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Applied and Environmental Microbiology, October 1998, p. 3813-3817, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Quantitative and Physiological Analyses of Chloride Dependence
of Growth of Halobacillus halophilus
Markus
Roeßler and
Volker
Müller*
Lehrstuhl für Mikrobiologie,
Ludwig-Maximilians-Universität München, 80638 Munich,
Germany
Received 16 March 1998/Accepted 27 July 1998
 |
ABSTRACT |
A quantitative analysis of the Cl
dependence of
growth of Halobacillus halophilus was performed. Optimal
growth rates were obtained at Cl concentrations of
between 0.5 and 2.0 M, and the final yield was also strictly dependent
on the Cl concentration. Br but not
I, SO42,
NO2, SO2,
OCN, SCN, BO2, or
BrO3 could substitute for Cl.
To analyze the function of chloride, chloride concentration was
determined. At low external Cl (Cle)
concentrations, the growth rate was low and Cl was
excluded from the cytoplasm; increasing the Cle
concentration led to an increase in the growth rate and an
energy-dependent uptake of Cl, thus decreasing the
Cle/internal Cli gradient from 
10
at 0.1 M Cle to a nearly constant value of 2 at
Cle concentrations which allowed optimal growth. Two
membrane proteins with apparent molecular masses of 31 and 16 kDa which
were identified to be specific for Cl-grown cultures are
possible candidates for a chloride uptake system.
| |
INTRODUCTION |
The North Sea coast of Germany is
characterized by intertidal flats (Wadden Sea) that are flooded
regularly with seawater. The foreland or salt marshes are established
by slowly sedimenting material and subsequent colonialization by
salt-tolerating plants. Salt marshes, due to their higher elevation,
are flooded only 100 to 200 times a year. One of the major factors in
this environment which undergoes frequent changes is the salt
concentration. Due to a more or less constant influx of salt via the
sea wind, as well as floodings with subsequent evaporation of seawater,
the salt concentration in the salt marshes can be as high as 10%, whereas the average salt concentration in the North Sea is
approximately 3.5%. On the other hand, after heavy rainfalls it can
decrease to freshwater concentrations.
Seawater contains 550 mM Cl, which is equivalent to 95%
of the anions present in seawater, and, therefore, chloride
concentration is one of the chemical factors in salt marshes which
undergo drastic changes. However, whether chloride is important for the
physiology of marine or salt marsh bacteria or whether it is even
involved in signaling and adaptation processes remains almost
unexplored. Already in 1956, MacLeod and coworkers reported the
isolation of Cl-dependent organisms from seawater
(9, 10); however, the organisms were not further
characterized with respect to the nature of chloride dependence, and,
unfortunately, they are no longer available in culture collections.
Claus et al. reported the isolation of an aerobic, endospore-forming
bacterium, Halobacillus halophilus (formerly
Sporosarcina halophila), from salt marshes. In their original description, they described that H. halophilus
grows optimally at approximately 0.5 to 0.9 M NaCl and that "chloride cannot be replaced by sulfate" (2). Unfortunately, it was
not determined whether growth failure was due to a lack of chloride or
to a growth inhibition by sulfate, quantitative measurements were not
done, and the physiology of the Cl dependence was not
analyzed. To unequivocally identify a chloride dependence of the salt
marsh bacterium H. halophilus, we performed a detailed
physiological characterization. This study unequivocally identifies chloride as an obligatory ion for H. halophilus, quantifies the requirements for chloride,
reveals Br and NO3 as a
substitute for Cl, indicates the presence of a chloride
uptake system, and identifies two membrane-bound polypeptides which are
possible candidates for the Cl pump.
| |
MATERIALS AND METHODS |
Organism, cultivation, and growth experiments.
