The fate of the BlaI repressor during the induction of the Bacillus licheniformis BlaP β-lactamase

Abstract

The induction of the Staphylococcus aureus BlaZ and Bacillus licheniformis 749/I BlaP β-lactamases by β-lactam antibiotics occurs according to similar processes. In both bacteria, the products of the blaI and blaR1 genes share a high degree of sequence homology and act as repressors and penicillin-sensory transducers respectively. It has been shown in S. aureus that the BlaI repressor, which controls the expression of BlaZ negatively, is degraded after the addition of the inducer. In the present study, we followed the fate of BlaI during β-lactamase induction in B. licheniformis 749/I and in a recombinant Bacillus subtilis 168 strain harbouring the pDML995 plasmid, which carries the B. licheniformis blaP, blaI and blaR1 genes. In contrast to the situation in B. licheniformis 749/I, β-lactamase induction in B. subtilis 168/pDML995 was not correlated with the proteolysis of BlaI. To exclude molecular variations undetectable by SDS-PAGE, two-dimensional gel electrophoresis was performed with cellular extracts from uninduced or induced B. subtilis 168/pDML995 cells. No variation in the BlaI isoelectric point was observed in induced cells, whereas the DNA-binding property was lost. Cross-linking experiments with dithiobis(succimidylpropionate) confirmed that, in uninduced recombinant B. subtilis cells, BlaI was present as a homodimer and that this situation was not altered in induced conditions. This latter result is incompatible with a mechanism of inactivation of BlaI by proteolysis and suggests that the inactivation of BlaI results from a non-covalent modification by a co-activator and that the subsequent proteolysis of BlaI might be a secondary phenomenon. In addition to the presence of this co-activator, our results show that the presence of penicillin stress is also required for full induction of β-lactamase biosynthesis.