©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Molecular Cloning and Characterization of BM1P1 and BM1P2 Proteins, Putative Positive Transcription Factors Involved in Barbiturate-mediated Induction of the Genes Encoding Cytochrome P450 of Bacillus megaterium(*)

(Received for publication, December 13, 1994; and in revised form, March 21, 1995)

Jian-Sen He Qianwa Liang Armand J. Fulco (§)

From the Department of Biological Chemistry and the Laboratory of Structural Biology and Molecular Medicine, School of Medicine, University of California, Los Angeles, California 90024-1737

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Analysis of a 1.3-kilobase segment of 5`-flanking DNA from the barbiturate-inducible P450 gene (CYP106) of Bacillus megaterium revealed two open reading frames. One, BM1P1, encodes 98 amino acids and is located 267 base pairs upstream from the sequence encoding cytochrome P450 but in the opposite orientation. The second, BM1P2 (88 amino acids), is 892 base pairs upstream from the P450 coding sequence and in the same coding strand. The expression of BM1P1 and BM1P2 was strongly stimulated in cells grown in the presence of pentobarbital, and the BM1P1 gene product exerted positive control on expression of P450. When a 177-base pair fragment encompassing the overlapping promoter regions of the P450 and BM1P1 genes was used as a probe in DNA binding assays, the BM1P1 and BM1P2 gene products and Bm3R1 (the repressor protein regulating the barbiturate-mediated expression of P450) could bind individually, but the addition of BM1P1 or BM1P2 to a binding mixture containing Bm3R1 completely prevented the appearance of a Bm3R1 binding band. When a 208-base pair fragment containing a Barbie box sequence and located upstream of the 177-base pair fragment was used as a probe, only a Bm3R1 binding band was detected. Although neither BM1P1 and BM1P2 appeared to bind to this 208-base pair fragment, their presence strongly inhibited the binding of Bm3R1 to the same probe. The evidence suggests that BM1P1 and BM1P2 may, in part, act as positive regulatory proteins involved in the expression of the P450 gene by interfering with the binding of the repressor protein, Bm3R1, to the regulatory regions of P450.


INTRODUCTION

Cytochrome P450 (CYP102), a catalytically self-sufficient fatty acid monooxygenase(1) , is strongly induced when Bacillus megaterium is grown in the presence of barbiturates (2, 3, 4) or related compounds such as disubstituted acetyl ureas (5) and peroxisome proliferators(6) . About 1 kilobase of 5`-flanking DNA is required for barbiturate-inducible expression of the P450 gene in B. megaterium(7) . Analysis of this region reveals an open reading frame immediately upstream of the B. megaterium cytochrome P450 structural gene that encodes a protein, Bm3R1, containing a helix-turn-helix DNA-binding motif(8) . The gene encoding Bm3R1 forms a barbiturate-inducible co-transcriptional unit with the P450 gene and Bm3R1 acts as a negative regulator repressing the expression of both genes at the transcriptional level by binding specifically to a segment of DNA containing the promoter-operator region of the Bm3R1-P450 genes. Thus, in a B. megaterium mutant carrying a point mutation that results in a G39E amino acid substitution in the beta-turn region of the DNA binding motif of Bm3R1, binding to the palindromic operator sequence is abolished and the expression of P450 becomes constitutive at an extremely high level(8) . The interaction between Bm3R1 repressor and its operator, in vitro, was also strongly inhibited by barbiturates that were strong in vivo inducers of P450 such as pentobarbital but not by the same concentrations of barbiturates that were weak inducers or non-inducers (9) .

Cytochrome P450 (CYP106), also from B. megaterium but clearly distinct in structure and function from P450(1, 10) is moderately induced by barbiturates. A DNA fragment from B. megaterium containing the first 14 bp (^1)of the P450structural gene and 504 bp of 5`-flanking sequence was studied in detail with respect to barbiturate-mediated regulation(11) . Although an initial comparison of the 5`-flanking regions of the P450 and P450 genes revealed no sequence in the P450 gene with a high degree of similarity to the 20-bp palindromic operator of P450, there was a string of 17 bp in each that shared a high degree of sequence identity. This element, designated a ``Barbie box'' sequence(9, 12) , was also present in the 5`-flanking regions of the barbiturate-inducible P450 genes (CYP2B1/2) of the rat (11) and has now been recognized in essentially all barbiturate-inducible genes whose regulatory sequences have been reported(12) . Barbie box sequences from the two B. megaterium and two rat P450 genes were used as probes in gel retardation assays with protein obtained from B. megaterium grown either in the presence or absence of barbiturates or with protein from nuclear extracts from livers of rats left untreated or injected with phenobarbital. Each of the four 17-mers bound strongly to a single protein from bacteria grown in the absence of barbiturates but this binding was dramatically reduced with protein from pentobarbital- or phenobarbital-grown cells. Conversely, the probes complexed weakly to one protein band from nuclear extracts from untreated rats but much more strongly with protein from phenobarbital-treated rats(11) . The results, in B. megaterium at least, were consistent with the concept that the induction of the P450 gene in response to barbiturates involved, in part, a release of repression caused by the binding of a protein at the Barbie box region. In support of this hypothesis, deletion analysis of the 5`-flanking region of the P450 gene indicated that a negative regulatory element was involved in barbiturate-mediated regulation of cytochrome P450 and that a positive regulatory sequence may also be implicated. Both elements were located within a 51-bp region that included the Barbie box element. More precisely, a segment of the putative positive regulatory region of the P450gene appeared to be located in the 27-bp segment just 5` to the Barbie box while a repressor binding site resided in a 24-bp region starting at the first base of this element. Deletion of this 24-bp putative repressor binding site resulted in very high constitutive expression of the P450 gene with no further stimulation of expression in cultures growth in the presence of barbiturates(11) .

We now describe the characterization of two genes, designated BM1P1 and BM1P2, located immediately upstream of the P450 structural gene. These encode proteins that appear to positively regulate the expression of the P450 gene. We also present evidence indicating that Bm3R1, the repressor protein that negatively regulates the expression of the P450 gene is also involved in the expression of the P450 gene.


EXPERIMENTAL PROCEDURES

Materials

Restriction endonucleases and nucleic acid modifying enzymes were obtained from New England Biolabs Inc., Promega Corp., or Life Technologies, Inc. Expression vector PKK223-3 was from Pharmacia Biotechnology Inc. Isotopes and scintillation fluids were from DuPont NEN Research Products or Amersham Corp. Oligonucleotides used in this work were obtained from Integrated DNA Technologies Inc. Ni-NTA affinity resin for protein purification and the QIAGEN-tip 500 for the purification total RNA were purchased from QIAGEN. Gradient polyacrylamide gels and protein standards were obtained from Bio-Rad. Other biochemicals and chemicals were obtained from Sigma or Bio-Rad. The Sequenase and nucleotide kit for DNA sequencing was from U. S. Biochemical Corp.