H.
halophilus (DSM 2266) was maintained on nutrient broth (NB)
(Difco) supplemented with 0.05 M MgSO4 and 0.5 M NaCl or
0.5 M NaNO3 (in case the NO3
dependence of growth was to be determined). Growth experiments were
done in 15-ml tubes filled with 5 ml of the indicated medium. After
inoculation (1%), the cultures were incubated on a rotary shaker at
30°C. The optical density at 600 nm (OD600) was
determined in a Bausch and Lomb photometer. To determine the pH
optimum, 0.05 M Tris (pH 7 to 10.5) or 0.05 M succinate (pH 5 to 6.5)
was added to the medium, and the pH was adjusted as indicated in the experiments by the addition of NaOH or H2SO4.
All data points given reflect the means of duplicate tubes from one
experiment, and experiments were performed at least three times.
Preparation of cell suspensions and determination of chloride
concentrations and internal volume.
For preparation of cell
suspensions, NB with the indicated NaCl concentrations was inoculated
(1%) from cultures maintained at 0.5 M NaCl. Fresh cell suspensions
were prepared for each experiment. Cells in the late logarithmic growth
phase were harvested by centrifugation and washed once with 0.05 M Tris
containing 0.05 M MgSO4 and NaCl as indicated. The cell
pellet was resuspended in the same buffer to a concentration of 2 to 5 mg/ml and stored on ice until use. The protein concentration of the
cell suspension was determined by the method described by Schmidt et
al. (20), with bovine serum albumin as a standard.
The experiments were carried out in Eppendorf tubes filled with 0.5 ml
of 0.05 M Tris (pH 7.8), 50 µl of the concentrated cell suspension
(15 to 25 mg of protein/ml), NaCl concentrations as indicated, and 10 µl of Na36Cl (specific activity, 50 µCi/mmol and 0.45 µCi). After incubation at room temperature on a rotary shaker for 15 min, seven samples of 50 µl each were taken and used to calculate
internal Cl (Cli) concentrations. The
cell suspension then received valinomycin (final concentration, 100 µM) and K2SO4 (final concentration, 0.125 M)
and was incubated for 10 min. This treatment dissipated the membrane
potential and led in any case to a collapse of the external
Cl (Cle)/Cli
gradient, indicating that only freely permeable and not
cell-surface-bound Cl was measured. Samples were filtered
through nitrocellulose filters (25 mm in diameter, pore size, 0.45 µm; Sartorius, Göttingen, Germany) and washed three times with
0.05 M Tris. Sampling and washing were done in less than 15 s.
Nonspecific binding of 36Cl was reduced by
incubation of the filters overnight in 0.05 M Tris (pH 7.8) containing
0.5 M NaCl; all values were corrected for nonspecific binding of
Cl to the filters. The filters were air dried, and
radioactivity was determined in a liquid scintillation counter type
PW4700 (Philipps, Hamburg, Germany) by using ultima gold (Packard,
Dreieich, Germany) as the scintillation cocktail.
The Cl
i concentration was calculated by using the
internal volume determined by silicone oil centrifugation as described
elsewhere
(
17).
3H
2O and
[
14C]dextrane were used as markers for the total and
external water
spaces, respectively. A 10-µCi amount of
3H
2O and 1 µCi of [
14C]dextrane
were added to 10 ml of cell suspension. It was confirmed
that the
sugars were not taken up by the cells.
Preparation of cell extracts and SDS-PAGE.
Precultures were
grown for at least two transfers with the anion at the concentration
indicated and then used to inoculate 200 ml of NB containing 50 mM
MgSO4 and the anion as sodium salt. The cells were
harvested at an OD600 of 0.5, washed twice with 0.05 M Tris
(pH 7.8), resuspended to a final concentration of 2.5 to 4 mg/ml in
0.05 M Tris (pH 7.8), and broken up in a French pressure cell. Cell
debris was removed by low-speed centrifugation, and the resulting cell
extract was then centrifuged at 110,000 × g and 4°C
for 1 h. The pellet was resuspended in 2 ml of 0.05 M Tris (pH
7.8) to a final protein concentration of 1.5 to 2.5 mg/ml. The protein
content was determined by the method described by Lowry et al.
(8), and 10 µg of protein was applied to a sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel according to
the method described by Schägger and von Jagow (19)
with 12.5% gels. Silver staining was done as described elsewhere
(1).
| |
RESULTS |
Dependence of growth on NaCl, Na+, and
Cl.
