Dimerization and DNA binding properties of the Bacillus licheniformis 749/I BlaI repressor.

Abstract In the absence of penicillin, the β-lactamase encoding gene blaP of Bacillus licheniformis749/I is negatively regulated by the transcriptional repressor BlaI. Three palindromic operator regions are recognized by BlaI: two in theblaP 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 β-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 interactsin 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, andK d OP3 = 10.5 ± 2.5 10−15 m 2. The role of the DNA binding properties of BlaI on the β-lactamase regulation is discussed.