title: The Bacillus subtilis mannose regulator, ManR, a DNA-binding protein regulated by HPr and its cognate PTS transporter ManP.
abstract: The transcriptional activator ManR of the Bacillus subtilis mannose utilization operon is composed of an N-terminal DNA-binding domain, two phosphotransferase system (PTS) regulation domains (PRDs), an EIIB(Bgl) - and an EIIA(Fru) -like domain. Site-specific mutagenesis of ManR revealed the role of conserved amino acids representing potential phosphorylation sites. This was investigated by β-galactosidase activity tests and by mobility shift assays after incubation with the PTS components HPr and EI. In analogy to other PRD-containing regulators we propose stimulation of ManR activity by phosphorylation. Mutations in PRD1 lowered ManR activity, whereas mutations in PRD2 abolished ManR activity completely. The Cys415Ala (EIIB(Bgl)) and the His570Ala mutations (EIIA(Fru)) provoked constitutive activities to different degrees, whereas the latter had the greater influence. Addition of EIIBA(Man) reduced the binding capability significantly in a wild-type and a Cys415Ala background, but had no effect on a His570Ala mutant. The different expression levels originating from the two promoters PmanR and PmanP could be ascribed to different 5'-untranslated mRNA regions. Sequences of 44 bp were identified and confirmed as the ManR binding sites by DNase I footprinting. The binding properties of ManR, in particular the equilibrium dissociation constant KD and the dissociation rate kdiss, were determined for both promoter regions.
authors.affiliation: Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
authors.forename: Marian
authors.initials: M
authors.lastname: Wenzel
authors.forename: Josef
authors.initials: J
authors.lastname: Altenbuchner
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D018983:DNA Footprinting / Q000379:methods; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D007763:Lac Operon; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C442131:ScoC protein, Bacillus subtilis; D014157:Transcription Factors; D010770:Phosphotransferases; D003850:Deoxyribonuclease I; D001616:beta-Galactosidase; D008358:Mannose