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Applied and Environmental Microbiology, February 1999, p. 560-568, Vol. 65, No. 2
Philipps University Marburg, Department of
Biology, D-35032 Marburg, Federal Republic of Germany
Received 17 August 1998/Accepted 10 November 1998
We report here that the naturally occurring choline ester
choline-O-sulfate serves as an effective compatible solute
for Bacillus subtilis, and we have identified a
high-affinity ATP-binding cassette (ABC) transport system responsible
for its uptake. The osmoprotective effect of this trimethylammonium
compound closely matches that of the potent and widely employed
osmoprotectant glycine betaine. Growth experiments with a set of
B. subtilis strains carrying defined mutations in the
glycine betaine uptake systems OpuA, OpuC, and OpuD and in the
high-affinity choline transporter OpuB revealed that
choline-O-sulfate was specifically acquired from the
environment via OpuC. Competition experiments demonstrated that
choline-O-sulfate functioned as an effective competitive inhibitor for OpuC-mediated glycine betaine uptake, with a
Ki of approximately 4 µM. Uptake studies with
[1,2-dimethyl-14C]choline-O-sulfate
showed that its transport was stimulated by high osmolality, and
kinetic analysis revealed that OpuC has high affinity for
choline-O-sulfate, with a Km value
of 4 ± 1 µM and a maximum rate of transport
(Vmax) of 54 ± 3 nmol/min · mg of protein in cells grown in minimal medium with 0.4 M NaCl. Growth studies utilizing a B. subtilis mutant defective in the
choline to glycine betaine synthesis pathway and natural abundance
13C nuclear magnetic resonance spectroscopy of whole-cell
extracts from the wild-type strain demonstrated that
choline-O-sulfate was accumulated in the cytoplasm and was
not hydrolyzed to choline by B. subtilis. In contrast, the
osmoprotective effect of acetylcholine for B. subtilis is
dependent on its biotransformation into glycine betaine.
Choline-O-sulfate was not used as the sole carbon,
nitrogen, or sulfur source, and our findings thus characterize this
choline ester as an effective compatible solute and metabolically inert stress compound for B. subtilis. OpuC mediates the
efficient transport not only of glycine betaine and
choline-O-sulfate but also of carnitine, crotonobetaine,
and Rapid fluxes of water along the
osmotic gradient are triggered by the exposure of microorganisms to
high osmolality environments, which in turn imposes considerable strain
on all the physiological processes of the cell (57).
Microorganisms actively respond to such increases in the osmolality of
their habitat to prevent dehydration of the cytoplasm and to maintain
turgor within acceptable boundaries. Since bacteria do not possess
active transport mechanisms for water, turgor is adjusted by
controlling the pool of osmotically active solutes within the cell
(12, 15, 34, 45, 54).
Most nonhalophilic bacteria do not use ions to permanently increase the
osmotic potential of their cytoplasm when they are faced by high
osmotic growth conditions (15, 67). Instead, large amounts
of organic osmolytes, the so-called compatible solutes, are
accumulated. These compounds are well tolerated by enzymes and cell
components and are thus highly congruous with the entire cellular
physiology, even when they are amassed at molar concentrations (72). The intracellular accumulation of compatible solutes
as a strategy of adaption to high osmolality has been widely adopted not only by Bacteria and Archaea but also by
fungal, plant, animal, and even human cells (4, 8, 39, 42,
58) and is thus a remarkable example of convergent evolution.
Moreover, the nature of the organic osmolytes that are amassed during
water stress is maintained across the kingdoms, reflecting fundamental
constraints on the kinds of solutes that are compatible with
macromolecular and cellular functions (39, 72).
One of the most effective and widely used osmoprotectants in both the
prokaryotic and eukaryotic world is the trimethylammonium compound
glycine betaine (12, 15, 58). It can be amassed by
microorganisms through either synthesis or uptake, and its intracellular accumulation confers a considerable degree of osmotic tolerance. A few bacteria produce this compatible solute by a stepwise
methylation of glycine (15, 60). However, in most bacterial
species glycine betaine synthesis proceeds via a two-step enzymatic
oxidation of choline with glycine betaine aldehyde as the intermediate
(6, 38, 61). Direct glycine betaine uptake from the
environment has been observed in a wide variety of gram-negative and
gram-positive Bacteria (12, 34, 45, 54) and a few Archaea (56). Molecular analysis of glycine
betaine uptake systems initially focused on the gram-negative
enterobacteria Escherichia coli and Salmonella
typhimurium, for which two osmoregulated high-affinity transporters have been characterized in detail. These are the secondary
transporter ProP and the binding-protein-dependent ATP-binding cassette
(ABC) transporter ProU (11).
Recently, the genetics and physiology of the osmostress response in
various gram-positive microorganisms have attracted wider attention
(3, 16, 29, 52, 55, 64, 68). Bacillus subtilis is
a ubiquitous inhabitant of the soil and is exposed to frequent osmotic
changes due to flooding and desiccation of the upper layers of its
habitat (45). Upon exposure to high-osmolality conditions,
this bacterium produces large amounts of proline via de novo synthesis
as an endogenous osmostress response (70). In addition,
B. subtilis makes considerable effort to acquire compatible
solutes or their precursors from the environment, and five
osmoregulated transport systems have so far been characterized at the
molecular level (34). OpuE (Opu denotes osmoprotectant uptake), a member of the sodium/solute symporter family, is used to
efficiently scavenge proline from the habitat for osmoprotective purposes (69). In addition, three systems serve as effective glycine betaine transporters: OpuA, OpuC, and OpuD (31, 33, 35). OpuA and OpuC are multicomponent systems and are members of
the ABC superfamily of transporters; both are related to the ProU
transporter from E. coli. The secondary transporter OpuD is
a representative of a new class of glycine betaine uptake systems (31) which also comprises BetP from the gram-positive soil
bacterium Corynebacterium glutamicum (52).
Acquisition of choline for the synthesis of glycine betaine (5,
6) is accomplished by both OpuC and the structurally closely
related, substrate-specific ABC transporter OpuB (32). The
OpuC system also serves for the high-affinity uptake of the betaines
L-carnitine, crotonobetaine, and The choline ester choline-O-sulfate (Fig. 1) is widespread
in nature and plays an important role in the microbial transformation of sulfur in the soil (1, 41). It is synthesized by a
variety of plants, lichens, algae, and fungi and by several bacterial species (10, 14, 20-24, 26). Its formation is catalyzed by sulfur transferases that use 3'-phosphoadenosine-5'-phosphosulfate and
choline as their substrates (59). Production of
choline-O-sulfate probably serves in the detoxification of
SO42 Bacterial strains.
Strain JH642 (trpC2 pheA1;
BGSC 1A96; gift from J. Hoch) is a derivative of the wild-type B. subtilis strain 168 and is the parent of all mutants used in the
study. The construction and properties of strains RMKB20
[ Growth conditions, media, and chemicals.
