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Microbiology. 2014 Jan;160(Pt 1):91-101. doi: 10.1099/mic.0.071233-0. Epub 2013 Nov 6.

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

Author information

1
Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.

Abstract

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.

PMID:
24196428
DOI:
10.1099/mic.0.071233-0
[Indexed for MEDLINE]

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