Abstract
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 [(3)H]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.
Highlights
Many bacteria transport D-mannitol and other carbohydrates via a phosphoenolpyruvate-dependent phosphotransferase system (PTS)1 [1,2,3]
PTS regulation domain (PRD)-I and PRD-II, are expected to contain the phosphorylation sites based on the homology of MtlR to antiterminators such as SacY and BglG and the DNA-binding regulators LicR and LevR
The current study shows that HPr phosphorylates both PRD-I and PRD-II
Summary
Many bacteria transport D-mannitol and other carbohydrates via a phosphoenolpyruvate-dependent phosphotransferase system (PTS)1 [1,2,3]. When favorable catabolites like glucose are utilized, HPr is phosphorylated by a kinase on a specific serine [5] that forms a complex with the CcpA repressor Binding of this complex to catabolite response element sites located in or near the promoter regions of catabolic operons will prevent expression of these operons [6]. In addition to catabolite repression, the expression of the mannitol operon is probably regulated by the mannitol regulator MtlR [7] Domains in this protein show similarity to domains of two types of transcriptional regulators: DNA-binding proteins and anti-terminators. The activity of most of these proteins can be regulated by phosphorylation of PRD-I and/or PRD-II by the PTS components HPr and/or IIB Based on these similarities, it was assumed that MtlR is a DNA-binding protein whose activity is regulated by the PTS [8]. In addition to PRD-I and PRD-II, a third phosphorylation domain is presented that is involved in the phosphorylation and dephosphorylation of MtlR by IICBmtl
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