Bacterial Strains and Plasmid Constructs

The Escherichia coli strains (DH5alpha or JM 101) used for cloning were grown in LB medium at 37 °C with shaking. B. megaterium ATCC 14581, the wild-type source of the genes encoding cytochrome P450 and the proteins designated BM1P1 and BM1P2, was grown in shake culture at 35 °C as described previously (10, 11) . Plasmids used in this study are listed in Table 1and their construction is described below. pBM1-1.9 (1) was subcloned as shown in Fig. 1A from pBM1-6.6, the original clone containing the cytochrome P450gene. The plasmid pUB, which was utilized in the analysis of the regulatory region of BM1P1, is an E. coli-B.megaterium shuttle vector containing a promoterless CAT gene; a detailed description of its construction and use has already been published(7) . For the construction of pUB the following procedure was used. A 504-bp DNA fragment was isolated by double cutting pBM1-1.9 using XmnI and SalI(10, 11) . The 504-bp fragment was subjected to dNTP and Klenow treatment, the HindIII linker and T4 DNA ligase were added and the preparation was treated with HindIII. The fragment, thus modified, was purified from an agarose gel, ligated into the HindIII site of pUB, and its orientation verified by DNA sequencing. As described previously (11) this 504-bp fragment, in the opposite orientation, was utilized in pUB to characterize the P450 gene regulatory region. For the construction of pBM1P1-6His, two PCR primers flanking the BM1P1 coding region were prepared. The first, 5`-CGCGAATTCATGAATCATCATCATCATCATCATCAAAAACAGCTAGATATTTTA-3` (designated P1-1 primer) contained an EcoRI site (underlined) at the 5` end plus a sequence from the start codon to the codon for the eighth amino acid of the BM1P1 gene (positions -267 to -293 of the P450 gene fragment; see Fig. 1B) except that codons for six histidines (CATCATCATCATCATCAT) were inserted between the codons for the second amino acid (Asn) and the third amino acid (Gln) to encode BM1P1 (Fig. 1C) as a histidine-tagged protein with an N-terminal amino acid sequence beginning MNHHHHHHQKQLDIL. The second primer 5`-GCGCAAGCTTACTTTCAGGAATT-3` (designated P1-2 primer) contained a HindIII site at its 5` end (underlined) and was complementary to the sequence -576 to -555 of the P450 gene fragment (see Fig. 1B). pBM1-6.6 was then used as template for PCR amplification in the presence of added P1-1 and P1-2 primers. The PCR products were treated with EcoRI and HindIII, gel-purified, and then ligated into the EcoRI and HindIII sites located downstream of the tac promoter in the expression vector pKK223-3. The procedures for the construction of pBM1P2-6His were those used for pBM1P1-6His except for the difference in primers. The first had the sequence 5`-CGCGAATTCATGTGGCATCATCATCATCATCATAAATTAGTTGTTTCCTATCTT 3` and was designated P2-1 primer. It contained an EcoRI site (underlined) at the 5` end plus a sequence from start codon to the codon for the ninth amino acid of the BM1P2 gene (see Fig. 1B) to encode BM1P2 (Fig. 1C) as a histidine-tagged protein with an N-terminal amino acid sequence beginning MWHHHHHHKLVVSYL. The primer spanned positions -1155 to -1129 of the BM1P2 gene fragment (Fig. 1B) except that six histidine codons (CATCATCATCATCATCAT) were added between the codons for the second amino acid (Trp) and the third amino acid (Lys). The second primer, designated P2-2 primer, had the sequence 5`-GCGCAAGCTTCGGTATTTAATAAGAAAACAG-3` and contained a HindIII site (underlined) at the 5` end. It was complementary to the sequence -883 to -903 of the P450 gene fragment (see Fig. 1B). For the construction of pBM1-385, two oligonucleotide primers were utilized. The first, designated primer B, had the sequence 5`-GCGAATTCGTAATGAGATAAGCAGTTCGC-3` and contained an EcoRI site (underlined) at the 5` end plus sequence -429 to -409 of the P450 regulatory region (Fig. 1B). The second, designated primer A, had the sequence 5`-GCAAGCTTACTAGCTACATAGCGCTCAGT-3`, contained a HindIII site (underlined) at the 5` end and was complementary to the sequence -44 to -64 of the P450 gene regulatory region (Fig. 1B). pBM1-6.6 was then used as template for PCR amplification in the presence of primers A and B. The PCR products were treated with EcoRI and HindIII, gel-purified, and then cloned into pUC19. The final product, pBM1-385, had a 385-bp insert that included the regulatory region of P450 and BM1P1 ( Fig. 1and Fig. 9). Plasmids pBM1-385A and pBM1-385B were constructed from pBM1-385. For pBM1-385A, a primer, designated primer C, with the sequence 5`-GCGAATTCTTATCTGCCTTTTCCTACGTG-3`, was utilized in conjunction with primer A. Primer C contained an EcoRI site (underlined) at the 5` end plus the sequence encompassing -221 to -201 of the P450 regulatory region (Fig. 1). Using pBM1-385 as the template for primers C and A, PCR amplification was performed. pBM1-385A has a 177-bp insert containing the operator region for P450 and BM1P1 including two inverted repeat sequences (Fig. 1B). One is a perfect 24-bp inverted repeat, the second is a 10-bp inverted repeat. For the construction of pBM1-385B a second oligonucleotide, designated primer D, with the sequence 5`-GCAAGCTTGCTATACTAAATAAAAAGTAA-3`, was utilized. Primer D contained a HindIII site (underlined) at the 5` end and was complementary to the sequence -222 to -242 of the P450 regulatory region (Fig. 1B). Using pBM1-385 as template for primers D and B, PCR amplification was carried out. The product was treated with EcoRI and HindIII, gel-purified, and then ligated into pUC19. pBM1-385B has a 208-bp insert containing a 17-bp consensus sequence (Fig. 1B) that binds one or more barbiturate-responsive proteins (11, 12) and has been designated a Barbie box sequence(9) . Plasmids BM1mp19 and BM1mp18 were prepared as follows: pBM1-6.6 was treated with PstI (Fig. 1A), the product gel-purified, and the 3.3-kb fragment ligated into the PstI sites of M13 mp19 or M13 mp18. DNA sequencing was performed on each construct to determine orientation.




Figure 1: Description of plasmids containing the P450, BM1P1, and BM1P2 genes. Panel A shows the construction of plasmids pBM1-6.6 and pBM1-1.9. pBM1-6.6 contains a 6.6-kb insert (in pUC19) cloned from B. megaterium DNA and is the original clone encoding cytochrome P450(10) . pBM1-1.9 was subcloned from pBM1-6.6(10) . The inserts in the sequencing plasmids, pBM1-mp19 and pBM1-mp18, were cloned from the 3.3-kb PstI-PstI DNA fragment (see ``Experimental Procedures''). Also shown (solid arrows) are the orientations of the P450, BM1P1, and BM1P2 genes. Panel B shows the annotated nucleotide sequence of that portion of the 6.6-kb insert of pBM1-6.6 that contained the complete BM1P1 and BM1P2 genes and included the overlapping regulatory regions of the P450 and BM1P1 genes. The numbering scheme for the sequence is determined by assigning +1 to the nucleotide ``A'' of the ATG translation initiation sequence of P450; the bases of the open reading frames encoding BM1P1, BM1P2, and the N-terminal portion of P450 are shown in bold letters. Start sites for transcription (open circle, base underlined) and translation (filled circle, base underlined) are indicated as are a variety of other features. These include Shine-Dalgarno sequences (bases labeled ``SD'' above and in underlined capital letters), a Barbie box sequence (bases labeled above and in underlined lower case letters), various inverted repeat sequences (bases in lower case letters), the locations of promoter sequences (bases labeled ``-10'' or ``-35'' above and underlined) and the termination points of the BM1P1 and BM1P2 open reading frames (filled triangle). Panel C shows the amino acid sequences, as deduced from the open reading frames, of BM1P1 and BM1P2.




Figure 9: DNA-protein binding assays using the 177-bp DNA fragment from pBM1-385A as a probe with proteins BM1P1, BM1P2, and Bm3R1. This probe, containing the shared regulatory region of the P450 and BM1P1 genes, was used in gel mobility shift assays to determine its binding affinity for 3 different barbiturate-responsive regulatory proteins. Lane 1, probe only; lane 2, probe plus 4 µg of partially purified Bm3R1 protein; lane 3, probe, 4 µg of partially purified Bm3R1 protein and 20-fold by weight of unlabeled DNA consisting of the structural gene encoding P450; lane 4, probe, 4 µg of partially purified Bm3R1 protein and 20-fold by weight of unlabeled DNA; lane 5, probe, 4 µg of partially purified Bm3R1 protein and 20-fold by weight of the unlabeled 208-bp DNA fragment from pBM1-385B; lane 6, probe, 4 µg of partially purified Bm3R1 protein and 5-fold by weight of the unlabeled 177-bp DNA fragment from pBM1-385A; lane 7, probe plus 2 µg of purified BM1P2 protein; lane8, probe, 2 µg of purified BM1P2 protein and 20-fold by weight of unlabeled DNA consisting of the structural gene encoding P450; lane 9, probe, 2 µg of purified BM1P2 protein and 20-fold by weight of the unlabeled 208-bp DNA fragment from pBM1-385B; lane 10, probe, 2 µg of purified BM1P2 protein and 5-fold by weight of the unlabeled 177-bp DNA fragment from pBM1-385A; lane 11, probe, 2 µg of purified BM1P2 protein and 20-fold by weight of the unlabeled 177-bp DNA fragment from pBM1-385A; lane 12, probe plus 2 µg of purified BM1P1 protein; lane 13, probe, 2 µg of purified BM1P1 protein and 20-fold by weight of the unlabeled DNA consisting of the structural gene encoding P450; lane 14, probe, 2 µg of purified BM1P1 protein and 20-fold by weight of the unlabeled 208-bp DNA fragment from pBM1-385B; lane 15, probe, 2 µg of purified BM1P1 protein and 5-fold by weight of the unlabeled 177-bp DNA fragment from pBM1-385A; lane 16, probe, 2 µg of purified BM1P1 protein and 20-fold by weight of the unlabeled 177-bp DNA fragment from pBM1-385A; lane 17, probe, 2 µg of purified BM1P1 protein and 4 µg of partially purified Bm3R1 protein; lane 18, probe, 2 µg of purified BM1P2 protein and 4 µg of partially purified Bm3R1 protein; lane 19, probe, 2 µg of purified BM1P1 protein, 2 µg of pure BM1P2 protein, and 4 µg of partially purified Bm3R1 protein.



