The fate of the BlaI repressor during the induction of the Bacillus licheniformis BlaP beta-lactamase.
The induction of the Staphylococcus aureus BlaZ and Bacillus licheniformis 749/I BlaP beta-lactamases by beta-lactam antibiotics occurs according to similar processes. In both bacteria, the products of the blaI and blaRl 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 beta-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 blaRl genes. In contrast to the situation in B. licheniformis 749/I, beta-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/pDML995cells. No variation in the Blal 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 beta-lactamase biosynthesis.
Filée
Patrice
P
Centre d'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart-Tilman, B4000, Belgium.
Benlafya
Kamal
K
Delmarcelle
Michaël
M
Moutzourelis
Georgios
G
Frère
Jean-Marie
JM
Brans
Alain
A
Joris
Bernard
B
D001407:Bacillus / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics; D004269:DNA, Bacterial / Q000378:metabolism; D019281:Dimerization; D015180:Electrophoresis, Gel, Two-Dimensional; D004790:Enzyme Induction / Q000235:genetics; D005798:Genes, Bacterial; D007525:Isoelectric Focusing; D008957:Models, Genetic; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D001618:beta-Lactamases