[en] 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 muM 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 muM) or in the same range as (75 muM) 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-dOP1=1.7+/-0.5 10(-15) M-2, K-dOP2=3.3+/-0.9 10(-15) M-2, and K-dOP3=10.5+/-2.5 10(-15) M-2. The role of the DNA binding properties of BlaI on the beta-lactamase regulation is discussed.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Filée, Patrice ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Vreuls, Christelle ; Université de Liège - ULiège > Département des sciences de la vie > Biologie et génétique moléculaire
Herman, Raphaël ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Thamm, Iris ; Université de Liège - ULiège > Département des sciences de la vie > Département des sciences de la vie
Aerts, T.
De Deyn, P. P.
Frère, Jean-Marie ; Université de Liège - ULiège > Département des sciences de la vie > Département des sciences de la vie
Joris, Bernard ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Language :
English
Title :
Dimerization and DNA binding properties of the Bacillus licheniformis 749/I BlaI repressor
Publication date :
May 2003
Journal title :
Journal of Biological Chemistry
ISSN :
0021-9258
eISSN :
1083-351X
Publisher :
Amer Soc Biochemistry Molecular Biology Inc, Bethesda, United States - Maryland
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