The Bacillus subtilis mannose regulator, ManR, a DNA‐binding protein regulated by HPr and its cognate PTS transporter ManP. Issue 3 (1st April 2013)

• Record Type: Journal Article
• Authors: Wenzel, Marian; Altenbuchner, Josef
• Is Part Of: Molecular microbiology. Volume 88:Issue 3(2013)
• Page Start: 562
• Page End: 576
• Publication Date: 2013-04-01
• Subjects: Molecular microbiology -- Periodicals; 572.829
• DOI: 10.1111/mmi.12209
• Languages: English
• ISSNs: 0950-382X

Abstract

<abstract abstract-type="main"> <title>Summary</title> <p>The transcriptional activator ManR of the <italic>Bacillus subtilis</italic> mannose utilization operon is composed of an N‐terminal DNA‐binding domain, two phosphotransferase system (PTS) regulation domains (PRDs), an EIIB<sup>Bgl</sup>‐ and an EIIA<sup>Fru</sup>‐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<sup>Bgl</sup>) and the His570Ala mutations (EIIA<sup>Fru</sup>) provoked constitutive activities to different degrees, whereas the latter had the greater influence. Addition of EIIBA<sup>Man</sup> 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 P<italic><sub>manR</sub></italic> and P<italic><sub>manP</sub></italic> 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 <italic>K</italic><sub>D</sub> and the dissociation rate <italic>k</italic><sub>diss</sub>, were determined for both promoter regions.</p> </abstract>