Dimerization and DNA binding properties of the Bacillus licheniformis 749/I BlaI repressor.
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.
Filée
Patrice
P
Centre d'ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart-Tilman, B4000 Liège, Belgium.
Vreuls
Christelle
C
Herman
Raphaël
R
Thamm
Iris
I
Aerts
Tony
T
De Deyn
Peter P
PP
Frère
Jean-Marie
JM
Joris
Bernard
B
D001407:Bacillus / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D011485:Protein Binding; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D001618:beta-Lactamases