The Bacillus subtilis mtl operon encodes the enzymes necessary for mannitol utilization. Its expression is controlled by MtlR, a transcriptional activator belonging to the DeoR family. MtlR contains a HTH domain followed by two PTS regulation domains (PRDs), an EIIBGat domain and an EIIAMtl-like domain.The general mechanism of the regulation of MtlR activity is based on its phosphorylation by components of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS). The phosphorylation of EIIBGat on cysteine 419 by P~EIIAMtl has a major negative effect on the activity of MtlR. The absence of EIIAMtl in a mtlF mutant therefore leads to constitutively active MtlR.In this study a new mechanism of MtlR regulation based on the interaction of the PTS component EIIBMtl with MtlR is presented.We observed that the deletion of the entire mtlAFD operon or of mtlF and only the 3’-part of mtlA (encoding the EIIBMtl domain) abolishes the constitutive MtlR activity of the mtlF mutant, suggesting that MtlR activity depends on functional EIIBMtl. By carrying out yeast two-hybrid experiments we could establish a direct, specific and bidirectional interaction between EIIBMtl and the EIIBGatEIIAMtl-like part of MtlR.Complementation of the above mutants was possible with entire MtlA, but not with the EIIBMtl domain. EIIBMtl is normally fused to the membrane protein EIICMtl; we therefore fused EIIBMtl to another membrane, which indeed restored MtlR function in the absence of EIICMtl. The EIICMtl domain is therefore not essential for the interaction between EIIBMtl and MtlR; it is rather the vicinity of the membrane which is required for the activation of MtlR.A regulation model of MtlR activity is proposed. In this model, the MtlR-mediated induction of the mtlAFD operon requires the phosphorylation of PRDII by P~His-HPr and the dephosphorylation of EIIBGat by EIIAMtl. The presence of unphosphorylated EIIBMtl, which prevails when the inducer mannitol is present, is also required. Under these conditions unphosphorylated EIIBMtl sequesters MtlR dephosphoryled on cysteine 419, but phosphorylated at His-342, to the membrane thereby activating the transcription activator, which leads to increased expression of the mtlAFD operon.