Preparation of RNA

Extraction of total RNA was performed basically as described (14) but with several modifications. B. megaterium cells grown in the presence or absence of 4 mM pentobarbital were harvested in log phase by centrifugation. The cell pellet was transferred to 8 ml of ice-cold GuSCN solution (4 M guanidine isothiocyanate, 50 mM Tris-HCl (pH 7.5), 25 mM EDTA plus 0.2 ml of 2-mercaptoethanol). The cell suspension was subjected to sonication and then 1 ml of 25% Triton X-100 and 8 ml of 3 M sodium acetate buffer (pH 6.0) were added and the mixture was incubated in an ice bath for 30-40 min. The preparation was centrifuged at 15,000 g, the supernatant was treated with isopropyl alcohol, and the resulting precipitate was collected. The pellet was dissolved in 20 mM Tris-HCl buffer (pH 8.0), undissolved particulate matter was removed and 2 M NaCl and 1 M MOPS (pH 7.0) were added to a final concentration of 400 mM NaCl and 50 mM MOPS (pH 7.0). Before applying this crude RNA preparation to a QIAGEN-tip 500 column, the column was equilibrated with a buffer (pH 7.0) containing 400 mM NaCl, 50 mM MOPS, 15% ethanol, and 0.15% Triton X-100. After the crude RNA preparation had been applied to the column, washing was continued with a buffer (pH 7.0) containing 400 mM NaCl, 50 mM MOPS, and 15% ethanol. Purified RNA was then eluted with a solution (pH 7.0) containing 900 mM NaCl, 50 mM MOPS, 15% ethanol, and 6 M urea and then precipitated from the buffer by the addition of isopropyl alcohol.

Primer Extension Analysis

To locate the 5` ends of the BM1P1 and BM1P2 mRNAs, two synthetic oligonucleotides were prepared. The first, a 21-base oligonucleotide (5`-TAGCTGTTTTTGATTCATTGC-3`) complementary to the sequence at the beginning of the BM1P1 coding region (Fig. 1B) was end labeled with P using [-P]ATP and T4 polynucleotide kinase and then co-precipitated with 100 µg of total RNAs. The primer extension reaction with avian myeloblastosis virus reverse transcriptase was then carried out by the method of Kingston(15) . The second oligonucleotide was a 20-base oligonucleotide (5`-AACAACTAATTTCCACATGA-3`) complementary to the sequence at the beginning of the BM1P2 coding region (Fig. 1B). The primer extension reaction was performed as described above. The sequence ladder corresponding to the mRNA sequence was generated by using pBM1-6.6 as a DNA template and the same oligonucleotides as primers.

Northern Hybridization Assay

Plasmids pBM1P1-6His and pBM1P2-6His were used as the source of hybridization probes. After treatment of the plasmids with HindIII and EcoRI, the released inserts were gel-purified and labeled DNA probes prepared using random oligonucleotide primers. Northern hybridization reactions were carried out as described by Sambrook et al.(17) .

Overproduction of BM1P1 and BM1P2 Proteins in E. coli and Their Purification

E. coli was transformed with plasmids pBM1P1-6His and pBM1P2-6His, respectively, and the cells were grown at 37 °C in 2-liter flasks each containing 1 liter of LB medium and 50 mg of ampicillin. When cultures reached an optical density of about 0.5 at 600 nm, IPTG was then added to final concentrations of 2, 1, 0.5, 0.2, and 0.0 mM to different flasks. The cultures were then incubated for an additional 4 h at 37 °C before cells were collected by centrifugation. The cell pellets were resuspended in buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl), and the cells were disrupted by sonication. Cell breakage was monitored by measuring the release of nucleic acid at A until it reached a maximum. The sonicated preparations were then centrifuged at 40,000 g and the supernatants analyzed on 4-20% gradient polyacrylamide gels with protein bands visualized by staining with Coomassie Blue R-250. Although BM1P1 and BM1P2 are small proteins, they were obtained chiefly in insoluble form when IPTG was used as an inducer, especially at high concentrations. The insoluble proteins were solubilized in 8 M urea or in 6 M guanidine hydrochloride for analysis by PAGE but were not further utilized. The 40,000 g supernatant solutions were applied to a Ni-NTA resin column, previously equilibrated with a buffer consisting of 50 mM sodium phosphate (pH 8.0) and 300 mM NaCl. Wash buffer (pH 6.0) containing 50 mM sodium phosphate, 300 mM NaCl, and 10% glycerol was first passed through the column before the protein was eluted with a gradient of 0-0.5 M imidazole in the wash buffer. Fractions (0.5 ml) were collected and analyzed on 4-20 or 10-20% gradient polyacrylamide gels. The fractions containing BM1P1 or BM1P2, both of which were eluted at approximately 0.20-0.25 M imidazole, were pooled, desalted, and concentrated to 2 ml or less using centrifugal concentrators (3K Macrosept® from Filtron, Inc.) and subjected to gel filtration chromatography. Pooled samples (10 mg of protein/ml) were loaded onto a Sephadex G-100 column (1.5 60 cm), equilibrated, and eluted by a buffer (pH 7.5) containing 20 mM potassium phosphate, 200 mM NaCl, 1 mM DTT, and 10% glycerol. After analysis of the eluted fractions by SDS-PAGE, the BM1P1- or BM1P2-containing peak fractions were pooled, concentrated, desalted, and stored at -70 °C in a 20 mM potassium phosphate buffer (pH 7.5) containing 1 mM DTT and 50% glycerol.

Gel Mobility Shift Assays

DNA binding assays were performed according to the method of Fried and Crothers(18) . Plasmids pBM1-385, pBM1-A, and pBM1-B were treated with EcoRI and HindIII, labeled with [alpha-P]dNTP by treatment with the Klenow fragment of DNA polymerase and the inserts purified on agarose gels to yield three different probes. BM1P1 and BM1P2 used for gel mobility shift assays were the purified proteins. Protein Bm3R1 (8, 9) was used for gel mobility shift assays in partially purified form; the partially purified Bm3R1 was obtained from E. coli cells containing the bm3R1 wild-type gene (8, 9) . After sonication of the cells and centrifugation of the broken cell preparation at 40,000 g for 2 h, ammonium sulfate was added slowly to the supernatant until 40% saturation was reached; 30 min later the preparation was centrifuged and the precipitate containing crude Bm3R1 was dissolved in 20 mM potassium phosphate (pH 7.5) containing 1 mM DTT and 20% glycerol and dialyzed in the cold for at least 8 h. Binding reactions were carried out in 25 µl of a mixture containing 0.5-1.0 ng of DNA probe, 2.5-3.0 µg of purified BM1P1 and/or BM1P2, or 4.0 µg of partially purified Bm3R1 in a buffer containing 50 mM Tris-HCl (pH 7.6), 1 mM DTT, 0.1 mM EDTA, 60 mM KCl, 6% glycerol, and 0.01% bovine serum albumin. The mixtures were incubated at room temperature for 15 min before being applied to the 5% polyacrylamide gel.

Other Procedures

DNA sequencing of BM1P1 and BM1P2 DNA was carried out by the enzymatic method of Sanger et al.(13) . CAT assays for measuring the rate of transfer of ^14C-labeled acetyl groups from acetyl-coenzyme A to unlabeled chloramphenicol and Western blot analyses were performed as described previously(7) . Sequence comparisons of the BM1P1 and BM1P2 proteins with other protein was carried out by the ``Blast'' program (^2)(via the NCBI BLAST E-mail server) developed by the National Center for Biotechnology Information at the National Library of Medicine(32) .


