Journal of General Microbiology (1982), 128,1057-1062.
Printed in Great Britain
1057
Na+-Dependent Active Transport Systems for Organic Solutes in an
Alkalophilic Bacillus
By A K I K A Z U A N D O , ' * I W A O K U S A K A 2 A N D S A K U S O F U K U 1 2
Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University, 648
Matsudo, Matsudo-city 2 71, Japan
Institute of Applied Microbiology, University of Tokyo, 1 -I -1 Yayoi, Bunkyo-ku, Tokyo 11 3,
Japan
(Received 26 August 1981; revised 13 October 1981)
Transport of nutrients (glutamate, glucose and acetate) into membrane vesicles of Bacillus sp.
A-007 was specifically dependent on the Na+ gradient (outside high). The nutrients were
co-transported with Na+, the process being stimulated by alkaline pH. In addition to the
transport process, binding of glutamate to membrane vesicles was also pH- and
Na+-dependent.
INTRODUCTION
Alkalophilic bacteria are distinguished by the uniqueness of their extracellular enzymes,
which function optimally at alkaline pH, i.e. from pH 9 to 11 (Horikoshi & Asakura, 1975;
Kitada & Horikoshi, 1976; Nakamura et al., 1975; Nakamura & Horikoshi, 1976). In a
previous study (Ando et al., 1981) we reported on some physiological properties of an
alkalophilic bacterium, strain A-007, which was isolated from soil and identified as Bacillus
sp. Respiration of glucose by the bacterium was maximal at pH 10.0, the optimum pH for the
growth of the bacterium, whereas respiration of endogenous substrate was highest at a neutral
pH. Furthermore, the proton concentration in the A-007 cells was measured to be
approximately 0.1 PM (pH 7.0), this value being similar to that in the cells of Bacillus subtilis
W-23, which grows most rapidly at near neutral pH. Thus, the alkaline preference of growth
might be associated with the transport activities.
In this paper we describe the general properties of energy-requiring systems for nutrient
transport into the cells of the alkalophilic Bacillus A-007, studied with membrane vesicles.
METHODS
Organism and cultivation. Bacillus sp. strain A-007, was grown aerobically at 42 OC in a medium containing (g
I-'): glucose, 10; polypeptone (Daigo Eiyo, Japan), 5 ; yeast extract (Difco), 5 ; K,HPO,, 1; MgSO,. 7H,O, 0.2;
and Na,CO,, 20 (pH 10.7). Cells in the mid-exponential phase of growth were collected by centrifuging (15 000 g,
5 min) and washed twice with 25 mM-HEPES/KOH buffer pH 7.4 containing 10 mM-MgC1,.
Preparation of membrane vesicles. Bacteria were resuspended in 0.1 M-sodium phosphate buffer pH 6.8
containing 0.4 M-sucrose, 10 mM-MgC1, and 0.2 mg lysozyme (Sigma) m1-I. and incubated at 37 "C. Within
10 min, more than 99 % of the cells changed into protoplasts. The protoplasts were harvested by centrifugation
(30000 g, 10 rnin), osmotically lysed by resuspending in 25 mM-HEPES/KOH buffer pH 7.4 containing
10 mM-MgC1, and 1 pg deoxyribonuclease (Sigma) m1-I. and homogenized with a Teflon homogenizer. After
removal of unbroken cells from the homogenate by low-speed centrifugation (800 g, 10 rnin), the membrane
fraction was collected by centrifugation (30000 g, 10 min). The membrane fraction was washed three times by
centrifugation with 25 mM buffer (Tris/HCI pH 7.4 or KHCO,/KOH pH 10.0) containing 0.4 M-choline chloride
Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; HEPES, N-2-hydroxyethylpiperazine"-2-ethanesulphonic acid.
0022-1287/82/0001-0131 $02.00 01982 SGM
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1058
A. A N D O , I. K U S A K A A N D S. F U K U I
and 10 mM-MgC1,. Protein concentration was determined by the Lowry method, using bovine serum albumin as
standard.