The B. subtilis strains were grown in Spizizen's minimal medium (SMM)
with 0.5% (wt/vol) glucose as the carbon source and supplemented with
L-tryptophan (20 mg/liter), L-phenylalanine (18 mg/liter), and a solution of trace elements (25). When it was used as the sole carbon source, choline-O-sulfate was
added to SMM to a final concentration of 30 mM and the growth yield of
the cultures was monitored spectrophotometrically (optical density at
578 nm [OD578]) after 24 h. Use by B. subtilis of choline-O-sulfate as the sole sulfur source
was tested by substituting
(NH4)2SO4 with NH4Cl
and substituting MgSO4 with MgCl2 in the SMM
salts. The (NH4)2SO4 present in SMM
was substituted with 30 mM K2SO4 to test the
use of choline-O-sulfate as the sole nitrogen source by
B. subtilis JH642. The cultures used to probe the function of choline-O-sulfate as the sole carbon, nitrogen, or sulfur
source were inoculated to an OD578 of approximately 0.05 from SMM-grown overnight cultures which had been carefully washed with
the modified SMM to remove residual traces of
(NH4)2SO4, MgSO4, and
glucose from the inoculum. The osmotic strength of SMM was increased by the addition of NaCl from stock solutions. The osmolality values of
these media were determined with a vapor pressure osmometer (model
5500; Wescor); the osmolality values of SMM, SMM with 0.4 M NaCl, and
SMM with 1.2 M NaCl were 340, 1,100, and 2,700 mosmol/kg of water,
respectively. Growth conditions for B. subtilis cells cultured in SMM and SMM with increased osmolality (1.2 M NaCl) and
testing for the osmoprotective effects of various compounds were as
described previously (30).
[1-methyl-14C]glycine betaine (2.03 GBq/mmol)
was custom synthesized by American Radiolabeled Chemicals Inc. (St.
Louis, Mo.).
[1,2-dimethyl-14C]choline-O-sulfate
was prepared from [1,2-dimethyl-14C]choline
(259 MBq/mmol) (New England Nuclear, Boston, Mass.) by the protocol of
Stevens and Vohra (63) and was a kind gift from D. Le
Rudulier and M. Østerås (Université de Nice, France) (50). [methyl-3H]acetylcholine
(3.15 TBq/mmol) was obtained from American Radiolabeled Chemicals Inc.
Choline, glycine betaine, acetylcholine, and phosphorylcholine were
purchased from Sigma (Steinheim, Germany). 2H2O
containing D4-3-(trimethylsilyl)propionate was obtained
from Aldrich (Deisenhofen, Germany).
Synthesis of choline-O-sulfate.
Nonradiolabeled
choline-O-sulfate was synthesized by heating 2 moles of
choline chloride with an excess (5.2 mol) of concentrated H2SO4 at 95°C overnight. The ester produced
was then precipitated by adding the reaction mixture (500 ml) to four
liters of 95% ethanol. The precipitated choline-O-sulfate
was collected by filtration and was recrystallized from 60% ethanol as
described by Bellenger et al. (2). Production of
choline-O-sulfate was confirmed by 1H nuclear
magnetic resonance (NMR) spectroscopy (10) by using a Bruker
AC300 spectrometer operating at 300 MHz. The purity of choline-O-sulfate was checked by elementary analysis in a
CHN-Rapid apparatus from Heraeus (Osterode, Germany) by the analytical
service laboratory of the Chemistry Department of the Philipps
University of Marburg, Germany.
Transport assays.
The uptake of
[1,2-dimethyl-14C]choline-O-sulfate
was measured at 37°C at various final substrate concentrations in
bacterial cultures grown to log phase (OD578 = 0.3 to 0.5)
in minimal media of high osmolality (SMM with 0.4 M NaCl) with glucose
as the carbon source, as detailed previously for the uptake of
radiolabeled glycine betaine (31, 33). The
choline-O-sulfate concentration in the uptake assay was
varied from 1 to 40 µM for kinetic studies. To determine the degree
of inhibition of glycine betaine transport via the OpuC system by
choline-O-sulfate, the concentration of the unlabeled
choline-O-sulfate was kept at 50 µM and the substrate concentration of the radiolabeled
[1-methyl-14C]glycine betaine was varied from
1 to 40 µM.
Preparation of cell extracts for 13C-NMR
spectroscopy.
Cultures (300 ml) of different strains (JH642 and
JBB5) were grown overnight in 1-liter Erlenmeyer flasks at 37°C on a
rotary shaker (220 rpm) in SMM or SMM with 1.2 M NaCl and 0.5%
(wt/vol) glucose as the carbon source in the absence or presence of
choline-O-sulfates; these stationary-phase cultures reached
OD578 between 2.5 and 3. The cells were harvested by
centrifugation and washed once with 150 ml of SMM containing 1.2 M NaCl
but no glucose, and the cell pellet was extracted with 10 ml of 80%
(vol/vol) ethanol. The cellular debris was removed by centrifugation,
and the supernatant was evaporated to dryness; ethanolic cell extracts
from two 300-ml cultures were then dissolved in 1 ml of
2H2O supplemented with 1.2 mg of
D4-3-(trimethylsilyl)propionate as a standard.
13C-NMR spectra were recorded with a Bruker AC300
spectrometer operating at 75 MHz (65). 13C-NMR
tracings from choline-O-sulfate, glycine betaine, choline, and acetylcholine relative to
D4-3-(trimethylsilyl)-propionate were recorded to identify
unambiguously resonances from these compounds present in the
13C-NMR spectra from the ethanolic cell extracts.
Choline-O-sulfate functions as an osmoprotectant.
To examine a possible osmoprotective effect of
choline-O-sulfate (Fig. 1) for
the proliferation of osmotically stressed B. subtilis cells,
we grew the wild-type strain JH642 in SMM (340 mosmol/kg) and
high-osmolality minimal medium (SMM with 1.2 M NaCl; 2,700 mosmol/kg)
in the absence or presence of 1 mM choline-O-sulfate. High
osmolality strongly impaired the growth of the cultures, and
choline-O-sulfate largely reversed this growth inhibition (Fig. 2A). A concentration as low as 20 µM of choline-O-sulfate was sufficient to exert a notable
osmoprotective effect on the growth of B. subtilis in the
high-osmolality medium, and approximately 100 to 200 µM provided full
osmoprotection (Fig. 3A). The
osmoprotective effects of choline-O-sulfate thus closely
match those of the potent osmoprotectant glycine betaine and its
biosynthetic precursor choline (Fig. 2 and 3) (5, 31). These
data establish that choline-O-sulfate can serve as an
effective osmoprotectant for B. subtilis.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
High-Affinity Transport of
Choline-O-Sulfate and Its Use as a Compatible Solute in
Bacillus subtilis
and
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
-butyrobetaine (R. Kappes and E. Bremer, Microbiology
144:83-90, 1998). Thus, our data underscore its crucial role in the
acquisition of a variety of osmoprotectants from the environment by
B. subtilis.
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
-butyrobetaine, all
highly effective osmoprotectants for B. subtilis
(30), and with low affinity (28), for the uptake of the frequently used osmoprotectant ectoine (15, 67).
