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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
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
-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.
*
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.
Applied and Environmental Microbiology, February 1999, p. 560-568, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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