RESULTS

Nucleotide Sequence Analysis of the BM1P1 and BM1P2 Genes

The nucleotide sequence of about 1.3 kb of the 5`-flanking region of the P450 structural gene of B. megaterium is shown in Fig. 1B. Sequence analysis revealed two open reading frames; one, with a coding capacity of 98 amino acids, is located 267 bp upstream of the P450 coding sequence and is designated BM1P1 in this report. BM1P1, which is located in a an orientation opposite that of P450, shares a portion of its regulatory region with that of P450. Since promoters for the transcription of P450 and BM1P1 partially overlap in this 267-bp region, coordinate regulation of expression of these two proteins would be facilitated. The BM1P1 open reading frame begins with a putative translation initiation sequence, ATG, that is preceded by a sequence, AGAGGAG, which could be expected to serve as a ribosome binding site(19, 20) . Also in this region are two 24-bp sequences that form a perfect inverted repeat but separated by a 5-bp sequence, TAATT (Fig. 1B). The P450 -10 sequence, TATACTA, and mRNA start site are both in this 24-bp inverted repeat region. Six bp (TAATTA) upstream from the first (24-bp) inverted repeat, another inverted repeat of 10 bp appears (Fig. 1B). The -35 sequences of both P450 and BM1P1 are located within this 10-bp inverted repeat region and, indeed, overlap on complementary strands as shown in Fig. 1B. The second open reading frame, designated BM1P2, is located 892 bp upstream from the P450 coding sequence and has the same orientation. The BM1P2 open reading frame begins with ATG and its ribosome binding site is AGGG. The amino acid sequences of BM1P1 and BM1P2 as deduced from the sequenced nucleotide are shown in Fig. 1C.

Positive Response of BM1P1 to Pentobarbital Induction

In a previous report(11) , a series of deletion derivatives of the first 504 bp of the 5`-flanking region of the P450 gene were subcloned into the promoter-probing vector, pUB, by transcriptional fusion to the CAT gene. In this study, we tested the reverse orientation of this 504-bp fragment (i.e. with the BM1P1 promoter in the correct orientation). Plasmid pUB was constructed as described under ``Experimental Procedures'' and B. megaterium transformed by this construct was grown in the presence or absence of 4 mM pentobarbital and assayed for CAT activity. Transformed cells grown in the presence of 4 mM pentobarbital showed a 4.5-fold increase in CAT activity over the basal level (data not shown), indicating that the BM1P1 gene is barbiturate-inducible.

Western Blotting Assays for P450 Proteins in B. megaterium

B. megaterium, transformed by plasmid pUB, was analyzed for cytochromes P450 and P450 by Western blotting. As Fig. 2shows, in B. megaterium transformed by pUB and grown in the absence of pentobarbital, the level of P450 protein (Fig. 2, lane 3) was dramatically increased compared to cells transformed by the pUB (vector only) grown in the absence of pentobarbital (Fig. 2, lane 1). The levels of cytochrome P450 (Fig. 2, lanes 5 and 7), however, appeared to be slightly enhanced. For B. megaterium transformed by pUB and grown in the presence of 4 mM pentobarbital, the level of P450 protein (Fig. 2, lane 4) was higher than in B. megaterium transformed by the vector only and grown in the presence of 4 mM pentobarbital (Fig. 2, lane 2). Again, the level P450 protein did not seem to change significantly (Fig. 2, lanes 6 and 8). Thus, although cells transformed by pUB and grown in the absence of barbiturates show a dramatic increase in cytochrome P450 levels relative to cells transformed the vector only, they are capable of a further significant increase when grown in the presence of pentobarbital. A comparison of the cells transformed by pUB grown in the absence or presence of 4 mM pentobarbital (Fig. 2, lanes 3 and 4), indicate pentobarbital can induce P450. Western blotting gave essentially the same results as the CAT assays, again suggesting that BM1P1 may be a positive regulatory protein.


Figure 2: Biosynthesis of cytochrome P450 in B. megaterium transformed by pUB. The biosynthesis of P450 in B. megaterium transformed by plasmid pUB was compared to that in B. megaterium transformed by the plasmid pUB (vector only), both grown in the presence or absence of 4 mM pentobarbital. Soluble protein (50 µg of the supernatant fraction from centrifugation, at 40,000 g, of a preparation obtained by sonication of the harvested B. megaterium cells) was subjected to electrophoresis on a 12% polyacrylamide gel, transferred to a nitrocellulose membrane, and immunoblotted with antiserum to P450 and to P450, respectively. Immunoreactive bands were visualized by a peroxidase-catalyzed color reaction(7) . Panel A shows the results of immunoblotting with antiserum to P450. The soluble protein was obtained from B. megaterium cells transformed by pUB (lanes 1 and 2) or pUB (lanes 3 and 4) and grown in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 4 mM pentobarbital. Fig. 3B shows the results of immunoblotting with antiserum to P450. The soluble protein was obtained from B. megaterium cells transformed by pUB (lanes 5 and 6) or by pUB (lanes 7 and 8) and grown in the absence (lanes 5 and 7) or presence (lanes 6 and 8) of 4 mM pentobarbital.




Figure 3: Northern blotting analysis of BM1P1 and BM1P2 transcription levels in wild type B. megaterium grown in the absence or presence of pentobarbital. Total RNA (125 µg/lane) was subjected to electrophoresis in an agarose-formaldehyde gel, blotted to nylon membranes, and hybridized to the BM1P1 or BM1P2 probes. Panel A, BM1P1 probe of RNA from cells grown in the absence (lane 1) or presence (lane 2) of 4 mM pentobarbital. Panel B, BM1P2 probe of RNA from cells grown in the absence (lane 1) or presence (lane 2) of 4 mM pentobarbital.



Expression of BM1P1 and BM1P2

Based on the nucleotide sequence analysis of the BM1P1 and BM1P2 genes and on the BM1P1 CAT activity results and Western blotting assays, we considered it likely that these two open reading frames encode two small proteins involved in the regulation of P450 expression. We therefore decided to study the effects of pentobarbital on the transcription levels of BM1P1 and BM1P2 in B. megaterium. utilizing Northern blotting analysis. The results (Fig. 3) showed that the expression of both BM1P1 and BM1P2 were strongly stimulated when the cells were grown in the presence of 4 mM pentobartital. Densitometric analysis of the band indicated an approximately 5-fold increase in the level of these transcripts in cells grown in the presence of pentobarbital.

Identification of the Transcriptional Start Site of BM1P1

A primer extension experiment was performed to identify the 5` end of the BM1P1 mRNA. To more precisely determine the transcription start site for the BM1P1 gene, we used a 21-base oligonucleotide primer complementary to the beginning of the BM1P1 coding region (``Experimental Procedures'') to prime DNA synthesis by incubation with total RNAs purified from B. megaterium grown in the absence or presence of 4 mM pentobartital. We observed a single cDNA band extending from the primer to a T in the sequence ladder (Fig. 4A). This T corresponded to an A (the transcription initiation site) in the BM1P1 coding strand. Upstream from the transcription start site, putative -10 and -35 sites similar to the consensus sequence of prokaryotic promoters (21) were identified (Fig. 1B). The BM1P1 and P450 genes appear to share the same regulatory region that includes, in a sequence of about 200 bp, a 24-bp perfect inverted repeat and a 10-bp inverted repeat. The P450 and BM1P1 genes also seem to share the same -35 site. In the primer extension experiment, there was no clean DNA band produced from hybridization with RNA purified from cells grown in the absence of 4 mM pentobarbital (Fig. 4A).


Figure 4: Primer extension analysis of BM1P1 and BM1P2. Total RNA was isolated and subjected to primer extension as described under ``Experimental Procedures.'' The primer extension products extracted from B.megaterium grown in the absence or presence of 4 mM pentobarbital and a sequencing ladder, prepared from the same primer, were subjected to electrophoresis. To the left of the sequence ladder in each panel, a sequence complementary to that read from the ladder is shown with the transcription start site is indicated by an asterisk. The primer extension products are indicated by arrows. Panel A shows the primer extension of BM1P1. Lanes 1 and 3, RNA extracted from cells grown in the present of 4 mM pentobarbital; lanes 2 and 4, RNA extracted from cells grown in the absence of pentobarbital. Panel B shows the primer extension of BM1P2. Lanes 1 and 2, RNA extracted from cells grown in the presence of 4 mM pentobarbital, lane 3, RNA from cells grown in the absence of pentobarbital.



Identification of the Transcription Start Site of BM1P2

A 20-base oligonucleotide complementary to the beginning of the BM1P2 coding region (see ``Experimental Procedures'') was used to prime DNA synthesis. We analyzed a band produced at the position of a T in the sequence ladder (Fig. 4B) that corresponded to an A, the expected transcription start site in the BM1P2 coding strand. As shown in the Fig. 4B, RNA from cells grown in the presence of 4 mM pentobarbital produced a stronger band than RNA from cells grown in its absence. Although no potential -10/-35 sites can be found at the expected locations upstream from the transcription initiation site (+1) of BM1P2, a sequence, TAATACT, spanning bases -41 to -35, is similar to the -10 consensus sequence of prokaryotic promoters. Eighteen bp upstream from this sequence, the sequence TTGTAT, spanning bases -65 to -60, shares similarity with the -35 consensus sequence of prokaryotic promoters. We repeated the BM1P2 primer extension experiment several times, always with the same results.