Uptake of organic nutrients by membrane vesicles. Nutrient uptake by membrane vesicles was measured at
37 "C by a filtration method. The uptake medium contained 25 mM-buffer (HEPES/KOH pH 7.4 or
KHCO,/KOH pH 10.0), 10 mM-MgCl,, membrane vesicles (0.25 mg protein), 0.4 M salt (NaCl, KCl, LiCl or
choline chloride) and I4C-labelled substrate, in 1.O ml total volume. The substrate concentration in the uptake
, 1 GBq mol-'; D-glucose - 1 10 p ~ 370
, GBq mol-'; acetate mixture was as follows: L-glutamate - 87.8 p ~ 85
57.3 p ~ 630GBq
,
mol-'. Uptake was started by the addition of substrate to pre-incubated uptake mixture at
37 "C. At suitable intervals, 100 pl samples were filtered through membrane filters (pore size 0.45 prn; Toyo
Roshi, Japan). Membrane vesicles on the filters were washed four times with 2-5 ml of the uptake mixture without
substrate and dried. Radioactivity on the filter was measured in a gas-flow counter. Uptake rates were calculated
from the linear portion of uptake curves (i.e. from the radioactivity taken up in 1 min) and expressed as nmol
substrate accumulated (mg protein)-' min-I.
L-Glutamate binding. Measurement of L-glutamate binding to membrane vesicles was done according to
Kennedy et al. (1974). Membrane vesicles, prepared as described above, were washed and suspended in
25 mM-potassium phosphate pH 7.5 or HEPES/KOH pH 7.0 buffer containing 10 mM-MgC1, and 0.4 M-choline
chloride. The membrane vesicles (0.3 mg protein) were added to a binding mixture (total 0.5 ml) containing
48 p ~ - ( ' ~ C I g l u t a m a(9-5
t e TBq mol-'), 10 mM-MgCI,, ionophores [20 pg valinomycin and 20 pg monensin ml-'
(see Fig. 5)1,40 p ~ - C C C P(see Fig. 6), 25 mM buffer [potassium phosphate pH 7.5 (see Fig. 5); HEPES/KOH for
pH 7.0 to 7-4, Tris/HCl for pH 8.0 to 8.9, KHCO,/KOH for pH 9.5 to 10-5 (see Fig. 6)l. After incubation at
37 "C for 20 min, the vesicles were precipitated by centrifugation (105000g, 30 min). The amount of
membrane-bound glutamate was calculated by subtracting the radioactivity of the supernatant after centrifugation
from that of the mixture before centrifugation.
Radioactive compounds and chemicals. L-I ''C1Glutamic acid and D-[ 14Clglucosewere purchased from New
England Nuclear; [l4Clacetate was from Daichi Kagaku (Japan); CCCP was from Sigma. Monensin was a
generous gift from Dr N Ootake (Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan). Other
chemicals were of the best grade commercially available.
RESULTS
Membrane vesicles prepared as described above were suspended in 25 mM-HEPES/KOH
buffer pH 7.4 or KHCO,/KOH buffer pH 10.0, containing 0 - 4 M cation (Na+, Li+, K+ or
choline). As shown in Fig. 1, glutamate was accumulated significantly into the vesicles in the
presence of the Na+ gradient (in < out), and the activity was higher at alkaline pH.
Accumulation of glucose was also dependent on the Na+ gradient (in < out), and the activity
was highest at alkaline pH (Fig. 2).
The effect of Nat concentration on the rate of glutamate transport was examined. The rate
was determined at different concentrations of NaCl added to the reaction mixture. The
dependence on Na+ was strictly hyperbolic, the apparent K , for Na+ being approximately
13 mM. The apparent K , for glutamate uptake when 0.1 M-NaCl was present in the assay
mixture was estimated to be approximately 26 IM.