Hence, like other microorganisms, B. subtilis can rely on a
spectrum of exogenously provided compatible solutes for osmoprotective purposes.
, and this ester can also function as a
source of choline and sulfur after hydrolysis by choline sulfatases
(41). An additional role for choline-O-sulfate as
an osmoprotective compound was suggested by the observation that the
intracellular pool of this betaine increased in various taxa of
halophytic plants subjected to salt stress (21, 22, 59).
Indeed, in bioassays choline-O-sulfate proved to be
osmoprotective both for several gram-negative bacterial species
(9, 19, 21, 44) and for the osmotolerant and xerotolerant
fungus Penicillium fellutanum (51). However, the wider function of this choline ester as an effective osmoprotectant for
the microbial world and its mechanisms for uptake from the environment
and its intracellular accumulation either as a compatible solute or as
a precursor for glycine betaine synthesis (50) have remained
largely unexplored. Here, we report on the identification of a
high-affinity choline-O-sulfate transport system at the
molecular level in B. subtilis and on the role of this
betaine as an effective compatible solute for this soil bacterium.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
(opuA::erm)4
opuC-20::Tn10(spc) (opuD::neo)2], RMKB22
[(opuA::erm)4
opuB-20::Tn10(spc)
(opuD::neo)2], RMKB25
[opuC-20::Tn10(spc)],
RMKB33
[(opuA::erm)4
(opuB::tet)23 opuC-20::Tn10(spc)], and RMKB34
[(opuB::tet)23
opuC-20::Tn10(spc) (opuD::neo)2] have been
described (30). Strain JBB5
[(gbsAB::neo)2] is
defective in glycine betaine synthesis since it lacks the GbsA and GbsB
enzymes (6).
RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
View larger version (37K):
[in a new window]
FIG. 1.
Osmoprotective compounds for B. subtilis and
chemically related substances.
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FIG. 2.
Osmoprotective effects of choline-O-sulfate
and acetylcholine for B. subtilis. The wild-type strain
JH642 (A) and its
(gbsAB::neo)2 derivative
JBB5 (B) were grown in SMM (
) and SMM with 1.2 M NaCl in the absence
(
) or presence of 1 mM glycine betaine (
), choline (
),
acetylcholine (
), or choline-O-sulfate (
). Cultures
(75 ml) were inoculated to an OD578 of 0.1 from overnight
cultures pregrown in SMM and were propagated in 500-ml Erlenmeyer
flasks in a shaking water bath (220 rpm) at 37°C. Cell growth was
monitored over time by measuring the OD578.
View larger version (17K):
[in a new window]
FIG. 3.
Uptake of choline-O-sulfate is mediated by
the ABC transport system OpuC. Strains RMKB22 (OpuA
OpuB OpuC+ OpuD) (A) and RMKB25
(OpuA+ OpuB+ OpuC
OpuD+) (B) were grown in high osmolality minimal medium
(SMM with 1.2 M NaCl) in the presence of various concentrations of
glycine betaine (
) and choline-O-sulfate (). The cells
were inoculated to an OD578 of 0.05 from an overnight SMM
culture and were grown in 20 ml of medium in a 100-ml Erlenmeyer flask
on an orbital shaker (220 rpm) at 37°C. The growth yield of each
culture was determined spectrophotometrically by measuring the
OD578 after 22-h incubation. Under these circumstances,
there is essentially no cell growth of strains inoculated in SMM with
1.2 M NaCl in the absence of an osmoprotectant, in contrast to cells
grown in 75-ml cultures in 500-ml Erlenmeyer flasks, conditions that
provide a much better aeration for the cells (30).
Identification of the choline-O-sulfate transport system. Bacteria frequently employ multiple transport systems with different substrate specificities to acquire compatible solutes from the environment (11, 34). We therefore investigated the involvement of the known B. subtilis transporters for glycine betaine (OpuA, OpuC, and OpuD) (30, 33) or its biosynthetic precursor choline (OpuB and OpuC) (32) in the uptake of choline-O-sulfate from the environment. In a preliminary growth experiment, we used an isogenic set of mutants expressing only one of these uptake systems (30). This survey revealed that only B. subtilis strains synthesizing the OpuC transporter (31) could use choline-O-sulfate as an osmoprotectant. OpuC exhibits the characteristic features of a bacterial binding-protein-dependent transport system (7) and is a member of the ABC superfamily of transporters. It consists of an evolutionarily highly conserved ATPase (OpuCA), two sequence-related integral cytoplasmic membrane proteins (OpuCB and OpuCD), and a hydrophilic lipoprotein (OpuCC). This latter polypeptide is likely to serve as an extracellular substrate-binding protein tethered by its N-terminal lipid modification to the cytoplasmic membrane (32).
Disruption of the OpuC transporter in an otherwise wild-type background completely abolished osmoprotection by choline-O-sulfate (Fig. 3B). In contrast, this mutant strain was still fully protected by glycine betaine (Fig. 3B), which can be accumulated not only by OpuC but also via the high-affinity ABC transporter OpuA (33) and the secondary uptake system OpuD (31). These growth experiments unequivocally demonstrate that B. subtilis possesses only one choline-O-sulfate uptake system and establish that the multicomponent ABC transporter OpuC mediates the uptake of this betaine from exogenous sources.Competitive inhibition of glycine betaine transport by
choline-O-sulfate.
Transport of radiolabeled glycine
betaine via OpuC is moderately osmotically stimulated and exhibits a
Km of 6 µM and a Vmax of 65 nmol/min · mg of protein in cultures grown in SMM in the presence of 0.4 M NaCl (31). To analyze the entry of
choline-O-sulfate into the B. subtilis cells
through the OpuC system, we performed competition experiments with
[1-methyl-14C]glycine betaine and unlabeled
choline-O-sulfate. We grew strain RMKB22 (OpuA
OpuB OpuC+ OpuD) in SMM with
0.4 M NaCl to mid-exponential phase and measured the uptake of
[1-methyl-14C]glycine betaine over a range (1 to 40 µM) of substrate concentrations in either the absence or the
presence of a fixed concentration (50 µM) of the competitor
choline-O-sulfate. In close agreement with the previously
reported data (31) we found a Km of
2.8 µM for glycine betaine uptake in the absence of
choline-O-sulfate, and an apparent Km
of 34 µM in its presence (Fig. 4A). From these kinetic data we
calculated a Ki value of 4.5 ± 1 µM. The
lines in the Hanes plot were linear and parallel and yielded very
similar Vmax values for
[1-methyl-14C]glycine betaine uptake in the
absence (Vmax was 89 nmol/min · mg of
protein) or presence (Vmax was 92 nmol/min
· mg of protein) (Fig. 4A) of the inhibitor. These transport
characteristics indicated that choline-O-sulfate behaves as
a competitive inhibitor (71) for glycine betaine uptake via
OpuC and that this ABC transporter serves as a high-affinity uptake
system for this osmoprotectant.
