Barbiturate-mediated regulation of expression of the cytochrome P450BM-3 gene of Bacillus megaterium by Bm3R1 protein.

In a previous publication (Wen, L.-P., Ruettinger, R. T., and Fulco, A.J. (1989) J. Biol. Chem. 264, 10996-11003), we reported that about 1 kilobase of 5' flanking sequence was required for barbiturate-inducible expression of the cytochrome P450BM-3 gene in Bacillus megaterium. We have now found, by analysis of various deletion and frameshift derivatives of this region, that an open reading frame immediately upstream of the B. megaterium cytochrome P450BM-3 structural gene encodes a protein, designated Bm3R1, which negatively controls the expression of the P450BM-3 gene at the transcriptional level. A helix-turn-helix DNA binding motif was found near the N-terminal portion of Bm3R1 protein. The 5' terminus of the bm3R1 transcript generated in vivo was determined by nuclease S1 mapping and primer extension analysis to be 44 base pairs upstream of the translation initiation sequence GTG of bm3R1. A putative promoter sequence with a high degree of similarity to the -35 and -10 consensus sequence recognized by the Bacillus subtilis sigma-43 factor was located at an appropriate distance from the transcription start site. A B. megaterium mutant which highly constitutively produced P450BM-3 protein was isolated and complementation of this cytochrome P450BM-3-constitutive mutant by a DNA fragment containing the wild-type bm3R1 gene indicated that the mutation in this locus was trans-dominant. Sequence analysis of the bm3R1 gene and its upstream region from this mutant, after amplification by the polymerase chain reaction, identified a single base change that resulted in a glycine to glutamate substitution in the beta-turn region of the DNA binding motif. By placing the bm3R1 gene under the control of a tac promoter and changing the translation initiation sequence from GTG to ATG, we succeeded in overproducing the Bm3R1 protein in Escherichia coli. A 20-bp perfect palindromic putative operator site, located between the presumed promoter sequences and the bm3R1 structural gene, was defined both by in vivo titration of Bm3R1 repressor and by gel mobility shift assays using the cell-free extracts containing the overproduced wild-type or mutant Bm3R1 protein. The barbiturate effect in mediating the induction of cytochrome P450BM-3 appears to be indirect but probably involves, in part, the release of inhibition by Bm3R1 repressor protein by interfering with its binding to the palindromic putative operator sequence and perhaps to other sites on the regulatory region of the gene encoding cytochrome P450BM-3.