Comparison of BM1P1 and BM1P2 with Other DNA-binding Proteins

A significant similarity was found between the derived amino acid sequence of BM1P1 and Bm3R1, a critical regulatory protein controlling the expression of P450 in B. megaterium(8, 9) . Analysis by the Blast program^2 reveals a 35% identity (58% similarity when conservative substitutions are counted) between the N-terminal regions of the two proteins (residues 4-54 of BM1P1 and 7-57 of Bm3R1). In prokaryotes most DNA-binding proteins use a helix-turn-helix structural motif to recognize target DNA sequences(22) . The MacVector® Protein Analysis Toolbox program for secondary structure predictions, based on the Chou-Fasman and Robson-Garnier methods(23, 24, 25) , was used to search BM1P1 for the helix-turn-helix structural motif. Two such motifs appeared, one located in the C-terminal portion of BM1P1 (residues 47-95), the second in the N-terminal region (residues 1-31). We also found a significant structural similarity between BM1P1 and Bm3R1 in the N-terminal helix-turn-helix regions (33% identity; 57% identities and conservative substitutions) when we compared residues 4-40 of BM1P1 to residues 7-43 of Bm3R1. A weak similarity between the BM1P1 C-terminal helix-turn-helix and the Bm3R1 N-terminal helix-turn-helix was also found. A sequence encompassing residues 32-73 of the BM1P2 also showed 23% identity, 59% positives with residues 217-258 of NIFA, a specific regulatory protein(26, 27, 28, 29, 30) . NIFA, as a transcriptional activator, is required for the activation of most of the NIF operons directly involved in nitrogen fixation. Its central region, encompassed by residues 217-258, contains an ATP-binding domain that interacts with -54 factor(26, 27, 28, 29, 30) .

Overproduction of BM1P1 and BM1P2 Proteins in E. coli

pBM1P1-6His and pBM1P2-6His were constructed as described under ``Experimental Procedures.'' In order to isolate and characterize the BM1P1 and BM1P2 gene products, expression vector pKK223-3 (Pharmacia) was used to construct expression plasmids. The BM1P1 and BM1P2 genes were individually cloned into the pKK223-3 vector so that the genes were under the control of the tac promoter. Before cloning into the expression vector, six repeat histidine codons were added at the N-terminal of each gene to obtain a histidine-tagged recombinant protein. The use of histidine-tagged recombinant proteins has recently become popular to facilitate their purification and for use in the study protein-protein interactions(31) . Since BM1P1 and BM1P2 are relatively small proteins (BM1P2 only has 88 amino acids, BM1P1, 98 amino acids), a 4-20% gradient polyacrylamide gel was used for their analysis. BM1P1, which appeared as a protein with an molecular mass of approximately 11 kDa, was found in the supernatant from cells containing pBM1P1-6His and grown in the presence of 0.5 mM IPTG (Fig. 5A, lane 2). Cells grown in the absence of IPTG yielded soluble protein that showed only a weak band at the same position (Fig. 5A, lane 3). Normally, for IPTG-inducible expression, IPTG was added to the final concentration of 1 or 2 mM. However, the expression of BM1P1 was very sensitive to high concentrations of IPTG; the induction levels were very high but, at the same time, more than 99% of the BM1P1 produced appeared as insoluble protein. Indeed, we could not easily detect BM1P1 protein in the 40,000 g supernatant when high concentrations of IPTG were used for induction. The insoluble BM1P1 protein pellet obtained after centrifugation of the sonicated product (see ``Experimental Procedures'') could be solubilized by treating the pellet with 8 M urea or 6 M guanidine hydrochloride, but sometimes this treatment had to be repeated several times for best results (see Fig. 5A lane 4, cells grown in 2 mM IPTG). Isolation of sufficient quantities of BM1P2 protein, with 10 amino acids less than BM1P1, was nevertheless, more difficult. Most of the BM1P2 gene product was insoluble, even from cultures induced with low levels of IPTG and we could not detect it in the 40,000 g supernatant, although it could be solubilized from the insoluble pellet by using denaturing conditions (see above). We eventually found that using not only lower concentrations of IPTG, but also lower growth temperatures (33-35 °C) and shorter induction times (2-3 h) we could obtain BM1P2 in the 40,000 g supernatant (Fig. 5B).


Figure 5: Overexpression of BM1P1 and BM1P2 in E.coli. E. coli (JM101) cells containing pBM1P1-6His or plasmid BM1P2-6His were grown in the presence or absence IPTG. Protein samples were subjected to electrophoresis on a 4-20% gradient polyacrylamide gel and visualized with Coomassie Blue R-250. Panel A: lane 1 contained protein standards; lane 2 contained soluble protein from cells transformed by pBM1P1-6His. The cells were grown at 37 °C to an optical density of 0.4 to 0.5 at 600 nm, IPTG was then added to a final concentration of 0.5 mM and the cultures were incubated for an additional 4 h. After the cells were broken by sonication and centrifuged at 40,000 g, the centrifugal supernatant was sampled. Lane 3 was the same as described for lane 2 except that IPTG was not added to the cells. Lane 4 contained protein from cells grown as described for lane 2 except that the final concentration of IPTG was 2 mM and the protein was derived not from the centrifugal supernatant but from the insoluble pellet by treatment with 6 M guanidine hydrochloride. Lane 5 contained BM1P1 fractions that had been solubilized from the pellet and then eluted from a Ni-NTA resin column. Panel B: lane1 contained protein standards; lane 2 contained soluble protein from cells transformed by pBM1P2-6His. The cells were grown at 37 °C to an optical density of 0.4 to 0.5 at 600 nm, IPTG was then added to a final concentration of 0.5 mM and the cultures were incubated for an additional 3 h. After the cells were broken by sonication and centrifuged at 40,000 g, the centrifugal supernatant was sampled. Lane 3 was the same as described for lane 2 except that IPTG was not added to the cells. Lane 4 was as described for lane 4 in panel A except that cells transformed by pBM1P2-6His were used. Lane 5 contained BM1P2 fractions that had been solubilized from the pellet and then eluted from a Ni-NTA resin column.



Purification of BM1P1 and BM1P2 Proteins

The procedures for the purification of BM1P1 and BM1P2 are described in detail under ``Experimental Procedures.'' After crude BM1P1 or BM1P2 preparations were eluted from a Ni-NTA resin column with a 0.0-0.5 M imidazole gradient, several minor impurities could still be detected in each preparation by SDS-PAGE followed by Coomassie Blue R-250 staining (Fig. 5, A, lane 5, and B, lane 5). However, after a second purification step on a Sephadex G-100 column, only one band in each of the two protein preparations could be detected by the same analytical procedure (Fig. 6, A and B). After analysis, the protein-containing fractions were pooled, concentrated, and desalted and then stored at -70 °C in 20 mM potassium phosphate (pH 7.5), 1 mM DTT, and 50% glycerol.


Figure 6: Final purification of BM1P1 and BM1P2 by gel filtration chromatography. After 40,000 g centrifugal supernatant preparations containing either BM1P1 or BM1P2 (see legend to Fig. 5) were eluted from a Ni-NTA resin column, the samples containing the desired protein were pooled and subjected to gel filtration chromatography on a Sephadex G-100 column. Samples from the protein-containing peak fractions eluted from this column were subjected to electrophoresis on a 4-20% polyacrylamide gradient gel and then visualized with Coomassie Blue. Panel A shows the BM1P1 band eluted from a Sephadex G-100 column while panel B shows the BM1P2 band eluted from the same column.