Kinetic parameters for OpuC-mediated choline-O-sulfate transport. To determine the parameters for choline-O-sulfate transport directly, we measured the initial uptake of [1,2-dimethyl-14C]choline-O-sulfate at a final substrate concentration of 10 µM in the wild-type strain JH642 in cultures grown in SMM or SMM with elevated osmolality (SMM with 0.4 M NaCl). Like the OpuC-mediated transport of glycine betaine and L-carnitine (30, 31), choline-O-sulfate uptake was moderately stimulated (three- to fourfold) by increases in the osmolality of the growth medium (Fig. 4B). Disruption of the OpuC system in an otherwise wild-type background by the opuC-20::Tn10(spc) mutation (strain RMKB25) completely abolished radiolabeled choline-O-sulfate transport at both low and high osmolality (Fig. 4B). This observation corroborates the finding that osmoprotection by choline-O-sulfate is eliminated in an OpuC-lacking strain that still possesses the OpuA, OpuB, OpuD, and OpuE osmoprotectant uptake systems (Fig. 3B).
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Osmoprotective effects of choline-O-sulfate are not
dependent on its enzymatic conversion into glycine betaine.
Choline-O-sulfate is a derivative of choline (Fig. 1), and
hydrolysis of the ester bond would liberate the precursor for glycine betaine biosynthesis (5). Consequently, the osmoprotective effects of choline-O-sulfate for B. subtilis
(Fig. 2A and 3A) could be indirect and dependent on its enzymatic
conversion to glycine betaine. To determine whether
choline-O-sulfate acted as a compatible solute per se or
whether its osmoprotective effects resulted from its biotransformation
into glycine betaine, we tested its osmoprotective capacity in strain
JBB5 [(gbsAB::neo)2].
This mutant cannot synthesize glycine betaine from choline because it
lacks the choline dehydrogenase (GbsB) and the glycine betaine aldehyde
dehydrogenase (GbsA) (6). Unlike choline, both
choline-O-sulfate and glycine betaine were fully
osmoprotective for strain JBB5 (Fig. 2B). Hence, osmoprotection of
B. subtilis by choline-O-sulfate clearly does not
depend on its conversion into glycine betaine.
Osmoprotection by acetylcholine requires its conversion into
glycine betaine.
We also observed that the choline ester
acetylcholine (Fig. 1) protected B. subtilis cells from the
detrimental effects of high osmolality with an efficiency similar to
those of choline and choline-O-sulfate (Fig. 2A).
Acetylcholine is found widely in plants and thus could reach soil
bacteria through decaying plant material. However, its concentration in
plants is rather low compared with that of choline (46, 66).
In contrast to choline-O-sulfate, acetylcholine could not
serve as an osmoprotectant for strain JBB5
[(gbsAB::neo)2] (Fig.
2B). Hence, for the gbsAB+ B. subtilis strain
JH642 (Fig. 2A), osmoprotection by acetylcholine was entirely dependent
on its hydrolysis to choline and acetate and the subsequent enzymatic
conversion of the choline moiety to glycine betaine. As observed
previously for choline, the presence of acetylcholine was detrimental
to cell growth for strain JBB5 (Fig. 2B), an effect possibly connected
with the accumulation of a large number of positively charged choline
molecules inside the cells (6). Thus, the mechanism of
osmoprotection by acetylcholine in B. subtilis is like that
of Pseudomonas aeruginosa, where acetylcholine serves as a
precursor for glycine betaine synthesis (40), but it differs
from that of the gram-positive bacterium Lactobacillus plantarum, where both acetylcholine and choline are accumulated as
moderately effective cationic osmolytes (37).
-substituted
acylesters acetylcarnitine and octanoylcarnitine by B. subtilis and their intracellular accumulation in the form of
L-carnitine in osmotically stressed cells (30). Although it is not clear where the hydrolysis of acetylcholine and the
acylesters of L-carnitine occurs, in its natural habitats B. subtilis might use such compounds as additional sources
for generating compatible solutes.
In addition, we also investigated a possible osmoprotective effect of
the choline ester phosphorylcholine (Fig. 1) for the growth of B. subtilis in high-osmolality media, since this trimethylammonium compound can serve as a precursor for glycine betaine synthesis in
P. aeruginosa under hyperosmotic conditions (13,
40). However, this negatively charged choline ester did not
display any osmoprotection for the B. subtilis wild-type
strain JH642. Hence, not all choline esters serve as a source of
precursor for glycine betaine synthesis in B. subtilis.
Choline-O-sulfate is accumulated in the cytoplasmic solute pool. To further substantiate the result that choline-O-sulfate serves as a compatible solute per se for B. subtilis, we monitored the fate of this compound in cultures grown at high osmolality by natural abundance 13C-NMR spectroscopy, a technique that is frequently used to assess the spectrum of compatible solutes inside bacterial cells (15, 30, 65, 70). Crude ethanol extracts were prepared from cells of strain JH642 grown in SMM or in SMM with 1.2 M NaCl in the presence or absence of choline-O-sulfate and were analyzed by 13C-NMR spectroscopy. As observed previously (28, 30, 70), glutamate and proline were the predominant osmolytes in salt-stressed cells grown in the absence of an osmoprotectant. The presence of 1 mM choline-O-sulfate in the growth medium resulted in the accumulation of this trimethylammonium compound in the cytoplasmic solute pool at both low and high osmolality (Fig. 5). Resonances from glycine betaine could not be detected in these spectra, although signals from this compound were readily found when it was added (1 mM) to the growth medium (data not shown).
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Choline-O-sulfate is metabolically inert in B. subtilis. We tested whether B. subtilis degrades choline-O-sulfate for use as a carbon, nitrogen, or sulfur source. Choline-O-sulfate did not support the growth of the B. subtilis wild-type strain JH642 when supplied at a concentration of 30 mM as the sole carbon source. Likewise, it did not serve as a nitrogen source (provided at a concentration of 30 mM) in a modified SMM medium in which (NH4)2SO4 had been replaced by K2SO4 at the equivalent concentration, whereas proline (30 mM) served as an effective nitrogen source in this (NH4)2SO4-depleted medium. In an SMM medium where (NH4)2SO4 and MgSO4 were replaced by NH4Cl and MgCl2 at equivalent concentrations, choline-O-sulfate (15 mM) did not support the growth of strain JH642, but methionine at a concentration of 15 mM readily served as a sole sulfur source. Hence, choline-O-sulfate is not used by B. subtilis for anabolic purposes.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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The use of a mixture of osmoprotectants by a given microorganism is frequently observed. It enhances the ability of bacteria to adapt flexibly to different ecological niches and to varying conditions in their habitats (12, 15, 34, 45). The data presented here show that choline-O-sulfate, which is produced by a wide variety of plants, lichens, algae, fungi, and bacteria, serves as an effective and metabolically inert compatible solute for B. subtilis. By using a set of strains with mutations in previously defined systems for uptake of osmoprotectants (34), we identified the osmotically stimulated ABC transporter OpuC as the uptake system for choline-O-sulfate. To our knowledge this is the first report in which the kinetic parameters and molecular details of a bacterial choline-O-sulfate uptake system have been characterized.