As shown in Figs 3 and 4, glutamate and glucose transport systems in Bacillus A-007
required an Na+ gradient. Uptake of both compounds could be shown with membrane
vesicles on which an Na+ gradient (in < out) was imposed. CCCP and valinomycin did not
inhibit the activities, rather a stimulation of glutamate transport was seen in the presence of
valinomycin. Monensin, an ionophore for Na+/H+ exchange, inhibited transport of both
glucose and glutamate (Table 1). Acetate transport was tested as an example of organic acid
transport in BaciZlus A-007. Acetate uptake was observed in vesicles on which an artificial
Na+ gradient (in < out) was imposed and the activity was inhibited by monensin (Table 1).
Glutamate binding to partially purified carrier of B. subtilis W23 was found to be
stimulated by Na+ (I. Kusaka, unpublished observation). Hence we also tested the
Na+-dependent binding of glutamate to the membranes of Bacillus A-007. As shown in Fig. 5 ,
in the absence of added Na+, glutamate was bound only in an amount of 20pmol (mg
protein)-' and the amounts increased when the Na+ concentration was increased up to 0.4 M.
Under these conditions about 120 pmol glutamate (mg protein)-' was bound. Glutamate
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Na+-dependent transport systems of an alkalophile
f
1
Time (min)
2
1059
2
1
Time (min)
Fig. 1
Fig. 2
Fig. 1. Effect of pH on Na+-driven glutamate transport into membrane vesicles. Vesicles were preloaded
with 0-4M-choline chloride containing 10 mM-MgC1, and 25 mM buffer (HEPES/KOH pH 7.4 or
KHCO,/KOH pH 10.0). The assay mixture contained 25 mM buffer (HEPES/KOH pH 7.4 or
KHCO,/KOH pH IO-O), 10 mM-MgCl,, 87.8 p ~ -''C]glutamate
[
(85 1 GBq mol-'), 0.4 M-NaCl and
membrane vesicles (0.25 mg protein) in a total volume of 1.0 ml (at 3 7 "C). H, pH,, = pH,,, = 7.4; 0,
PH,, = 10.0, pH,,, = 7.4; 0 ,pH,, = 7.4, pH,,, = 10.0; 0,
pHi, = pH,,, = 10.0.
Fig. 2. Effect of pH on Na+-driven glucose transport into membrane vesicles. Assay conditions were
similar to those for Fig. 1, except ['4C]glucose (1 10 p ~ 370
; GBq mol-I) was used as substrate.
Symbols as in Fig. 1.
binding to the membranes was pH-dependent, like the transport reaction. An example of
glutamate binding at different pH values is shown in Fig. 6. In this case, Na+ was not added.
The optimum pH for the binding was 10.0.
DISCUSSION
Na+-dependent transport systems for amino acids have been reported in several kinds of
bacteria (Sprott & MacLeod, 1972; Fein & MacLeod, 1975; Pearce et al., 1977) and
Na+-dependent transport of glutamate has been found in enteric bacteria (MacDonald et al.,
1977; Tsuchiya et al., 1977; Hassan & Tsuchiya, 1977). However, in these bacteria the
transport of neutral and basic amino acids was primarily H+-dependent (Harold, 1977). On
the other hand, in halophilic bacteria, especially Halobacterium, most of the transport
systems, including those of organic nutrients, have been shown to be Na+-dependent (Lanyi,
1978). This may suggest a convergent evolution between halophilic bacterial membranes and
membranes of eukaryotic cells.
