively moderate (120 mM; Table 1) and that K
is accumulated in the cells
at high pH in an NtpJ-independent manner where the external
K
concentration is more than 12 mM. The
expected mechanism for K
transport in the ntpJ-disrupted strain is described under
``Discussion.''
Figure 5:
The KtrII activities of various E.
hirae strains. Cells were grown in NaTY medium (pH 7.5), loaded
with Na, and suspended in 50 mM Na
-CHES buffer (pH 9.0) at a cell density of 1 mg
(dry weight)/ml. The suspension was supplemented with 20 µM tetrachlorosalicylanilide and with (closed symbols) or
without (open symbols) 10 mM glucose at 0 min;
K
uptake was initiated by the addition of 1 mM KCl at 10 min as indicated by arrows. The cellular
contents of K
(circles) and Na
(squares) were determined by flame photometry. A, strain 9790; B, mutant Nak1; C, mutant
JEM2.
Figure 6:
Sodium
pumping activity of mutant JEM2. Cells were grown in NaTY medium,
harvested at late log phase, and suspended at 4 mg of cell (dry
weight)/ml in 50 mM K-HEPES buffer (pH 7.0)
containing 100 mM maleate-KOH. Sodium chloride (20
mM, 2.3125 MBq/mmol) was then added, and the cell suspension
was incubated at 25 °C for 60 min. The cell suspension was divided
into aliquots, and at 0 min, 0.4 mM DCCD, 5 µM carbonyl cyanide m-chlorophenylhydrazone, and 5
µM valinomycin were added. Sodium extrusion was initiated
by the addition of 10 mM glucose at 10 min as indicated by the arrows. Open circles, without glucose; closed
circles, with glucose.
Figure 7:
Western blotting of denatured
polyacrylamide gel electrophoresis of the cell membranes from various E. hirae strains. The cell membranes were prepared from the
parent strain 9790 (A) and JEM2 (B) grown in KTY
medium at pH 7.4 (lane 2), NaTY medium containing 0.5 M NaCl at pH 7.4 (lane 3), and NaTY medium containing 0.5 M NaCl at pH 10.0 (lane 4), respectively. Ten
micrograms of each membrane and 0.5 µg of purified
V-ATPase (lane 1) were electrophoresed and
immunoblotted with antiserum against purified V
-ATPase
(dilution 1:3000) and visualized by using the alkaline phosphatase
system.
In bacteria, the transports of K and
Na
are mediated by separate transport systems usually
linked to the chemiosmotic proton
circulation(1, 2, 3) . In E. hirae,
in addition to two Na
extrusion systems
(Na
-ATPase and Na
/H
antiporter), two distinct potassium uptake systems have been
recognized. The major one, KtrI, is thought to be constitutive and
resembles the Trk system of E. coli(37) , dependent on
the proton motive force and ATP. The second one, KtrII, which was
internal Na
-dependent, but not dependent on the
electrochemical potentials of either H
or
Na
, required ATP(30, 31) . This
system stoichiometrically exchanged Na
for
K
. The mechanism of the KtrII system has not been
clearly understood. However, since (i) the mutant that lacked the
Na
-ATPase also lacked KtrII and (ii) the KtrII and the
Na
-ATPase were induced in parallel when cells were
grown in media rich in Na
, particularly under the
conditions that limit the generation of the proton motive force,
Kakinuma and Harold proposed the simplest hypothesis, that the sodium
ATPase is KtrII itself(30) . Therefore, we expected a new
Na
,K
-ATPase in this bacterium before
finding out that the molecular structure of this
Na
-ATPase belonged to that of the vacuolar type
ATPase, an electrogenic proton
pump(24, 25, 26) . This speculation should be
now withdrawn and replaced by the following. First, the
Na
-ATPase transports Na
electrogenically, not obligatorily linked with potassium ion
transport(41) ; even in the absence of the potassium ion, the
membrane potential was generated via the electrogenic Na
flux by the Na
-ATPase. In this context, it is
noteworthy that the 16-kDa proteolipid, whose amino acid sequence is
homologous to those of several eukaryotic V-ATPase proteolipids, is the
subunit of E. hirae Na
-ATPase complex, and
the DCCD-reactive glutamic acid residue (Glu
) is present
in the fourth membrane-spanning region(21) . The
Na
-ATPase activity was inhibited by DCCD, (
)suggesting that the 16-kDa proteolipid is the electrogenic
Na
pathway. Second, in the ntpJ-disrupted
mutant in which the KtrII activity was negative (Fig. 5C), the activity of Na
-ATPase
was normal ( Fig. 6and Fig. 7). In other words, NtpJ
protein is essential for the KtrII but not functionally essential for
the Na
-ATPase reaction. Finally, the ATP hydrolytic
activity of this enzyme is activated by Na
but not
K
. The activity was not synergistically activated by
Na
and K
(30) . Although both
Na
-ATPase and NtpJ protein are essential for the KtrII
activity, these results suggest that the KtrII activity is not a part
of the Na
-ATPase activity.