Compatible solutes are highly soluble molecules; they do not carry a net charge at physiological pH, and often they also stabilize proteins in vitro and protect against denaturation in solutions of high ionic strength (72). Choline-O-sulfate fulfills the criteria for a compatible solute. This zwitterion (Fig. 1) is amassed in osmotically stressed E. coli and S. typhimurium cells to levels exceeding 0.5 M (21). Such high concentrations do not inhibit the enzymatic function of purified barley leaf malate dehydrogenase, and they protect the enzyme against inhibition by NaCl (53). Depending on the degree of osmotic stress, osmoregulating B. subtilis cells amass glycine betaine from exogenous sources at intracellular levels ranging between 0.5 and 1 M (47, 70). Since choline-O-sulfate and glycine betaine have similar osmoprotective effects for B. subtilis when accumulated via OpuC (Fig. 3), one can expect that both compounds are amassed to comparable intracellular concentrations.
In addition to the chemical and biophysical properties of compatible
solutes, the kinetic parameters of their transport systems greatly
determine their effectiveness as osmoprotectants for bacteria. A
telling case is the uptake of the cyclic amino acid derivative ectoine,
an effective compatible solute for a wide range of bacterial species
(15, 67). An exogenous supply (1 mM) of ectoine is only
moderately osmoprotective for B. subtilis due to its
inefficient import by OpuC (Ki = 1.5 mM)
(28). However, this tetrahydropyrimidine has an
osmoprotective capacity similar to that of glycine betaine for E. coli (27) and the plant pathogen Erwinia
chrysanthemi (17), bacteria which possess high-affinity
ectoine and glycine betaine uptake systems. Although OpuC shows only
low affinity for ectoine, this ABC transporter exhibits high-affinity
uptake for the compatible solutes choline-O-sulfate, glycine
betaine, L-carnitine, crotonobetaine, and
-butyrobetaine, with Km and Ki values in the low micromolar range (4 to 7 µM) and a substantial transport capacity (Vmax
of 54 to 71 nmol/min · mg of protein) (30, 31). With
the exception of glycine betaine (31, 33), these compounds
are taken up only via OpuC. Thus, this transporter plays a pivotal role
in supplying B. subtilis with a variety of structurally
related osmoprotectants.
Choline-O-sulfate, L-carnitine, crotonobetaine,
and -butyrobetaine each serve as effective inhibitors for glycine
betaine uptake via OpuC (Fig. 4A) (30), suggesting that
these trimethylammonium compounds compete for the same binding site(s).
The substrate-binding proteins of binding-protein-dependent ABC
transporters serve as the primary site for high-affinity substrate
recognition (7). The OpuC system possesses a lipoprotein
(OpuCC) that is likely to function as an extracellular
substrate-binding protein which is tethered by its N-terminal lipid
tail to the cytoplasmic membrane (32). Unfortunately, it has
not yet been possible to isolate the OpuCC protein in a functional form
(36), which precludes a direct biochemical analysis of
substrate binding. We have successfully performed such experiments with
purified OpuAC, which is the lipid-modified substrate-binding protein
from the glycine betaine ABC transporter OpuA (35).
Consideration of the chemical nature of the substrates transported with
high affinity via OpuC reveals that all of them possess a fully
methylated ammonium group and that the main carbon chains exhibit
considerable variation with respect to length, chemical bonds, and
branching (Fig. 1). Each of these substrates is a zwitterion at
physiological pH, whereas uptake of the positively charged choline via
OpuC proceeds with a somewhat reduced affinity (Km = 38 µM) (32).
Choline-O-sulfate is a major product of sulfate metabolism
in a variety of plant, fungal, and bacterial species (14, 22, 24,
26) and is thus likely to be released from decaying cells of
these primary producers into the soil. Several species of the genus
Pseudomonas are even known to excrete
choline-O-sulfate into the medium (14). Like
other osmoprotectants, this betaine is probably present only at low and
variable concentrations in the natural habitats of B. subtilis. Therefore, the ability to scavenge compatible solutes
efficiently from the environment and to accumulate them to very high
intracellular levels against a steep concentration gradient is a
distinctive requirement for a transport system involved in the
acquisition of osmoprotectants. The ABC transporter OpuC meets these
physiological demands, and the osmoprotective effects of
choline-O-sulfate (Fig. 3A), glycine betaine,
L-carnitine, crotonobetaine, and -butyrobetaine were clearly noticeable when these compounds were supplied at concentrations as low as 20 µM to an OpuC-positive B. subtilis strain
(30). The presence of choline-O-sulfate in
physiologically relevant concentrations in the soil is substantiated by
reports demonstrating the presence of transport systems for this ester
in barley roots (49), in soil fungi (2, 24), and
in strains of Corynebacterium and of the rhizosphere
bacteria Pseudomonas, Agrobacterium, and Rhizobium (48). Although the details on the
type(s) of transport system involved and the kinetic parameters for
choline-O-sulfate uptake by most of these organisms have not
been documented, in barley roots (49), Neurospora
crassa (43), and Penicillium notatum
(2) choline-O-sulfate transport activities with
Km values of 7.7, 25, and 300 µM,
respectively, have been reported. Choline-O-sulfate from the
environment is accumulated in each of these cells as a source of sulfur
which becomes available after intracellular hydrolysis of
choline-O-sulfate by choline sulfatases. The OpuC-mediated
choline-O-sulfate transport in B. subtilis for osmoprotective purposes reported here compares favorably
(Km value of 4 µM) with that described for
anabolic functions.