DNA Binding Ability of the BM1P1 and BM1P2 Gene Products

In order to determine the DNA binding properties of the BM1P1 and BM1P2 gene products in vitro, three plasmids, pBM1-385, pBM1-385A, and pBM1-385B, were constructed as described under ``Experimental Procedures,'' and used as a source of three probes of different sizes as shown in Fig. 7. The first, an 177-bp fragment of the pBM1-385A insert incorporating the shared regulatory regions of the P450 and BM1P1 genes, contained two inverted repeat sequences, one a perfect 24-bp inverted repeat, the other a 10-bp inverted repeat. The second probe was a 208-bp fragment of the pBM1-385B insert containing a Barbie box sequence that can bind one or more barbiturate-responsive proteins(11, 12) . The third probe, a 385-bp fragment of the pBM1-385 insert combined the 177-bp fragment of the pBM1-385A insert and the 208-bp fragment of pBM1-385B insert as shown in Fig. 7. Two purified proteins (BM1P1 and BM1P2) and a preparation of Bm3R1, partially purified as described under ``Experimental Procedures,'' were assayed for DNA binding properties with the three probes described above. The results revealed that BM1P1, BM1P2, and Bm3R1 can all bind to the 385-bp fragment of the pBM1-385 insert (Fig. 8). The finding that Bm3R1 could bind to a 5`-flanking sequence of the P450 gene (and hence play a putative role in its expression) was especially intriguing since Bm3R1 was previously characterized as the repressor controlling the barbiturate-mediated expression of the P450 gene(8, 9) . This hypothesis is corroborated by our previous finding (^3)that a mutant constitutive for expression of P450 also exhibited a dramatic increase in P450 expression. The lesion in this mutant was later shown to involve the substitution of a glutamate for glycine at residue 39 of Bm3R1, an alteration that disrupted its helix-turn-helix DNA binding motif so that it no longer could bind as a repressor to its operator sequence(8) . Equally interesting is our finding that using Bm3R1 in the presence of either BM1P1 or BM1P2 caused the Bm3R1 binding band to disappear completely (Fig. 8, lanes 14-20). Thus, the two small proteins encoded in the 5`-flanking region of the P450 gene seemed to strongly compete with Bm3R1 for one or more specific binding sites in this region. To determine which portion of the 385-bp fragment were critical to binding by these proteins, inserts from plasmids pBM1-385A and pBM1-385B (Fig. 7) were utilized. The results indicate that the 177-bp fragment from pBM1-385A contains one or more important binding sites for all three proteins (BM1P1, BM1P2, and Bm3R1) since they all bind strongly in this region (Fig. 9, lanes 2-16). As already noted, this 177-bp fragment contains the shared regulatory region of the P450 and the BM1P1 genes and includes 2 inverted repeats (Fig. 1B). Again, the competitive effects of BM1P1 and BM1P2 on the DNA binding of Bm3R1 are apparent; at the protein concentrations employed, no Bm3R1 binding to the 177-bp fragment can be detected in the presence of either or both of these proteins (Fig. 9, lanes 17-19). On the other hand, the 208-bp fragment from pBM1-385B, containing the Barbie box sequence that binds one or more barbiturate-responsive proteins(11, 12) , has a binding site for Bm3R1 (Fig. 10, lanes 2-7), but not for BM1P1 or BM1P2 (Fig. 10, lanes 10 and 11). Nevertheless, the competitive effect of these two proteins on Bm3R1 binding is still evident (Fig. 10, lanes 8 and 9). Thus, although BM1P1 and BM1P2 do not appear to bind to the 208-bp fragment, they still prevent Bm3R1 from binding to this probe.


Figure 7: Derivation of probes used for gel mobility shift assays. The first probe, containing the shared P450 and BM1P1 regulatory regions, consisted of a 177-bp fragment derived from pBM1-385A. This probe spanned the -221 to -44-bp segment of the 5`-flanking region of the P450 gene as shown and annotated in detail in the legend to Fig. 1B. The second probe, containing a 17-bp Barbie box sequence known to bind several barbiturate-responsive proteins (12) consisted of a 208-bp fragment from pBM1-385B. This probe spanned the -429 to -222-bp segment of the 5`-flanking region of the P450 gene (see Fig. 1B). The third probe, containing both a 17-bp Barbie box sequence and the shared P450 and BM1P1 regulatory regions, consisted of the 385-bp insert from pBM1-385. This probe spanned the -429 to -44-bp segment of the 5`-flanking region of the P450 gene (see Fig. 1B).




Figure 8: DNA-protein binding assays using the 385-bp DNA fragment from pBM1-385 as a probe with proteins BM1P1, BM1P2, and Bm3R1. This probe, containing both a 17-bp Barbie box sequence and the shared P450 and BM1P1 regulatory regions (see Fig. 1B and 7) was used in gel mobility shift assays to determine its binding affinity for 3 different barbiturate-responsive regulatory proteins. Lane 1, probe only; lane 2, probe plus 2 µg of purified BM1P1 protein; lane 3, probe, 2 µg of purified BM1P1 protein and 20-fold by weight of unlabeled DNA consisting of the structural gene encoding P450; lane 4, probe, 2 µg of purified BM1P1 protein and 20-fold by weight of unlabeled DNA; lane 5, probe, 2 µg of purified BM1P1 protein and 20-fold by weight of unlabeled DNA consisting of the 177-bp fragment from pBM1-385A (Fig. 7); lane 6, probe plus 2 µg of purified BM1P2 protein; lane 7, probe, 2 µg of purified BM1P2 protein and 20-fold by weight of unlabeled DNA consisting of the structural gene encoding P450; lane 8, probe, 2 µg of purified BM1P2 protein and 20-fold by weight of unlabeled DNA; lane 9, probe, 2 µg of purified BM1P2 protein and 20-fold by weight of unlabeled DNA consisting of the 177-bp fragment from pBM1-385A (Fig. 7); lane 10, probe plus 4 µg of partially purified Bm3R1 protein; lane 11, probe, 4 µg of partially purified Bm3R1 protein and 20-fold by weight of unlabeled DNA consisting of the structural gene encoding P450; lane 12, probe, 4 µg of partially purified Bm3R1 protein and 20-fold by weight of unlabeled DNA; lane 13, probe, 4 µg of partially purified Bm3R1 protein and 20-fold by weight of unlabeled DNA consisting of the 177-bp fragment from pBM1-385A (Fig. 7); lane 14, probe, 2 µg of purified BM1P1 protein and 4 µg of partially purified Bm3R1 protein; lane 15, probe, 2 µg of BM1P1 purified protein and 4 µg of partially purified Bm3R1 protein; lane 16, probe, 2 µg of purified BM1P1 protein, 2 µg of purified BM1P2 protein, and 4 µg of partially purified Bm3R1 protein; lane 17, probe, 2 µg of purified BM1P2 protein, 4 µg of partially purified Bm3R1 protein, and 20-fold by weight of unlabeled DNA consisting of the structural gene encoding P450; lane 18, probe, 2 µg of purified BM1P1 protein, 4 µg of partially purified Bm3R1 protein and 20-fold by weight of unlabeled DNA consisting of the structural gene encoding P450; lane 19, probe, 2 µg of purified BM1P1 protein, 4 µg of partially purified Bm3R1 protein, and 20-fold by weight of unlabeled DNA; lane 20, probe, 2 µg of purified BM1P1 protein, 4 µg of partially purified Bm3R1 protein, and 20-fold by weight of unlabeled DNA consisting of the 177-bp fragment from pBM1-385A.




Figure 10: DNA-protein binding assays using the 208-bp DNA fragment from pBM1-385B as a probe with proteins BM1P1, BM1P2, and Bm3R1. This probe, containing a 17-bp Barbie box sequence, was used in gel mobility shift assays to determine its binding affinity for 3 different barbiturate-responsive regulatory proteins. Lane 1, probe only; lane 2, probe plus 4 µg of partially purified Bm3R1 protein; lane 3, probe, 4 µg of partially purified Bm3R1 protein and 20-fold by weight unlabeled DNA consisting of the structural gene encoding P450; lane 4, probe, 4 µg of partially purified Bm3R1 protein and 20-fold by weight of unlabeled DNA; lane 5, probe, 4 µg of partially purified Bm3R1 protein and 5-fold by weight of the unlabeled 177-bp fragment from pBM1-385A; lane 6, probe, 4 µg of partially purified Bm3R1 protein and 20-fold by weight of the unlabeled 177-bp fragment from pBM1-385A; lane 7, probe, 4 µg of partially purified Bm3R1 protein and 20-fold by weight of the unlabeled 208-bp fragment from pBM1-385B; lane 8, probe, 4 µg of partially purified Bm3R1 protein and 2 µg of purified BM1P1 protein; lane 9, probe, 4 µg of partially purified Bm3R1 protein and purified BM1P2 protein; lane 10, probe plus 2 µg of purified BM1P1 protein; lane 11, probe plus 2 µg of purified BM1P2 protein.