To perform a quantitative analysis of the
dependence of the growth of H. halophilus on salt
concentration, NB was supplemented with different concentrations of
NaCl. Growth was impaired in the absence of salt, but the growth rate
increased sharply with increasing NaCl concentrations. The highest
growth rates were obtained between 0.5 and 2.0 M NaCl, but even
concentrations of as much as 2.5 and 3.0 M were tolerated with growth
rates of 50 and 38%, respectively. In addition, the final yield was
dependent on the NaCl concentration (data not shown). To determine the
dependence on salt in more detail, single components of the medium were
substituted, while the ion concentration in the medium was kept
constant. First, the Na+ concentration was varied while the
total salt concentration was kept at least at 0.5 M by the appropriate
addition of KCl. Growth was strictly dependent on Na+, and
maximal growth rates were obtained at concentrations of between 0.5 and
2.0 M (data not shown). Na+ could not be substituted by
Li+ or K+. Of particular interest was the
effect of Cl on the growth of H. halophilus. To determine Cl dependence, the NaCl
concentration was varied from 0 to 1.0 M, while the total salt
concentration was kept at least at 0.5 M by the appropriate addition of
NaNO3. As can be seen from Fig. 1, growth was impaired in the absence of
Cl, but increasing Cl concentrations led to
increasing growth rates. Saturation was obtained at 0.5 to 1.0 M
Cl. Interestingly, the final yield was also strictly
dependent on the Cl concentration, but the dependence
showed a sigmoidal character; at 0.2 M Cl, the final
yield was only 1/10 of the maximal yield obtained at 0.5 M
Cl (Fig. 1).
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FIG. 1.
Cl dependence of growth of H. halophilus. (A) Growth determined in NB (pH 7.8), containing 0.05 M MgSO4 and 0 ( ), 0.1 ( ), 0.15 ( ), 0.2 ( ), 0.25 ( ), 0.3 ( ), 0.4 ( ), or 0.5 ( ) M NaCl. Salt concentration
was maintained at least at 0.5 M by appropriate addition of
NaNO3. (B) Growth rates plotted against Cl
concentration. (C) Cell yields (final OD600) plotted
against the Cl concentration. Precultures were maintained
in NB containing 0.5 M NaCl.
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|
To confirm that the observed dependence was indeed due to a stimulation
by chloride and not an inhibition by nitrate (which
was used to keep
the osmolarity constant), the following controls
were performed. First,
addition of 0.5 M NaCl to a culture containing
0.5 M NaNO
3
led to an immediate onset of growth indistinguishable
from that of
cultures maintained at 0.5 M NaCl. Second, when the
total anion
concentration was kept constant at 1.0 M by the appropriate
addition of
Na
2SO
4, the same chloride dependence was
observed.
Again, addition of NaCl to a culture containing
Na
2SO
4 led to
growth indistinguishable from
that of cultures maintained at 0.5
M NaCl. These experiments
unequivocally demonstrate a dependence
on chloride for growth and
biomass production of
H. halophilus.
The amounts of
chloride, NaCl, and Na
+ that were required for growth as
well as that were tolerated
were in the same range and reflect the
chemical composition of
the salt marshes.
To investigate the dependence on chloride in more detail, we analyzed
whether other halides or anions could support growth.
Of the compounds
tested, only Br
and Cl
but not
I
, SO
42,
NO
2, SO
2,
OCN
, SCN
, BO
2, or
BrO
3 supported growth of
H. halophilus. The growth rates and final
yields obtained with
chloride or bromide were alike. Therefore,
H. halophilus is the first aerobic bacterium for which a chloride
dependence has been shown unequivocally, and, most interestingly,
chloride can be substituted by another halide, bromide.
Determination of Cli concentrations.
To
analyze the function of chloride in H. halophilus,
Cli concentrations at different Cle
concentrations were determined. In order to calculate intracellular concentrations accurately, the intracellular volumes of the cells at
different osmolarities were determined. As can be seen from Fig.