The NtpJ protein is
probably an integral membrane protein having at least 10
membrane-spanning domains, whose amino acid sequence is similar to
those of the presumptive K/H
symporter proteins such as Trk1 and Trk2 of S. cerevisiae(22, 42) . Therefore, it is likely that the NtpJ
protein is the co-transporter itself. The K
gradient
(in
out) of 230 was generated in strain 9790 growing at pH 10.0
in NaTY medium containing 1 mM K
(Table 1), where the membrane potential of about -70
mV (inside negative) was generated; the intracellular pH of these cells
should be acidified to about 8.2 (43) . Since the Na
gradient (out
in) of 10 was generated in these cells (Table 1), the magnitude of the Na
electrochemical potential of -130 mV, but not the
H
gradient, is sufficient to generate the K
gradient shown in Table 1, suggesting that the NtpJ protein
may be the Na
/K
symporter. However,
in the KtrII assay (Fig. 5A), the K
gradient of about 200 was generated in strain 9790 where the
sodium potential was negligible; the Na
gradient (in
out) of 5 and the membrane potential of less than -70 mV
across the cell membrane were generated. In a previous
paper(30) , the K
gradient of at least 800 was
generated by the H
-ATPase mutant under the same assay,
where the size of the Na
potential was negligible.
These in vitro results are not consistent with the simple
secondary co-transport mechanism and suggest the possibility of the
primary pump mechanism; in this case, there should be gene(s) encoding
the other component(s), such as the ATPase catalytic subunit(s) of
KtrII, whose expression should also be regulated by the same signal for
the ntp operon. At the moment, it is hard to propose the
molecular mechanism of the NtpJ-dependent KtrII K
uptake system. To know more details of the KtrII system, we are
now working toward the isolation of the NtpJ-independent K
uptake mutant and the functional expression of the ntpJ gene in the K
transport mutant of E.
coli.
It is important to point out that the KtrII may not be
the only route of K uptake at high pH. In growing
JEM2, the K
concentration gradient of about
10-17 was generated whether or not the medium was supplemented
with additional 10 mM K
(Table 1). This
NtpJ-independent K
accumulation is enough for the
growth of this bacterium at high pH where the external K
is moderate (Fig. 4C). The action of the
Na
-ATPase is important for this K
uptake, because the Na
-ATPase mutant did not
grow under the same growth conditions as described above. We think that
the membrane potential generated by the Na
-ATPase is
the driving force for the NtpJ-independent K
accumulation, since the membrane potential of about -70 mV
was generated in JEM2; K
accumulation by JEM2 was
limited in the KtrII assay (Fig. 5C).
Thus, the
physiological function of E. hirae Na-ATPase
is to extrude Na
from cytoplasm and generate the
sodium potential, which drives active K
transport
systems at high pH where the proton motive force is minimal. It is
notable that two important genes for the cation homeostasis of E.
hirae at high pH form an operon.