Although the data presented in Fig. 8and Fig. 9provided strong evidence that Bm3R1 could bind to the regulatory regions of the P450 and BM1P1 genes, we sought to confirm this conclusion by determining the effect of anti-Bm3R1 antiserum^3(6) on DNA-protein complex formation in gel retardation assays employing Bm3R1 protein and the labeled 177- and 385-bp regulatory region probes. As shown in Fig. 11and 12, the addition of anti-Bm3R1 antiserum led to complete inhibition of complex formation (Fig. 11, lane 4; Fig. 12, lane 4). When anti-cytochrome P450 antiserum was used no effect was observed (Fig. 11, lane 3; Fig. 12, lane 3). Neither anti-Bm3R1 nor anti-cytochrome P450 affected BM1P1 and BM1P2 binding in this region (Fig. 11, lanes 6, 7, 9, and 10; Fig. 12, lanes 6, 7, 9, and 10). The difference in the observed mobility of the Bm3R1 complexes formed with the two probes (i.e. the 385- and 177-bp DNA fragments) can be explained by the presence of two binding regions in the 385-bp probe but only one binding site in the 177-bp probe. The same reasoning can also explain why the Bm3R1 complex bands migrate more slowly than those of the BM1P1/2 complexes when the 385-bp probe is used ( Fig. 8and Fig. 11) but run ahead of the BM1P1/2 complexes when the 177-bp probe is employed ( Fig. 9and Fig. 12).


Figure 11: The effects of antibody to Bm3R1 on DNA-protein binding assays using the 385-bp DNA fragment from pBM1-385 as a probe with proteins BM1P1, BM1P2, and Bm3R1. This probe, containing both a 17-bp Barbie box sequence and the shared P450 and BM1P1 regulatory regions (see Fig. 1B and 7), was used in gel mobility shift assays to determine the effect Bm3R1 had on antibody activity. Lane 1, probe only; lane 2, probe plus 1 µg of partially purified Bm3R1 protein; lane 3, same as lane 2 but in the presence of 1 µl of serum containing antibody to cytochrome P450; lane 4, same as lane 2 but in the presence of 1 µl of serum containing antibody to Bm3R1; lane 5, probe plus 1 µg of purified BM1P1 protein; lane 6, same as lane 5 but in the presence of 1 µl of serum containing antibody to cytochrome P450; lane 7, same as lane 5 but in the presence of 1 µl of serum containing antibody to Bm3R1; lane 8, probe plus 1 µg of purified BM1P2 protein; lane 9, same as lane 8 but in the presence of 1 µl of serum containing antibody to cytochrome P450; lane 10, same as lane 8 but in the presence of 1 µl of serum containing antibody to cytochrome P450.




Figure 12: The effects of antibody to Bm3R1 on DNA-protein binding assays using the 177-bp DNA fragment from pBM1-385A as a probe with proteins BM1P1, BM1P2, and Bm3R1. This probe, the shared P450 and BM1P1 regulatory regions, was used in gel mobility shift assays to determine the effect Bm3R1 antibody on binding activity. Lane 1, probe only; lane 2, probe plus 1 µg of partially purified Bm3R1 protein; lane 3, same as lane 2 but in micrograms of partially purified Bm3R1 protein; lane 3, same as lane 2 but in the presence of 1 µl of serum containing antibody to cytochrome P450; lane 4, same as lane 2 but in the presence of 1 µl of serum containing antibody to Bm3R1; lane 5, probe plus 1 µg of purified BM1P1 protein; lane 6, same as lane 5 but in the presence of 1 µl of serum containing antibody to cytochrome P450; lane 7, same as lane 5 but in the presence of 1 µl of serum containing antibody to Bm3R1; lane 8, probe plus 1 µg of purified BM1P2 protein; lane 9, same as lane 8 but in the presence of 1 µl of serum containing antibody to cytochrome P450; lane 10, same as lane 8 but in the presence of 1 µl of serum containing antibody to cytochrome P450.




DISCUSSION

The 1.3-kb nucleotide sequence 5` to the P450 structural gene in B. megaterium 14581 contains two open reading frames (Fig. 1B). Specific features of the sequence as well as our experimental results strongly suggest that the first open reading frame (BM1P1) encodes a protein that positively regulates P450 gene expression in B. megaterium. For example, the BM1P1 and P450 genes, transcribed in opposite directions, share the same regulatory region with the two promoters partially overlapping (Fig. 1B). Two inverted repeats are located in this region. One is a perfect 24-bp inverted repeat sequence; the second, located 6 bp upstream, is a 10-bp inverted repeat. Both the -10 sequence (TATACTA) and the transcription start site of the P450 gene are located in the 24-bp inverted repeat region while P450 and BM1P1 have overlapping -35 sequences located in the 10-bp inverted repeat region. One might thus expect that the binding of regulatory proteins to the shared regulatory region would repress or derepress the BM1P1 and P450 genes coordinately. A regulatory role for the BM1P1 protein is also suggested by the similarity of its primary and secondary structure (including a helix-turn-helix DNA-binding motif) to that of the N-terminal region of Bm3R1, the critical regulatory protein that represses P450 gene expression in B. megaterium by binding to its operator region(8, 9) . Our experimental results reinforce the inferences derived from computer analyses of the 5`-flanking region of the P450 gene. Thus, we found that in B. megaterium transformed by plasmid pUB (i.e. a construct containing the BM1P1 promoter plus most of the BM1P1 open reading frame in the same orientation as the CAT reporter gene), the cells grown in the presence of 4 mM pentobarbital showed a 4.5-fold increase in CAT activity over those grown in the absence of barbiturates (data not shown), indicating that one or more important regulatory sites mediating the increase of CAT expression in response to pentobarbital induction reside in this 0.5-kb region. Furthermore, B. megaterium cells transformed by plasmid pUB produced a significant increase of P450 protein when compared to cells transformed by plasmid pUB (i.e. the vector with no insert) when both were grown in the absence of pentobarbital (Fig. 2). This finding is compatible with the inference that BM1P1 protein (in this case expressed from pUB) acts as a positive regulatory factor in the expression of P450. A regulatory role for BM1P1 is also suggested by our results from gel mobility shift assays which demonstrated that pure BM1P1 protein could form specific DNA-protein complexes with 385- and 177-bp DNA fragments incorporating the overlapping promoter regions of the P450 and BM1P1 genes and containing 24- and 10-bp inverted repeat sequences (see Fig. 7-9). Finally, we showed by both Northern blotting analysis (Fig. 3A) and by primer extension analysis (Fig. 4A) that pentobarbital induces the expression of the BM1P1 gene in B. megaterium.

These same experiments indicate that BM1P2, a second protein encoded in the 5`-flanking region of the P450 gene (see Fig. 1B), could also form specific DNA-protein complexes with fragments containing the shared regulatory region of the BM1P1 and P450 genes (Fig. 7-9) and that the expression of the BM1P2 gene at the transcriptional level could also be induced in B. megaterium by pentobarbital (Fig. 3B and 4B). Such results suggest that BM1P2, like BM1P1, may also function as a positive regulatory factor involved in stimulating transcription or modulating expression of the P450 gene. Although it shows no obvious sequence similarity to either BM1P1 or to the P450 repressor, Bm3R1, it is similar in sequence to NIFA, a transcriptional activator required for activation of most NIF operons(26, 27, 28, 29, 30) .