2, the intracellular volumes of
H. halophilus varied with the external salt
concentration by 40%, from 2.6 to 1.6 µl/mg of protein. To determine
the Cli concentration in H. halophilus, cell suspensions were incubated with
Na36Cl and allowed to equilibrate, and the
Cli concentration was then determined during the
steady state of endergonic respiration. In the presence of 0.5 M
Na36Cl, the Cli concentration was
determined to be 0.08 M; upon dissipation of the membrane potential by
the addition of K2SO4 plus valinomycin, Cl entered the cells immediately within the time
resolution of the experiment until Cle and
Cli equilibrated (Cli = 0.472 M
[mean of seven experiments]). These experiments demonstrate that
Cl was freely permeable across the cytoplasmic membrane,
and the low Cli concentrations measured are therefore
not due to a restricted permeability of the
36Cl tracer used.
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FIG. 2.
Internal volume of H. halophilus at
increasing external NaCl concentrations. For explanations, see
Materials and Methods. The data points reflect the means of three
independent experiments.
|
|
It is evident from Fig.
3 that at low
Cl
concentrations which do not support growth (0.1 and
0.2 M), Cl
is excluded from the cytoplasm
(Cl
e/Cl
i = 10). The
Cl
e/Cl
i gradient of 10 has to be
considered minimal, since the values
determined for
Cl
i at low Cl
e are very close to
unspecific binding and, therefore, might be
less than those determined.
However, increasing the Cl
e concentration from 0.1 to
0.5 M led to growth stimulation and,
concomitantly, to an uptake of
Cl
into the cytoplasm, thereby decreasing
Cl
e/Cl
i continuously up to a value
of 2 at a Cl
e of ca. 1.2 M. A further increase of
Cl
e to as much as 2.0 M led to a nearly proportional
uptake of Cl
, thereby keeping
Cl
e/Cl
i fairly constant at 2. These
experiments demonstrate that chloride
is excluded from the cytoplasm at
low Cl
e. Increasing the Cl
e
concentration resulted not only in a stimulation of growth but,
concomitantly, in an uptake of chloride which, based on thermodynamic
calculations, was energy dependent (see Discussion).
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FIG. 3.
Cli concentrations () and
Cle/Cli gradients () at
increasing external NaCl concentrations. For explanations, see
Materials and Methods.
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|
Adaptation of H. halophilus to chloride-free
media.
After heavy rainfalls, the salt concentration in salt
marshes can decrease to freshwater concentrations and, therefore, we tested whether H. halophilus is able to adapt to salt-
or chloride-free conditions. Even after incubations for as much as 4 weeks, H. halophilus did not grow in the absence of
NaCl, indicating an obligate requirement for salt. However, when NaCl
was replaced by NaNO3, approximately 90% of the cultures
did grow after a variable adaptation time of approximately 30 h
(Fig. 4). The protein compositions of 10 cultures examined were identical, as judged by SDS-PAGE, and
microscopic examination of spores embedded in agar revealed that growth
in nitrate was not restricted to few cells but rather was a common
event (4a), indicating adaptation rather than mutation and
selection as a basis for this effect. Growth of
NO3-adapted cells was still strictly
dependent on the salt concentration, but at any given NaNO3
concentration the growth rate and the final yield were smaller in
NaNO3- than in NaCl-containing medium (Fig. 4). Upon
retransfer of nitrate-adapted cells into Cl-containing
medium, cells grew without any lag phase and the growth rate was less
than that in Cl-maintained cells but greater than that in
NO3-adapted cells (data not shown). Under the
same conditions, the cultures did not adapt to sulfate. These results
demonstrate the potential of H. halophilus to adapt to
changing environmental conditions, although the nitrate concentration
in the salt marshes might be too low to sustain growth.
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FIG. 4.
Adaptation of H. halophilus to
Cl-free but NO3-containing
media. (A) Cells grown in NB (pH 7.8) containing 0.05 M
MgSO4 and 0.5 M NaCl (squares) or NaNO3
(circles); (B) growth rates of NO3-maintained
cultures plotted against NO3
concentrations.
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|
Cl-induced proteins in H. halophilus.
