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J. Biol. Chem., Vol. 277, Issue 42, 39809-39814, October 18, 2002
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From the Institut für Mikrobiologie und Weinforschung,
Johannes Gutenberg-Universität Mainz, Becherweg 15, 55099 Mainz, Germany
The two-component regulatory system DcuSR of
Escherichia coli controls the expression of genes of
C4-dicarboxylate metabolism in response to extracellular
C4- dicarboxylates such as fumarate or succinate. DcuS is
a membrane-integral sensor kinase, and the sensory and kinase domains
are located on opposite sides of the cytoplasmic membrane. The intact
DcuS protein (His6-DcuS) was overproduced and isolated in
detergent containing buffer. His6-DcuS was reconstituted
into liposomes made from E. coli phospholipids. Reconstituted His6-DcuS catalyzed, in contrast to the
detergent-solubilized sensor, autophosphorylation by
[ This work is dedicated to the late Prof. A. Kröger (Frankfurt).
Function of DcuS from Escherichia coli as a
Fumarate-stimulated Histidine Protein Kinase in Vitro*
-33P]ATP with an approximate KD
of 0.16 mM for ATP. Up to 7% of the
reconstituted DcuS was phosphorylated. Phosphorylation was stimulated
up to 5.9-fold by C4-dicarboxylates, but not by other
carboxylates. The phosphoryl group of DcuS was rapidly transferred to
the response regulator DcuR. Upon phosphorylation, DcuR bound specifically to dcuB promoter DNA. The reconstituted DcuSR
system therefore represents a defined in vitro system,
which is capable of the complete transmembrane signal transduction by
the DcuSR two-component system from the stimulus (fumarate) to the DNA, including signal transfer across the phospholipid membrane.
*
The work was supported by grants from the Deutsche
Forschungsgemeinschaft and the Fonds der Chemischen Industrie.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Johannes Gutenberg
Universität Mainz, Inst. für Mikrobiologie und
Weinforschung, Becherweg 15, 55099 Mainz, Germany. Tel.:
49-6131-3923550; Fax: 49-6131-3922695; E-mail:
unden@mail.uni-mainz.de.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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