In the absence of penicillin, the beta-lactamase encoding gene blaP of Bacillus licheniformis 749/I is negatively regulated by the transcriptional repressor BlaI. Three palindromic operator regions are recognized by BlaI: two in the blaP promoter (OP1 and OP2) and one (OP3) in the promoter of the blaI-blaR1 operon. In this study, the dissociation constant of the purified BlaI dimer was estimated at 25 microm by equilibrium ultracentrifugation. Quantitative Western blot analysis indicates that the intracellular concentrations of BlaI in B. licheniformis 749/I and Bacillus subtilis transformed by a multicopy plasmid harboring the beta-lactamase locus (blaP-blaI-blaR1) were lower than (1.9 microm) or in the same range as (75 microm) the dissociation constant, respectively. This suggests that BlaI is partially dimeric in the cytoplasm of these strains and interacts in vivo with its operators as a preformed dimer. This hypothesis is supported by band shift assays on an operator containing a randomized half-operator sequence. The global dissociation constants of the operator-BlaI dimer complexes were measured by band shift assays and estimated as K(d)(OP1) = 1.7 +/- 0.5 10(-15) m(2), K(d)(OP2) = 3.3 +/- 0.9 10(-15) m(2), and K(d)(OP3) = 10.5 +/- 2.5 10(-15) m(2). The role of the DNA binding properties of BlaI on the beta-lactamase regulation is discussed.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1074/jbc.M210887200 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Analysis of the stability and affinity of BlaR-CTD protein to β-lactam antibiotics based on docking and mutagenesis studies.
J Biol Eng
March 2019
1National Reference Laboratory of Veterinary Drug Residues (HZAU) and MOA Key Laboratory for the Detection of Veterinary Drug Residues in Foods, Huazhong Agricultural University, Wuhan, 430070 China.
Owing to the thermal instability and low affinity of BlaR-CTD to some β-lactams, the receptor assay based on BlaR-CTD is limited in the detection of abundant variety of drugs and the result is often unstable. In this study, the three-dimensional structure of BlaR-CTD from ATCC14580 was constructed by homologous modeling based on the crystal structure of BlaR-CTD from 749/I, and the binding sites of this protein to 40 β-lactams were also obtained by molecular docking. To improve the stability and affinity of the protein, 23 mutant proteins were designed based on docking and homologous alignment results as well as by inserting disulfide bond and building the salt bridge.
View Article and Find Full Text PDFBacillus licheniformis BlaR1 L3 loop is a zinc metalloprotease activated by self-proteolysis.
PLoS One
September 2012
Centre for Protein Engineering, Department of Life Sciences, University of Liège, Liège, Belgium.
In Bacillus licheniformis 749/I, BlaP β-lactamase is induced by the presence of a β-lactam antibiotic outside the cell. The first step in the induction mechanism is the detection of the antibiotic by the membrane-bound penicillin receptor BlaR1 that is composed of two functional domains: a carboxy-terminal domain exposed outside the cell, which acts as a penicillin sensor, and an amino-terminal domain anchored to the cytoplasmic membrane, which works as a transducer-transmitter. The acylation of BlaR1 sensor domain by the antibiotic generates an intramolecular signal that leads to the activation of the L3 cytoplasmic loop of the transmitter by a single-point cleavage.
View Article and Find Full Text PDFEffects of monopropanediamino-β-cyclodextrin on the denaturation process of the hybrid protein BlaPChBD.
Biochim Biophys Acta
September 2011
Centre d'Ingénierie des Protéines, Université de Liège, Institut de Chimie B6, Sart-Tilman, Liège, Belgium.
Irreversible accumulation of protein aggregates represents an important problem both in vivo and in vitro. The aggregation of proteins is of critical importance in a wide variety of biomedical situations, ranging from diseases (such as Alzheimer's and Parkinson's diseases) to the production (e.g.
View Article and Find Full Text PDFGuanidinium chloride denaturation of the dimeric Bacillus licheniformis BlaI repressor highlights an independent domain unfolding pathway.
Biochem J
November 2004
Centre d'ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart-Tilman, B4000 Liège, Belgium.
The Bacillus licheniformis 749/I BlaI repressor is a prokaryotic regulator that, in the absence of a beta-lactam antibiotic, prevents the transcription of the blaP gene, which encodes the BlaP beta-lactamase. The BlaI repressor is composed of two structural domains. The 82-residue NTD (N-terminal domain) is a DNA-binding domain, and the CTD (C-terminal domain) containing the next 46 residues is a dimerization domain.
View Article and Find Full Text PDFCrystal structure of the sensor domain of the BlaR penicillin receptor from Bacillus licheniformis.
Biochemistry
November 2003
Institut de Physique B5, Université de Liège, B-4000 Sart Tilman, Belgium.
As in several staphylococci, the synthesis of the Bacillus licheniformis 749/I beta-lactamase is an inducible phenomenon regulated by a signal-transducing membrane protein BlaR. The C-terminal domain of this multimodular protein is an extracellular domain which specifically recognizes beta-lactam antibiotics. When it binds a beta-lactam, a signal is transmitted by the transmembrane region to the intracellular loops.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!