Abstract
In a previous publication (Wen, L.-P., and Fulco, A. J. (1987) J. Biol. Chem. 262, 6676-6682), we described the cloning of the gene encoding cytochrome P-450BM-3, a catalytically self-sufficient fatty acid monooxygenase induced by barbiturates in Bacillus megaterium. We have now subcloned a 1.6-kilobase segment of DNA from this cloned gene that includes the barbiturate-responsive regulatory region as well as 88 bases encoding the NH2-terminal portion of cytochrome P-450BM-3. From this, we generated two series of 5' and 3' deletion derivatives and examined their effects on gene expression. When the 1.6-kilobase fragment or the 1.3-kilobase 5'----3' deletion derivative is inserted into Escherichia coli on a vector containing a promoterless chloramphenicol acetyltransferase (CAT) gene with the sequence encoding the NH2-terminal portion of P-450BM-3 placed immediately in front of the CAT gene, CAT activity is constitutive and unaffected by pentobarbital. On the other hand, the basal level of CAT is low in B. megaterium transformed by the same construct but is strongly inducible by pentobarbital. Furthermore, the multicopy plasmid containing this regulatory region causes a dramatic decrease in both the basal and pentobarbital-induced expression of chromosomally encoded P-450BM-3 in B. megaterium. This competition effect, unlike CAT expression, is independent of the orientation of the regulatory DNA segment in the plasmid. Removal of 0.3 kilobase or more from the 3' end of the 1.6-kilobase segment of DNA or 0.6 kilobase from the 5' end abolishes the competition effect and also eliminates basal and inducible CAT expression in B. megaterium. In transformed E. coli, constitutive CAT expression is maintained when as little as 0.3 kilobase of DNA from the 3' end of the 1.6-kilobase segment is inserted in the correct orientation in front of the CAT gene. The data are consistent with the hypothesis that the synthesis of cytochrome P-450BM-3 in B. megaterium is under positive control and requires gene interaction with at least one trans-acting factor, presumably a protein, to activate transcription from the P-450BM-3 promoter. The binding of this putative protein is mediated by at least two regulatory regions (R1 and R2) that span about 1 kilobase of the 5'-flanking region of the gene.
Highlights
From the Departmentof Biological Chemistry, School of Medicine, and the Laboratory of Biomedical and Environmental Sciences, Universityof California, Los Angeles, California 90024
Strains of B. rnegaterium that we examined [27] were positive for the A series of 5' +3' deletion derivatives were generated from plasmid presence of pentobarbital-inducible P-450BM.3, a finding more con- BM3-6A.Here, the 3' end of the 1.6-kb fragment corresponded to the sistent with encoding by chromosomal rather than by plasmid-borne NHZ-terminal portion of the P-450~,~.s3tructural gene while the 5'
B. megaterium transformed by plasmid pBCAT.1 produced a significant level of CAT activity in the absence of barbiturates, and this activity was increased about 8-fold when the cells were grown in the presence of 4 mM pentobarbital (Table I)
Summary
The 1.6-kbHind111 fragment (with one Hind111 site derived from the pUCl9 vector, seeFig. 2 B ) was Throughout this paper, we refer to the naturally occurring gene isolated from a gel and inserted into plasmid pT~T3.19in both direcencoding P - 4 5 0 ~ "i~n B. rnegateriurn ATCC 1458a1s "chromosomally tions to form plasmids designated BM3.6A and B M ~ . Strains of B. rnegaterium that we examined [27] were positive for the A series of 5' +3' deletion derivatives were generated from plasmid presence of pentobarbital-inducible P-450BM., a finding more con- BM3-6A.Here, the 3' end of the 1.6-kb fragment corresponded to the sistent with encoding by chromosomal rather than by plasmid-borne NHZ-terminal portion of the P-450~,~.s3tructural gene while the 5'.
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