To determine chloride-induced proteins, cell extracts were prepared
from cultures grown in Cl or
NO3 media, separated into cytoplasm and
membrane fractions, and subjected to SDS-PAGE. As can be seen from Fig.
5, Cl-grown cultures
exhibited a polypeptide pattern different from those of
NO3-grown cultures. The cytoplasm of
Cl-grown cultures contained a 32-kDa polypeptide
apparently absent in NO3-grown cells. In
addition, membranes of Cl-grown cells exclusively
contained two polypeptides with molecular masses of 31 and 16 kDa. The
26- and 23-kDa polypeptides did not appear consistently in
Cl-grown cells, indicating the necessity of other
induction factors; however, if present, synthesis of at least the
23-kDa polypeptide was regulated by Cl. Apparently, there
are no indications for polypeptides exclusively found in
NO3-grown cells, as revealed by SDS-PAGE. An
increase in the Cle concentration led to a steady
increase in the membrane-bound 16-kDa polypeptide, whereas the
concentration of the 32-kDa cytoplasmic polypeptide reached a constant
level at 1.0 M Cl. On the other hand, the membrane-bound
31-kDa polypeptide was maximal at 0.25 and 0.5 M Cl but
steadily decreased with further increasing Cl
concentrations. These results clearly show the presence of
chloride-induced proteins (two membrane bound and one cytoplasmic)
whose synthesis is regulated by the Cle
concentration.
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FIG. 5.
SDS-PAGE of cytoplasm and membranes of H. halophilus. Cells were grown in NB plus 0.05 M MgSO4
(pH 7.8) containing 0.5 M NaNO3 (lanes 2 and 7), or 0.25 (lanes 3 and 8), 0.5 (lanes 4 and 9), 1.0 (lanes 5 and 10), or 2.0 (lanes 6 and 11) M NaCl. Cultures were grown for at least two transfers
in the medium indicated. Standards are shown in lanes 1 and 12. Chloride-induced proteins and their apparent molecular masses are
indicated by arrows.
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|
| |
DISCUSSION |
Living organisms depend on a variety of ions for growth,
metabolism, and energy generation, and a requirement for a special ion
might result from the adaptation of a microorganism to its environment.
NaCl is present in almost all environments, and a biological role of
Na+ is well documented in all kingdoms of life (4,
13). Chloride, on the other hand, is essential for eucaryotes, in
which it is required for solute transport, osmoregulation, and
maintaining electrical balances (11), but there are only a
few examples showing that chloride is involved in cellular functions in
procaryotes. Microorganisms growing at elevated salt concentrations are
faced with the problem of keeping the water activity of their cytoplasm high (7, 15), and two types of osmoadaptation are known
(6). First, compatible solutes which do not interfere with
the central metabolism are synthesized. Second, salts are accumulated
from the medium until the internal salt concentration equals the
external one (the so called salt-in-cytoplasm mode of osmoadaptation). The major cations taken up are K+ and Na+,
along with Cl as the major anion. Uptake of the
negatively charged chloride against the electrical field (inside
negative) is energy dependent, and halobacteria employ two modes for
Cl uptake, i.e., the light-driven Cl pump
halorhodopsin (14, 21) and a light-independent
Na+/Cl symporter (5). Since
H. halophilus is also slightly halophilic, one could
speculate that chloride is taken up in the course of osmoadaptation by
the salt-in-cytoplasm type. However, studies by Trüper, Galinski
and coworkers revealed that H. halophilus, grown at
10% salinity (1.7 M NaCl) in synthetic or complex medium, synthesizes
different compatible solutes at concentrations of 0.8 µmol/mg (dry
weight) (6, 22). Taking into account the determined internal
volume, this would correspond to an internal concentration of about 1.0 M. Although a quantitative discussion is always difficult in view of
the inherent problems with the measurements, this calculation makes a
major role of chloride in osmoadaptation highly unlikely.