In recent years, several kinds of alkalophilic bacteria have been isolated from several
sources and the character of their amino acid transport systems has been studied. Guffanti et
al. (1978) reported that the a-aminoisobutyric acid (AIB) transport system in Bacillus
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1060
A. ANDO, I. KUSAKA AND S. FUKUI
150 I
2
1
3
2
1
Time (min)
Time (min)
Fig. 3
Fig. 4
3
Fig. 3. Specificity of cations in driving glutamate transport. Membrane vesicles were prepared in a
medium containing 25 mM-KHCO,/KOH pH 10.0, 10 mM-MgCl,, 0.1 M-NaCl and 0-3 M-choline
chloride. The assay mixture (1.0ml) contained 25 mM-KHCO,/KOH buffer pH 10.0,10 mM-MgCl,,
1'4C1glutamate (87.8 p ~ 851
; GBq mol-I) and salts in a total concentration of 0.4 M. Salts in the
external medium were as follows: 0-4M-NaCl, 0;0.3 M-NaCl and 0.1 M-choline chloride, A; 0.2
M-NaCl and 0.2M-choline chloride, U; 0.4M-KCI, W; 0.4M-LiCl, A; 0-4M-choline chloride and HCI
to acidify the mixture to pH 7.0, a,,
Fig. 4.Specificity of cations as a drive force of glucose transport. The assay mixture (1-0 ml) contained
25 mM-KHCO,/KOH pH 10.0,10 mM-MgCl,, ['4Clglucose (1 10 p ~ 370
; GBq mol-'), membrane
vesicles (0.25mg protein) and 0.4M salt. Membrane vesicles were prepared in the same way as for
Fig. 3. Salts in the external medium were as follows: 0.4M-NaCl, 0; 0.4M-KCl, A; 0.4M-LiCl, A;
0.4M-choline chloride and HCl to acidify the mixture to pH 7.0, 0.
Table 1 . Effect of ionophores on transport of L-glutamate, D-glucose and acetate into
membrane vesicles driven by an Na+ gradient
The control uptake rates (100%) were [nmol (mg protein)-' min-'I: glutamate, 0.32;glucose, 0.42;
acetate, 0.25.Transport at pH 10.0was driven by 0.4M-NaCI.
Relative activity (%)
,
\
Ionophore
L-Glutamate
D-Glucose
Acetate
None
CCCP
Valinom ycin
Monensin
100
116
198
8.1
100
82
84
0.1
100
64
60
14
alkalophilus was Na+-dependent whereas that of P-galactoside was ATP-dependent.
Na+-dependent AIB and amino acid transport systems in alkalophilic bacteria were also
reported by Kitada & Horikoshi (1977, 1980a, b). As shown in the present paper, a number
of active transport systems in the alkalophilic Bacillus A-007 appear to be Na+-dependent.
The acetate transport in Bacillus A-007 was somewhat complicated, judging from its
sensitivity to several ionophores. However, the data in Table 1 indicate that more than 50%
of the acetate was accumulated by an Na+ gradient system.
Binding of L-glutamate to membrane vesicles of Bacillus A-007 was found to be an Na+and pH-dependent phenomenon. It needs further investigation to verify whether the binding
observed in the present study reflects the process of active transport. Substrate binding to the
carrier protein is one of the important steps in the active transport process and the glutamate
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Na f-dependent transport systems of an alkalophile
I"
0-1
0.3
NaCl concn (M)
0.5
7
1061
8
9
10
PH
Fig. 5
Fig. 6
Fig. 5 . Na+ dependency or' glutamate binding to membrane vesicles. Assay mixture (0.5 ml) contained
membrane vesicles (0.3 mg protein), 25 mM-potassium phosphate buffer pH 7.5, valinomycin (20 pg
ml-I), monensin (20 pg ml-I), 48 p~-['~CIglutamate
(9.5 TBq mol-') and 0.5 M salt (NaCl and choline
chloride). Membrane vesicles were prepared in medium containing 25 mM-potassium phosphate buffer
pH 7.5, 10 mM-MgC1, and 0.5 M-chohe chloride.
Fig. 6. pH dependency of glutamate binding to membrane vesicles. Assay mixture (0.5 ml) contained
membrane vesicles (0.3 mg protein), 10 mM-MgCI,, 0.4 M-choline chloride, 25 mM buffer (HEPES/
KOH for a pH range of 7.0 to 7.4, Tris/HCl for a pH range of 8.0 to 8-9, KHCOJKOH for a pH
range of 9 - 5 to l0-5), 40 p ~ - C C C Pand 48 p ~ - [ ~ ~ C I g l u t a m(9.5
a t e TBq mol-I). Membrane vesicles
were prepared in medium containing 25 mM-HEPES/KOH pH 7.4, 10 mM-MgC1, and 0.4 M-choline
chloride.
binding to the membranes observed in the present study may include the specific
glutamate-carrier protein binding. In this respect, it is of interest to note the Na+ stimulation
of the binding.
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