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J Biol Chem, Vol. 275, Issue 10, 7037-7044, March 10, 2000

Multiple Phosphorylation Events Regulate the Activity of the Mannitol Transcriptional Regulator MtlR of the Bacillus stearothermophilus Phosphoenolpyruvate-dependent Mannitol Phosphotransferase System^*

Sytse A. Henstra, Ria H. Duurkens, and George T. Robillard

From the Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

D-Mannitol is taken up by Bacillus stearothermophilus and phosphorylated via a phosphoenolpyruvate-dependent phosphotransferase system (PTS). Transcription of the genes involved in mannitol uptake in this bacterium is regulated by the transcriptional regulator MtlR, a DNA-binding protein whose affinity for DNA is controlled by phosphorylation by the PTS proteins HPr and IICB^mtl. The mutational and biochemical studies presented in this report reveal that two domains of MtlR, PTS regulation domain (PRD)-I and PRD-II, are phosphorylated by HPr, whereas a third IIA-like domain is phosphorylated by IICB^mtl. An involvement of PRD-I and the IIA-like domain in a decrease in affinity of MtlR for DNA and of PRD-II in an increase in affinity is demonstrated by DNA footprint experiments using MtlR mutants. Since both PRD-I and PRD-II are phosphorylated by HPr, PRD-I needs to be dephosphorylated by IICB^mtl and mannitol to obtain maximal affinity for DNA. This implies that a phosphoryl group can be transferred from HPr to IICB^mtl via MtlR. Indeed, this transfer could be demonstrated by the phosphoenolpyruvate-dependent formation of [³H]mannitol phosphate in the absence of IIA^mtl. Phosphoryl transfer experiments using MtlR mutants revealed that PRD-I and PRD-II are dephosphorylated via the IIA-like domain. Complementation experiments using two mutants with no or low phosphoryl transfer activity showed that phosphoryl transfer between MtlR molecules is possible, indicating that MtlR-MtlR interactions take place. Phosphorylation of the same site by HPr and dephosphorylation by IICB^mtl have not been described before; they could also play a role in other PRD-containing proteins.

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