Choline sulfatases are present in various microbial species (18, 23, 43, 62). They may play a role in providing the precursor for glycine betaine synthesis. However, biotransformation of choline-O-sulfate into glycine betaine is clearly not the cause for its osmoprotective effects in B. subtilis. As we have demonstrated here, choline-O-sulfate was fully osmoprotective for a B. subtilis mutant defective in the glycine betaine biosynthetic pathway (Fig. 2B) and was readily detected as an osmolyte in the cytoplasmic solute pool of osmotically stressed cells (Fig. 5). These findings contrast with the observations made with acetylcholine, whose osmoprotective effects for B. subtilis were entirely dependent on its enzymatic conversion into glycine betaine (Fig. 2). The accumulation of choline-O-sulfate as a metabolically inert stress compound is common to the bacteria B. subtilis, E. coli, and S. typhimurium (21), and its amassing at high intracellular levels is also observed in the fungus P. fellutanum (51). However, the osmoprotective effect of choline-O-sulfate does not always depend on its accumulation in an unmodified form. Recently, Østerås et al. (50) reported the presence of a choline sulfatase activity (BetC) in the rhizosphere bacterium Sinorhizobium meliloti that catalyzed the hydrolysis of choline-O-sulfate into choline and sulfate; the produced choline was then further oxidized into glycine betaine by the BetA and BetB enzymes. The structural gene (betC) for this choline sulfatase is part of the S. meliloti betICBA operon. The fact that both choline-O-sulfate and choline serve as inducers for this gene cluster thus further underscores the physiological relevance of the choline-O-sulfate to glycine betaine biosynthetic pathway. In contrast to B. subtilis (5), S. meliloti can use both glycine betaine and choline-O-sulfate not only for osmoprotection but also as sole carbon and nitrogen (sulfur) sources for growth (50).
The previously reported osmoprotective effects of choline-O-sulfate for several plant, fungal, and bacterial species (9, 19, 21, 44, 51) and the function of this choline ester as a precursor for glycine betaine synthesis in S. meliloti (50) coupled with our discovery of a high-affinity choline-O-sulfate transport system in B. subtilis for osmoprotective purposes all suggest that this betaine plays a much wider and physiologically more important role for osmoprotection than has previously been assumed.
| |
ACKNOWLEDGMENTS |
|---|
We are grateful to Daniel Le Rudulier and Magne Østerås for generously providing us with a sample of radiolabeled choline-O-sulfate and for communicating data prior to publication. The help of V. Koogle in editing the manuscript is greatly appreciated.
Financial support for this study was provided by the Deutsche Forschungsgemeinschaft through SFB-395 and the Graduiertenkolleg Enzymchemie and the Fonds der Chemischen Industrie. J. A. Le Good participated in an exchange program between the University of Oxford and the University of Marburg, and her stay in the Laboratory for Microbiology (Marburg) was supported by a grant through the ERASMUS program from the EC.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Philipps University Marburg, Department of Biology, Laboratory for Microbiology, Karl-von-Frisch Str., D-35032 Marburg, Federal Republic of Germany. Phone: (49) 6421-281529. Fax: (49) 6421-288979. E-mail: bremer{at}mailer.uni-marburg.de.
Present address: Washington University, Department of Biology, One
Brooking Dr., St. Louis, MO 63130.
Present address: Imperial Cancer Research Fund, Protein
Phosphorylation Laboratory, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
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REFERENCES |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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| 1. | Alexander, M. 1977. Microbial transformation of sulfur, p. 350-367. In Introduction to soil microbiology. John Wiley & Sons, New York, N.Y. |
| 2. |
Bellenger, N.,
P. Nissen,
T. C. Wood, and I. H. Segel.
1968.
Specificity and control of choline-O-sulfate transport in filamentous fungi.
J. Bacteriol.
96:1574-1585 |
| 3. | Bernard, T., M. Jebbar, S. Himidi-Kabbab, J. Hamelin, and C. Blanco. 1993. Ectoine accumulation and osmotic regulation in Brevibacterium linens. J. Gen. Microbiol. 139:129-136. |
| 4. | Blomberg, A. 1997. Osmoresponsive proteins and functional assessment strategies in Saccharomyces cerevisiae. Electrophoresis 18:1429-1440[Medline]. |
| 5. |
Boch, J.,
B. Kempf, and E. Bremer.
1994.
Osmoregulation in Bacillus subtilis: synthesis of the osmoprotectant glycine betaine from exogenously provided choline.
J. Bacteriol.
176:5364-5371 |
| 6. |
Boch, J.,
B. Kempf,
R. Schmid, and E. Bremer.
1996.
Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes.
J. Bacteriol.
178:5121-5129 |
| 7. | Boos, W., and J. M. Lucht. 1996. Periplasmic binding protein-dependent ABC transporters, p. 1175-1209. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, vol. 1. ASM Press, Washington, D.C. |
| 8. | Burg, M., E. Kwon, and D. Kültz. 1997. Regulation of gene expression by hypertonicity. Annu. Rev. Physiol. 59:437-455[Medline]. |
| 9. |
Cánovas, D.,
C. Vargas,
L. N. Csonka,
A. Ventosa, and J. J. Nieto.
1996.
Osmoprotectants in Halomonas elongata: high-affinity betaine transport system and choline-betaine pathway.
J. Bacteriol.
178:7221-7226 |
| 10. | Catalfomo, P., J. H. Block, G. H. Constantine, and P. W. Kirk. 1972. Choline sulfate (ester) in marine higher fungi. Mar. Chem. 1:157-162. |
| 11. | Csonka, L. N., and W. Epstein. 1996. Osmoregulation, p. 1210-1223. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, vol. 1. ASM Press, Washington, D.C. |
| 12. | Csonka, L. N., and A. D. Hanson. 1991. Prokaryotic osmoregulation: genetics and physiology. Annu. Rev. Microbiol. 45:569-606[Medline]. |
| 13. |
D'Souza-Ault, M. R.,
L. T. Smith, and G. M. Smith.
1993.
Roles of N-acetylglutaminylglutamine amide and glycine betaine in adaptation of Pseudomonas aeruginosa to osmotic stress.
Appl. Environ. Microbiol.
59:473-478 |
| 14. | Fitzgerald, J. W., and P. C. Luschinski. 1977. Further studies on the formation of choline sulfate by bacteria. Can. J. Microbiol. 23:483-490[Medline]. |
| 15. | Galinski, E. A., and H. G. Trüper. 1994. Microbial behaviour in salt-stressed ecosystems. FEMS Microbiol. Rev. 15:95-108. |
| 16. |
Glaasker, E.,
W. N. Konings, and B. Poolman.
1996.
Glycine betaine fluxes in Lactobacillus plantarum during osmostasis and hyper- and hypo-osmotic shock.
J. Biol. Chem.
271:10060-10065 |
| 17. |
Gouesbet, G.,
A. Trautwetter,
S. Bonnassie,
L. F. Wu, and C. Blanco.
1996.
Characterization of the Erwinia chrysanthemi osmoprotectant transporter gene ousA.
J. Bacteriol.
178:447-455 |
| 18. | Gravel, R. A. 1976. Choline-O-sulphate utilization in Aspergillus nidulans. Genet. Res. 28:261-276[Medline]. |
| 19. |
Gutierrez, J. A., and L. N. Csonka.
1995.
Isolation and characterization of adenylate kinase (adk) mutations in Salmonella typhimurium which block the ability of glycine betaine to function as an osmoprotectant.
