Analysis of the interaction between the global regulator Mlc and EIIBGlc of the glucose-specific phosphotransferase system in Escherichia coli.
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.
Seitz
Sabine
S
Department of Biology, University of Konstanz, Germany.
Lee
Sung-Jae
SJ
Pennetier
Carole
C
Boos
Winfried
W
Plumbridge
Jacqueline
J
D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism
D029968:Escherichia coli Proteins; C111065:Mlc protein, E coli; D012097:Repressor Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C031629:phosphoenolpyruvate-glucose phosphotransferase