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J. Biol. Chem., Vol. 278, Issue 12, 10744-10751, March 21, 2003
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From the Mlc is a global regulator acting as a
transcriptional repressor for several genes and operons of
Escherichia coli encoding sugar-metabolizing enzymes and
uptake systems. The repressing activity of Mlc is inactivated by
binding to the dephosphorylated form of EIICBGlc (PtsG),
which is formed during the transport of glucose. Here, we demonstrate
that EIIBGlc, the cytoplasmic domain of PtsG, alone is
sufficient to inactivate Mlc but only when EIIBGlc is
attached to the membrane by a protein anchor, which can be unrelated to
PtsG. Several EIIBGlc mutants, which were altered in and
around the phosphorylation site (Cys-421) of EIIBGlc, were
tested for their ability to bind Mlc and to affect transcriptional repression by Mlc. The exchange of Cys-421 with serine or aspartate still allowed binding to Mlc, and in addition, derepression became constitutive, i.e. independent of
phosphoenolpyruvate-dependent phosphotransferase system
(PTS) phosphorylation. Mutations were made in the surface-exposed
residues in the vicinity of Cys-421 and identified Arg-424 as essential
for binding to Mlc. Binding of Mlc to the EIIBGlc
constructs in membrane preparations paralleled their ability to
derepress Mlc-dependent transcription in vivo.
These observations demonstrate that it is not the charge change at
Cys-421, produced by PTS phosphorylation, that allows Mlc binding but
rather the structural change in the environment surrounding Cys-421
that the phosphorylation provokes. Native Mlc exists as a tetramer. Deleting 18 amino acids from the C-terminal removes a putative amphipathic helix and results in dimeric Mlc that is no longer able to repress.
Analysis of the Interaction between the Global Regulator Mlc and
EIIBGlc of the Glucose-specific Phosphotransferase System
in Escherichia coli*
,,, and Department of Biology, University of
Konstanz, D-78457 Konstanz, Germany and the § Institut de
Biologie Physico-Chimique (UPR9073), 13, rue Pierre et Marie Curie,
75005 Paris, France
*
This work was supported by grants from the Deutsche
Forschungsgemeinschaft and the Fonds der Chemischen Industrie (to
W. B.) and from the CNRS and the Université Paris 7 (to
UPR9073).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.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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