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Volume 160, Issue 1

Regulation of the mannitol utilization genes: promoter structure and transcriptional activation by the wild-type regulator (MtlR) and its mutants Free

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.

  • Received:
  • Accepted:
  • Published Online:
Funding
This study was supported by the:
  • the German Academic Exchange Service (DAAD)
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2014-01-01
2023-12-01
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Regulation of the Bacillus subtilis mannitol utilization genes: promoter structure and transcriptional activation by the wild-type regulator (MtlR) and its mutants
Microbiology 160, 91 (2014); https://doi.org/10.1099/mic.0.071233-0
/content/journal/micro/10.1099/mic.0.071233-0
/content/journal/micro/10.1099/mic.0.071233-0
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