J. Bacteriol.
177:390-400 |
| 20. | Hanson, A. D., and D. A. Gage. 1991. Identification and determination by fast atom bombardment mass spectrometry of the compatible solute choline-O-sulfate in Limonium species and other halophytes. Aust. J. Plant Physiol. 18:317-327. |
| 21. |
Hanson, A. D.,
B. Rathinasabapathi,
B. Chamberlin, and D. A. Gage.
1991.
Comparative physiological evidence that -alanine betaine and choline-O-sulfate act as compatible osmolytes in halophytic Limonium species.
Plant Physiol. (Rockville)
97:1199-1205 |
| 22. |
Hanson, A. D.,
B. Rathinasabapathi,
J. Rivoal,
M. Burnet,
M. O. Dillon, and D. A. Gage.
1994.
Osmoprotective compounds in the plumbaginaceae: a natural experiment in metabolic engineering of stress tolerance.
Proc. Natl. Acad. Sci. USA
91:306-310 |
| 23. | Harada, T. 1964. The formation of sulphatases by Pseudomonas aeruginosa. Biochim. Biophys. Acta 81:193-196. |
| 24. | Harada, T., and B. Spencer. 1960. Choline sulphate in fungi. J. Gen. Microbiol. 22:520-527. |
| 25. | Harwood, C. R., and A. R. Archibald. 1990. Growth, maintenance and general techniques, p. 1-26. In C. R. Harwood, and S. M. Cutting (ed.), Molecular biological methods for Bacillus. John Wiley & Sons, Inc., Chichester, United Kingdom. |
| 26. | Ikawa, M., A. Chakrvarti, and R. F. Taylor. 1972. Occurrence of choline in Lactobacillus plantarum. Can. J. Microbiol. 18:1241-1245[Medline]. |
| 27. |
Jebbar, M.,
R. Talibart,
K. Gloux,
T. Bernard, and C. Blanco.
1992.
Osmoprotection of Escherichia coli by ectoine: uptake and accumulation characteristics.
J. Bacteriol.
174:5027-5035 |
| 28. | Jebbar, M., C. von Blohn, and E. Bremer. 1997. Ectoine functions as an osmoprotectant in Bacillus subtilis and is accumulated via the ABC-transport system OpuC. FEMS Microbiol. Lett. 154:325-330. |
| 29. |
Jewell, J. B., and E. R. Kashket.
1991.
Osmotically regulated transport of proline by Lactobacillus acidophilus IFO 3532.
Appl. Environ. Microbiol.
57:2829-2833 |
| 30. |
Kappes, R., and E. Bremer.
1998.
Response of Bacillus subtilis to high osmolarity: uptake of carnitine, crotonobetaine and -butyrobetaine via the ABC transport system OpuC.
Microbiology
144:83-90 |
| 31. |
Kappes, R. M.,
B. Kempf, and E. Bremer.
1996.
Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD.
J. Bacteriol.
178:5071-5079 |
| 32. | Kappes, R. M., B. Kempf, S. Kneip, J. Boch, J. Gade, J. Meier-Wagner, and E. Bremer. Two evolutionarily closely related ABC-transporters mediate the uptake of choline for synthesis of the osmoprotectant glycine betaine in Bacillus subtilis. Mol. Microbiol., in press. |
| 33. |
Kempf, B., and E. Bremer.
1995.
OpuA, an osmotically regulated binding protein-dependent transport system for the osmoprotectant glycine betaine in Bacillus subtilis.
J. Biol. Chem.
270:16701-16713 |
| 34. | Kempf, B., and E. Bremer. 1998. Uptake and synthesis of compatible solutes as microbial stress responses to high osmolality environments. Arch. Microbiol. 170:319-330[Medline]. |
| 35. |
Kempf, B.,
J. Gade, and E. Bremer.
1997.
Lipoprotein from the osmoregulated ABC transport system OpuA of Bacillus subtilis: purification of the glycine betaine binding protein and characterization of a functional lipidless mutant.
J. Bacteriol.
179:6213-6220 |
| 36. | Kempf, B., J. Gade, and E. Bremer. 1998. Unpublished data. |
| 37. | Kets, E. P. W., M. N. Groot, E. A. Gallinski, and J. A. M. De Bont. 1997. Choline and acetylcholine: novel cationic osmolytes in Lactobacillus plantarum. Appl. Microbiol. Biotechnol. 48:94-98. |
| 38. | Lamark, T., I. Kaasen, M. W. Eshoo, P. Falkenberg, J. McDougall, and A. R. Strøm. 1991. DNA sequence and analysis of the bet genes encoding the osmoregulatory choline-glycine betaine pathway of Escherichia coli. Mol. Microbiol. 5:1049-1064[Medline]. |
| 39. |
Le Rudulier, D.,
A. R. Strøm,
A. M. Dandekar,
L. T. Smith, and R. C. Valentine.
1984.
Molecular biology of osmoregulation.
Science
224:1064-1068 |
| 40. | Lisa, T. A., C. H. Casale, and C. E. Domenech. 1994. Cholinesterase, acid phosphatase, and phospholipase C of Pseudomonas aeruginosa under hyperosmotic conditions in a high-phosphate medium. Curr. Microbiol. 28:71-76. |
| 41. | Markham, P., G. D. Robson, B. W. Bainbridge, and A. P. Trinci. 1993. Choline: its role in the growth of filamentous fungi and the regulation of mycelial morphology. FEMS Microbiol. Rev. 10:287-300[Medline]. |
| 42. | Martins, L., R. Huber, H. Huber, K. Stetter, M. Da Costa, and H. Santos. 1997. Organic solutes in hyperthermophilic Archaea. Appl. Environ. Microbiol. 63:896-902[Abstract]. |
| 43. | Marzluf, G. A. 1972. Genetic and metabolic control of sulfate metabolism in Neurospora crassa: a specific permease for choline-O-sulfate. Biochem. Genet. 7:219-233[Medline]. |
| 44. | Mason, T., and G. Blunden. 1989. Quaternary ammonium and tertiary sulphonium compounds of algal origin as alleviators of osmotic stress. Bot. Mar. 32:313-316. |
| 45. | Miller, K. J., and J. M. Wood. 1996. Osmoadaptation by rhizosphere bacteria. Annu. Rev. Microbiol. 50:101-136[Medline]. |
| 46. | Miura, G. A., and T.-M. Shih. 1984. Cholinergic constituents in plants: characterization and distribution of acetylcholine and choline. Physiol. Plant. 61:417-421. |
| 47. | Moses, S., C. von Blohn, E. Bakker, and E. Bremer. 1998. Unpublished data. |
| 48. | Nissen, P. 1968. Choline sulfate permease: transfer of information from bacteria to higher plants? Biochem. Biophys. Res. Commun. 32:696-703[Medline]. |
| 49. |
Nissen, P., and A. A. Benson.
1964.
Active transport of choline sulfate by barley roots.
Plant Physiol.
39:586-589 |
| 50. |
Østerås, M.,
E. Boncompagni,
N. Vincent,
M.-C. Poggi, and D. Le Rudulier.