Perhaps the most tantalizing evidence that BM1P1 and BM1P2 are involved in the barbiturate-responsive positive regulation of P450 gene expression is provided from gel retardation experiments involving their effect on Bm3R1 binding to putative regulatory sites. In a previous publication (11) we described the binding of a putative repressor protein to a 17-bp (Barbie box) sequence situated upstream from the P450 coding region and also discovered in the 5`-flanking region of the P450 gene. In other work from this laboratory (8, 9) , we also reported the characterization and function of Bm3P1, a protein encoded in an open reading frame immediately upstream of the B. megaterium P450 structural gene. Bm3P1 acts to repress the expression of both its own gene (bm3R1) and the P450 gene at the transcriptional level by binding to a bicistronic operator site, a 20-bp perfect palindromic sequence located between the promoter and the bm3R1 structural gene (the effects of barbiturates on this process is summarized in the Introduction of this paper). Gel retardation experiments reported here show that Bm3R1 can also bind to sequences in the 5`-flanking region of the P450 gene. Thus, the regulatory region of the BM1P1 and P450 genes contains a second binding site for Bm3R1 (see Fig. 9, lanes 2-6) while a third binding site is located in a 208-bp DNA fragment upstream from this regulatory region (Fig. 10, lanes 2-7). This DNA fragment contains a Barbie box sequence shown previously to bind Bm3R1(12) . However, when BM1P1 or BM1P2 are included in the binding reaction mixtures with Bm3R1, a DNA-protein binding band for Bm3R1 can no longer be detected (see Fig. 8, lanes 14-20; Fig. 9, lanes 17-19; Fig. 10, lanes 8 and 9). Since the 385- and 177-bp fragments from plasmids pBM1-385 and pBM1-385A, respectively, each contain one or more important binding sites for all three proteins ( Fig. 8and Fig. 9), it is not surprising that BM1P1 and BM1P2 compete effectively with Bm3R1 for binding to these fragments. Both contain the shared regulatory region of the P450 and the BM1P1 genes and include 2 inverted repeats (Fig. 1B). On the other hand, the 208-bp fragment from pBM1-385B, containing a Barbie box sequence that can bind one or more barbiturate-responsive proteins(11, 12) , has a binding site for Bm3R1 (Fig. 10, lanes 2-7) but not for BM1P1 or BM1P2 (Fig. 10, lanes 10 and 11). Thus, although BM1P1 and BM1P2 do not appear to bind to the 208-bp fragment, they still prevent Bm3R1 from binding to this probe (Fig. 10, lanes 8 and 9). The conclusion that BM1P1 and BM1P2 interfered with Bm3R1 binding to both sites was confirmed by ``supershift'' experiments ( Fig. 11and Fig. 12). The results therefore suggest that BM1P1 and/or BM1P2 may interact directly with Bm3R1 to alter its binding properties to the Barbie box site and may effectively compete with Bm3R1 for binding to the shared promoter region site. Regardless of the mechanisms involved in the competitive effects of BM1P1/2 on Bm3R1 binding, if Bm3R1, in binding to these sites acts to repress the expression of the P450 gene then it would seem to follow that BM1P1 and BM1P2 mediate derepression (activation) of the P450 gene. As already noted (see ``Results''), evidence that Bm3R1 may indeed be a repressor for the P450 gene was deduced from our previous finding that in the G39E Bm3R1 mutant of B. megaterium that is constitutive for cytochrome P450, the synthesis of cytochrome P450 relative to the wild type strain is also dramatically increased. (^4)


FOOTNOTES

*
This work was supported by National Institutes of Health Research Grant GM23913 and by the Director of the Office of Energy Research, Office of Health and Environmental Research, Contract DE-FC03-ER06015. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests or inquiries should be addressed: University of California, Laboratory of Structural Biology and Molecular Medicine, 900 Veteran Ave., Los Angeles, CA 90024-1786. Tel.: 310-825-8750; Fax: 310-825-9433; fulco{at}lbes.medsch.ucla.edu.

^1
The abbreviations used are: bp, base pair(s); kb, kilobase pair(s); CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol.

^2
The BLAST algorithm is a heuristic for finding ungapped, locally optimal sequence alignments. The BLAST family of programs employs this algorithm to compare an amino acid query sequence against a protein sequence data base or a nucleotide query sequence against a nucleotide sequence data base, as well as other combinations of protein and nucleic acid (32).

^3
Anti-Bm3R1 antiserum was a gift from Dr. C. Roland Wolf of the Biomedical Research Centre, Ninewells Hospital & Medical School, Dundee, Scotland.

^4
L.-P. Wen and A. J. Fulco, unpublished experiments.


ACKNOWLEDGEMENTS

We thank Keynes Tong from this laboratory for his excellent technical assistance in several of the experiments reported here. We are also grateful to Dr. C. Roland Wolf for his generous gift of anti-Bm3R1 antiserum that we used in several of the experiments reported here.


REFERENCES

  1. Fulco, A. J. (1991)Annu. Rev. Pharm. Toxicol.31,177-203 [CrossRef][Medline] [Order article via Infotrieve]
  2. Narhi, L. O., and Fulco, A. J.(1982)J. Biol. Chem.257,2147-2150 [Abstract/Free Full Text]
  3. Fulco, A. J., Kim, B-H., Matson, R. S., Narhi, L. O., and Ruettinger, R. T.(1983) Mol. Cell. Biochem.53/54,155-162
  4. Kim, B-H., and Fulco, A. J.(1983)Biochem. Biophys. Res. Commun. 116,843-850 [Medline] [Order article via Infotrieve]
  5. Ruettinger, R. T., Kim, B-H., and Fulco, A. J.(1984)Biochim. Biophys. Acta 801,372-380 [Medline] [Order article via Infotrieve]
  6. English, N., Hughes, V., and Wolf, C. R.(1994)J. Biol. Chem.269,26836-26841 [Abstract/Free Full Text]
  7. Wen, L.-P., Ruettinger, R. T., and Fulco, A. J.(1989)J. Biol. Chem. 264,10996-11003 [Abstract/Free Full Text]
  8. Shaw, G.-C., and Fulco, A. J.(1992)J. Biol. Chem.267,5515-5526 [Abstract/Free Full Text]
  9. Shaw, G.-C., and Fulco, A. J.(1993)J. Biol. Chem.268,2997-3004 [Abstract/Free Full Text]
  10. He, J.-S, Ruettinger, R. T., Liu, H.-M., and Fulco, A. J.(1989)Biochim. Biophys. Acta1009,301-303 [Medline] [Order article via Infotrieve]
  11. He, J.-S., and Fulco, A. J.(1991)J. Biol. Chem.266,7864-7869 [Abstract/Free Full Text]
  12. Liang, Q., He, J.-S., and Fulco, A. J.(1995)J. Biol. Chem.270,4438-4450 [Abstract/Free Full Text]
  13. Sanger, F., Nicklen, S., and Coulson, A. R.(1977)Proc. Natl. Acad. Sci. U. S. A.74,5463-5467 [Abstract]
  14. The Qiagenologist (1990) Application Protocols, 3rd Ed., Diagen, Gmbh, Dsseldoff, Federal Republic of Germany
  15. Kingston, R. E. (1987) in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds) Vol. 1, pp. 4.8.1-4.8.3. Wiley Interscience, Boston
  16. Brosius, J., and Holy, A.(1984)Proc. Natl. Acad. Sci. U. S. A. 81,6929-6933 [Abstract]
  17. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  18. Fried, M., and Crothers, D. M.(1981)Nucleic Acids Res. 9,6505-6525 [Abstract]
  19. Moran, C. P., Jr., Lang, N., LeGrice, S. F. J., Lee, G., Stephens, M., Sonenshein, A. L., Pero, J., and Losick, R. L.(1982)Mol. & Gen. Genet. 186,339-346
  20. McLaughin, J. R., Murray, C. L., and Rabinowitz, J. C.(1981)J. Biol. Chem.256,11283-11291 [Abstract/Free Full Text]
  21. Hawley, D. K., and McClure, W. R.(1983)Nucleic Acids Res.11,2237-2255 [Abstract]
  22. Pabo, C. O., and Sauer, R. T.(1984)Annu. Rev. Biochem.53,293-321 [CrossRef][Medline] [Order article via Infotrieve]
  23. Chou, P. Y., and Fasman, G. D.(1974)Adv. Enzymol. Relat. Areas Mol. Biol. 47,45-148
  24. Chou, P. Y., and Fasman, G. D.(1978)Annu. Rev. Biochem.47,251-276 [CrossRef][Medline] [Order article via Infotrieve]
  25. Garnier, J., Osguthorpe, D. J., and Robson, B.(1978)J. Mol. Biol. 120,97-120 [Medline] [Order article via Infotrieve]
  26. Agron, P. G., Ditta, G. S., and Helinski, D. R.(1992)J. Bacteriol. 174,4120-4129 [Abstract]
  27. Cannon, W., and Buck, M. (1992)J. Mol. Biol.225,271-286 [Medline] [Order article via Infotrieve]
  28. Surza, E. M., Funayama, S., Rigo, L. U., Yates, M. G., and Pedrosa, F. O.(1991) J. Gen. Microbiol.137,1511-1522 [Medline] [Order article via Infotrieve]
  29. Cannon, W., Charlton, W., and Buck, M.(1991)J. Mol. Biol.220,915-931 [Medline] [Order article via Infotrieve]
  30. Tripathi, A. K., Kreutzed, R., and Klingmuller, W.(1991)Mol. & Gen. Genet.227,86-90
  31. Hoffmann, A., and Roeder, R. G.(1991)Nucleic Acids Res.19,6337-6338 [Medline] [Order article via Infotrieve]
  32. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J.(1990) J. Mol. Biol.215,403-410 [CrossRef][Medline] [Order article via Infotrieve]

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