Expression of mannitol utilization genes in Bacillus subtilis is directed by PmtlA, the promoter of the mtlAFD operon, and PmtlR, the promoter of the MtlR activator. MtlR contains phosphoenolpyruvate-dependent phosphotransferase system (PTS) regulation domains, called PRDs. The activity of PRD-containing MtlR is mainly regulated by the phosphorylation/dephosphorylation of its PRDII and EIIB(Gat)-like domains. Replacing histidine 342 and cysteine 419 residues, which are the targets of phosphorylation in these two domains, by aspartate and alanine provided MtlR-H342D C419A, which permanently activates PmtlA in vivo. In the mtlR-H342D C419A mutant, PmtlA was active, even when the mtlAFD operon was deleted from the genome. The mtlR-H342D C419A allele was expressed in an Escherichia coli strain lacking enzyme I of the PTS. Electrophoretic mobility shift assays using purified MtlR-H342D C419A showed an interaction between the MtlR double-mutant and the Cy5-labelled PmtlA and PmtlR DNA fragments. These investigations indicate that the activated MtlR functions regardless of the presence of the mannitol-specific transporter (MtlA). This is in contrast to the proposed model in which the sequestration of MtlR by the MtlA transporter is necessary for the activity of MtlR. Additionally, DNase I footprinting, construction of PmtlA-PlicB hybrid promoters, as well as increasing the distance between the MtlR operator and the -35 box of PmtlA revealed that the activated MtlR molecules and RNA polymerase holoenzyme likely form a class II type activation complex at PmtlA and PmtlR during transcription initiation.