However, a contribution of chloride to the mechanism of
osmoadaptation cannot be excluded, and it is noteworthy in this
connection that certain halophiles such as Pseudomonas
halosaccharolytica and Bacillus
haloalkaliphilus, which produce compatible solutes, also
take up chloride. P. halosaccharolytica keeps its internal
chloride concentration fairly constant at 0.7 to 1.0 M at
Cle values of 1 to 3 M (12). On the other
hand, B. haloalkaliphilus, like H. halophilus, excludes chloride from the cytoplasm at low Cle concentrations but takes it up at higher
Cle concentrations, thereby decreasing
Cle/Cli from 5.8 at 0.85 M
Cle to 1.3 at 3.4 M Cle
(23).
The observation that cell yield is also dependent on chloride
concentration indicates a structural role of Cl. A
possible function could be a stabilization of enzymes or protein complexes, as seen, for example, with the photosystem II of plants or
cyanobacteria (3). It is noteworthy that photosystem II is
stabilized not only by chloride but also by bromide or nitrate, a
striking similarity to the anion dependence of H. halophilus. In addition, it was reported that photosynthesis in
cyanobacteria (and plants as well) is dependent on chloride (3,
16), and an active, ATP-dependent uptake of Cl was
demonstrated (24).
It is evident from the data presented that chloride is taken up upon
increase of the growth rate. This uptake is energy dependent. The
magnitude of the membrane potential of H. halophilus is
approximately 
180 mV (16a), and if chloride is in
equilibrium with the membrane potential, it would sustain a
Cle/Cli gradient of 1,000. However,
we determined a Cle/Cli gradient of
approximately 2 under optimal growth conditions; therefore, this is
clear evidence that chloride is taken up against the electrical field
in an energy-dependent fashion. The nature of the driving force is
unknown, but N-terminal sequencing of the membrane proteins found
specifically in halide-grown cells will facilitate identification of
the transporters.
Whether chloride dependence is a striking feature of salt marsh or
other halotolerant bacteria and whether chloride is involved in
signaling of and adaptation to environmental changes such as salt
concentration remain open questions. Recently, chloride-inducible genes
in Lactococcus lactis were identified; however, the
functions of the gene products are unknown and it is not known whether
chloride is essential for growth of L. lactis
(18). Future studies are aimed at identifying the role of
chloride in the physiology of H. halophilus in light of
its environment, i.e., salt marshes.
| |
ACKNOWLEDGMENTS |
We are indebted to D. Claus, Göttingen, Germany, for
drawing our attention to H. halophilus, for fruitful
and stimulating discussions, and for advice. The hospitality and
helpful advice with the 36Cl experiments of
the staff of the Zentrales Isotopenlabor der Universität Göttingen are gratefully acknowledged.
This work was supported by a grant from the Fonds der Chemischen
Industrie.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Lehrstuhl
für Mikrobiologie, Ludwig-Maximilians-Universität
München, Maria-Ward-Str. 1a, 80638 Munich, Germany. Phone:
49-8917919836. Fax: 49-8917919855. E-mail:
v.mueller{at}lrz.uni-muenchen.de.
| |
REFERENCES |
| 1.
|
Blum, H.,
H. Beier, and H. J. Gross.
1987.
Improved silver staining of plant proteins, RNA, and DNA in polyacrylamide gels.
Electrophoresis
8:93-98.
|
| 2.
|
Claus, D.,
F. Fahmy,
H. J. Rolf, and N. Tosunoglu.
1983.
Sporosarcina halophila sp. nov., an obligate, slightly halophilic bacterium from salt marsh soils.
Syst. Appl. Microbiol.
4:496-506.
|
| 3.
|
Critchley, C.
1985.
The role of chloride in photosystem II.
Biochim. Biophys. Acta
811:33-46.
|
| 4.
|
Dimroth, P.
1997.
Primary sodium ion translocating enzymes.
Biochim. Biophys. Acta
1318:11-51[Medline].
|
| 4a.
| Dohrmann, A.-B., and V. Müller. Unpublished
data.
|
| 5.
|
Duschl, A., and G. Wagner.
1986.
Primary and secondary chloride transport in Halobacterium halobium.
J. Bacteriol.
168:548-552[Abstract/Free Full Text].
|
| 6.
|
Galinski, E. A., and H. G. Trüper.
