1887

Abstract

Expression of mannitol utilization genes in is directed by P, the promoter of the operon, and P, 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-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 P. In the -H342D C419A mutant, P was active, even when the operon was deleted from the genome. The -H342D C419A allele was expressed in an 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 P and P 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 P-P hybrid promoters, as well as increasing the distance between the MtlR operator and the −35 box of P revealed that the activated MtlR molecules and RNA polymerase holoenzyme likely form a class II type activation complex at P and P during transcription initiation.

Funding
This study was supported by the:
  • the German Academic Exchange Service (DAAD)
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2014-01-01
2021-05-16
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