1998.
Presence of a gene encoding choline sulfatase in Sinorhizobium meliloti bet operon: choline-O-sulfate is metabolized into glycine betaine.
Proc. Natl. Acad. Sci. USA
95:11394-11399 |
| 51. |
Park, Y.-I., and J. E. Gander.
1998.
Choline derivatives involved in osmotolerance of Penicillium fellutanum.
Appl. Environ. Microbiol.
64:273-278 |
| 52. |
Peter, H.,
A. Burkovski, and R. Krämer.
1996.
Isolation, characterization, and expression of the Corynebacterium glutamicum betP gene, encoding the transport system for the compatible solute glycine betaine.
J. Bacteriol.
178:5229-5234 |
| 53. | Pollard, A., and R. G. Wyn Jones. 1979. Enzyme activities in concentrated solutions of glycine betaine and other solutes. Planta 144:291-298. |
| 54. | Poolman, B., and E. Glaasker. 1998. Regulation of compatible solute accumulation in bacteria. Mol. Microbiol. 29:397-407[Medline]. |
| 55. |
Pourkomailian, B., and I. R. Booth.
1994.
Glycine betaine transport by Staphylococcus aureus: evidence for feedback regulation of the activity of the two transport systems.
Microbiology
140:3131-3138 |
| 56. | Proctor, L. M., R. Lai, and R. P. Gunsalus. 1997. The methanogenic archaeon Methanosarcina thermophila TM-1 possesses a high-affinity glycine betaine transporter involved in osmotic adaptation. Appl. Environ. Microbiol. 63:2252-2257[Abstract]. |
| 57. | Record, M. T., Jr., E. S. Courtenay, D. S. Cayley, and H. J. Guttman. 1998. Responses of E. coli to osmotic stress: large changes in amounts of cytoplasmic solutes and water. Trends Biochem. Sci. 23:143-148[Medline]. |
| 58. | Rhodes, D., and A. D. Hanson. 1993. Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44:357-384. |
| 59. | Rioval, J., and A. D. Hanson. 1994. Choline-O-sulfate biosynthesis in plants. Identification and partial characterization of a salinity-inducible choline sulfotransferase from species of Limonium (Plumbaginaceae). Plant Physiol. (Rockville) 106:1187-1193[Abstract]. |
| 60. | Robinson, P. M., and M. F. Roberts. 1997. Effects of osmolyte precursors on the distribution of compatible solutes in Methanohalophilus portucalensis. Appl. Environ. Microbiol. 63:4032-4038[Abstract]. |
| 61. |
Rozwadowski, K. L.,
G. G. Khachatourians, and G. Selvaraj.
1991.
Choline oxidase, a catabolic enzyme in Arthrobacter pascens, facilitates adaptation to osmotic stress in Escherichia coli.
J. Bacteriol.
173:472-478 |
| 62. | Spencer, B., E. C. Hussey, B. A. Orsi, and J. M. Scott. 1968. Mechanism of choline-O-sulphate utilization in fungi. Biochem. J. 106:461-469[Medline]. |
| 63. | Stevens, C. M., and P. Vohra. 1955. Occurrence of choline sulfate in Penicillium chrysogenum. J. Am. Chem. Soc. 77:4935-4936. |
| 64. |
Stimeling, K. W.,
J. E. Graham,
A. Kaenjak, and B. J. Wilkinson.
1994.
Evidence for feedback (trans) regulation of, and two systems for, glycine betaine transport by Staphylococcus aureus.
Microbiology
140:3139-3144 |
| 65. |
Talibart, R.,
M. Jebbar,
G. Gouesbet,
S. Himdi-Kabbab,
H. Wróblewski,
C. Blanco, and T. Bernard.
1994.
Osmoadaptation in rhizobia: ectoine-induced salt tolerance.
J. Bacteriol.
176:5210-5217 |
| 66. | Tretyn, A., and R. E. Kendrick. 1991. Acetylcholine in plants: presence, metabolism and mechanism of action. Bot. Rev. 57:33-73. |
| 67. |
Ventosa, A.,
J. J. Nieto, and A. Oren.
1998.
Biology of moderately halophilic aerobic bacteria.
Microbiol. Mol. Biol. Rev.
62:504-544 |
| 68. |
Verheul, A.,
E. Glaasker,
B. Poolman, and T. Abee.
1997.
Betaine and L-carnitine transport by Listeria monocytogenes Scott A in response to osmotic signals.
J. Bacteriol.
179:6979-6985 |
| 69. | von Blohn, C., B. Kempf, R. M. Kappes, and E. Bremer. 1997. Osmostress response in Bacillus subtilis: characterization of a proline uptake system (OpuE) regulated by high osmolarity and the alternative transcription factor sigma B. Mol. Microbiol. 25:175-187[Medline]. |
| 70. |
Whatmore, A. M.,
J. A. Chudek, and R. H. Reed.
1990.
The effects of osmotic upshock on the intracellular solute pools of Bacillus subtilis.
J. Gen. Microbiol.
136:2527-2535 |
| 71. |
Xavier, K. B.,
L. O. Martins,
R. Peist,
M. Kossmann,
W. Boos, and H. Santos.
1996.
High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis.
J. Bacteriol.
178:4773-4777 |
| 72. | Yancey, P. H. 1994. Compatible and counteracting solutes, p. 81-109. In K. Strange (ed.), Cellular and molecular physiology of cell volume regulation. CRC Press, Inc., Boca Raton, Fla. |
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(2003). Role of Ectoine in Vibrio cholerae Osmoadaptation. Appl. Environ. Microbiol.
69: 5919-5927
[Abstract] [Full Text] -
Kuhlmann, A. U., Bremer, E.
(2002). Osmotically Regulated Synthesis of the Compatible Solute Ectoine in Bacillus pasteurii and Related Bacillus spp.. Appl. Environ. Microbiol.
68: 772-783
[Abstract] [Full Text] -
Hoffmann, T., Schutz, A., Brosius, M., Volker, A., Volker, U., Bremer, E.
(2002). High-Salinity-Induced Iron Limitation in Bacillus subtilis. J. Bacteriol.
184: 718-727
[Abstract] [Full Text] -
Boncompagni, E., Dupont, L., Mignot, T., Østeräs, M., Lambert, A., Poggi, M.-C., Le Rudulier, D.
(2000). Characterization of a Sinorhizobium meliloti ATP-Binding Cassette Histidine Transporter Also Involved in Betaine and Proline Uptake. J. Bacteriol.
182: 3717-3725
[Abstract] [Full Text] -
Cánovas, D., Vargas, C., Kneip, S., Morón, M.-J., Ventosa, A., Bremer, E., Nieto, J. J.
(2000). Genes for the synthesis of the osmoprotectant glycine betaine from choline in the moderately halophilic bacterium Halomonas elongata DSM 3043. Microbiology
146: 455-463
[Abstract] [Full Text]
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