1994.
Microbial behaviour in salt-stressed ecosystems.
FEMS Microbiol. Rev.
15:95-108.
|
| 7.
|
Lanyi, J. K.
1990.
Light-driven primary ionic pumps, p. 55-86.
In
T. A. Krulwich (ed.), Bacterial energetics. Academic Press, San Diego, Calif.
|
| 8.
|
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr, and R. J. Randall.
1951.
Protein measurement with the folin-phenol reagent.
J. Biol. Chem.
193:265-275[Free Full Text].
|
| 9.
|
MacLeod, R. A., and E. Onofrey.
1956.
Nutrition and metabolism of marine bacteria II. Observations on the relation of sea water to the growth of marine bacteria.
J. Bacteriol.
71:661-667[Free Full Text].
|
| 10.
|
MacLeod, R. A., and E. Onofrey.
1957.
Nutrition and metabolism of marine bacteria VI. Quantitative requirements for halides, magnesium, calcium, and iron.
Can. J. Microbiol.
3:753-759.
|
| 11.
|
Mannino, J.
1995.
Human biology.
Mosby, St. Louis, Mo.
|
| 12.
|
Masui, M., and S. Wada.
1973.
Intracellular concentrations of Na+, K+, and Cl of a moderately halophilic bacterium.
Can. J. Microbiol.
10:1181-1186.
|
| 13.
|
Müller, V.,
M. Blaut,
R. Heise,
C. Winner, and G. Gottschalk.
1990.
Sodium bioenergetics in methanogens and acetogens.
FEMS Microbiol. Rev.
87:373-377.
|
| 14.
|
Oesterhelt, D.
1995.
Structure and function of halorhodopsin.
Isr. J. Chem.
35:475-494.
|
| 15.
|
Oren, A.
1994.
The ecology of the extremely halophilic archaea.
FEMS Microbiol. Rev.
13:415-439.
|
| 16.
|
Putnam-Evans, C., and T. M. Bricker.
1997.
Site-directed mutagenesis of the basic residues 321K to 321G in the CP47 protein of photosystem II alters the chloride requirement for growth and oxygen-evolving activity in Synechocystis 6803.
Plant Mol. Biol.
34:455-463[Medline].
|
| 16a.
| Roeßler, M., and V. Müller. Unpublished data.
|
| 17.
|
Rottenberg, H.
1979.
The measurement of membrane potential and  pH in cells, organelles and vesicles.
Methods Enzymol.
55:547-569[Medline].
|
| 18.
|
Sanders, J. W.,
K. Leenhouts,
J. Burghoorn,
J. R. Brands,
G. Venema, and J. Kok.
1998.
A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation.
Mol. Microbiol.
27:299-310[Medline].
|
| 19.
|
Schägger, H., and G. von Jagow.
1987.
Tricine-sodium dodecylsulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.
Anal. Biochem.
166:369-379.
|
| 20.
|
Schmidt, K.,
S. Liaanen-Jensen, and H. G. Schlegel.
1963.
Die Carotinoide der Thiorodaceae.
Arch. Mikrobiol.
46:117-126.
|
| 21.
|
Schobert, B., and J. K. Lanyi.
1982.
Halorhodopsin is a light-driven chloride pump.
J. Biol. Chem.
257:10306-10313[Abstract/Free Full Text].
|
| 22.
|
Severin, J.
1993.
Kompatible Solute und Wachstumskinetik bei halophilen aeroben heterotrophen Eubakterien. Ph.D. thesis
University of Bonn, Germany.
|
| 23.
|
Weisser, J., and H. G. Trüper.
1985.
Osmoregulation in a new haloalkaliphilic Bacillus from the Wadi Natrun (Egypt).
Syst. Appl. Microbiol.
6:7-11.
|
| 24.
|
Zdrou, I., and H. W. Tromballa.
1981.
Active transport of chloride by Anacystis nidulans.
Arch. Microbiol.
129:325-330.
|
Applied and Environmental Microbiology, October 1998, p. 3813-3817, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
