Evidence against the Bm1P1 protein as a positive transcription factor for barbiturate-mediated induction of cytochrome P450(BM-1) in Bacillus megaterium

Abstract

The Bm1P1 protein was previously proposed to act as a positive transcription factor involved in barbituratemediated induction of cytochrome P450(BM-1) in Bacillus megaterium. We now report that the bm1P1 gene encodes a protein of 217 amino acids, rather than the 98 amino acids as reported previously. In vitro gel shift assays indicate that the Bm1P1 protein did not interact with probes comprising the regulatory regions of the P450(BM-1) gene. Moreover, disruption of the bm1P1 gene did not markedly affect barbiturate induction of P450(Bm-1) expression. A multicopy plasmid harboring only the P450(BM-1) promoter region could increase expression of the chromosome-encoded P450(BM-1). The level of expression is comparable with that shown by a multicopy plasmid harboring the P450(BM-1) promoter region along with the bm1P1 gene. These results strongly suggest that the Bm1P1 protein is unlikely to act as a positive regulator for barbiturate induction of P450(BM-1) expression. Finally, deletion of the Barbie box did not markedly diminish the effect of pentobarbital on expression of a reporter gene transcriptionally fused to the P450(BM-1) promoter. This suggests that the Barbie box is unlikely to be a key element in barbiturate-mediated induction of P450(BM-1).