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Originally published In Press as doi:10.1074/jbc.M210887200 on March 3, 2003

J. Biol. Chem., Vol. 278, Issue 19, 16482-16487, May 9, 2003
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Dimerization and DNA Binding Properties of the Bacillus licheniformis 749/I BlaI Repressor*

Patrice FiléeDagger §, Christelle Vreuls, Raphaël HermanDagger , Iris ThammDagger , Tony Aerts||, Peter P. De Deyn||, Jean-Marie FrèreDagger , and Bernard JorisDagger **

From the Dagger  Centre d'ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart-Tilman, B4000 Liège, Belgium, the  Laboratoire de Physique Biomédicale, Institut de Physique B5, Université de Liège, Sart-Tilman, B4000 Liège, Belgium, and the || Department of Biomedical Sciences, University of Antwerp, B-2610 Antwerp, Belgium

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 µM 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 µM) or in the same range as (75 µM) 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 KdOP1 = 1.7 ± 0.5 10-15 M2, KdOP2 = 3.3 ± 0.9 10-15 M2, and KdOP3 = 10.5 ± 2.5 10-15 M2. The role of the DNA binding properties of BlaI on the beta -lactamase regulation is discussed.

* This work was supported by Grant P5/33 from the Belgian Program on Interuniversity Poles of Attraction initiated by the Federal Office for Scientific, Technical and Cultural Affaires and Fond National de la Recherche Scientifique Grant 1.5201.02, and Fonds de la Recherche Fondamentale Collective Grant 2.4530.03.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Fellow of the Fonds pour la Formation à la Recherche dans l'Industrie et l'Agriculture.

** Research Associate of the Fond National de la Recherche Scientifique. To whom correspondence should be addressed. Tel.: 32-4-366-2954; Fax: 32-4-366-3364; E-mail: bjoris@ulg.ac.be.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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