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J. Biol. Chem., Vol. 278, Issue 12, 10744-10751, March 21, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/12/10744    most recent M212066200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Seitz, S. Articles by Plumbridge, J. Search for Related Content PubMed PubMed Citation Articles by Seitz, S. Articles by Plumbridge, J.

Analysis of the Interaction between the Global Regulator Mlc and EIIB^Glc of the Glucose-specific Phosphotransferase System in Escherichia coli^*

Sabine Seitz, Sung-Jae Lee, Carole Pennetier^§, Winfried Boos, and Jacqueline Plumbridge^§¶

From the  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

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 EIICB^Glc (PtsG), which is formed during the transport of glucose. Here, we demonstrate that EIIB^Glc, the cytoplasmic domain of PtsG, alone is sufficient to inactivate Mlc but only when EIIB^Glc is attached to the membrane by a protein anchor, which can be unrelated to PtsG. Several EIIB^Glc mutants, which were altered in and around the phosphorylation site (Cys-421) of EIIB^Glc, 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 EIIB^Glc 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.

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