(Received for publication, December 13, 1994; and in revised form, March 21, 1995)
From the
Analysis of a 1.3-kilobase segment of 5`-flanking DNA from the
barbiturate-inducible P450
Cytochrome P450 Cytochrome
P450 We now describe the characterization of
two genes, designated BM1P1 and BM1P2, located
immediately upstream of the P450
Figure 1:
Description of plasmids
containing the P450
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
Figure 2:
Biosynthesis of cytochrome P450
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.
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.
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
Figure 6:
Final purification of BM1P1 and BM1P2 by
gel filtration chromatography. After 40,000
Figure 7:
Derivation of probes used for gel mobility
shift assays. The first probe, containing the shared P450
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
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
Although the data presented in Fig. 8and Fig. 9provided strong evidence that Bm3R1 could bind to the
regulatory regions of the P450
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
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
The 1.3-kb nucleotide sequence 5` to the P450 These same
experiments indicate that BM1P2, a second protein encoded in the
5`-flanking region of the P450 Perhaps the most tantalizing evidence that BM1P1 and BM1P2 are
involved in the barbiturate-responsive positive regulation of P450
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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
.
(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
-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) .
(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 (
)of the P450
structural
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 P450
gene 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) .
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.
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 (DH5 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 P450
gene. 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.
, 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.
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 [-
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 C-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 (
)(via the
NCBI BLAST E-mail server) developed by the National Center for
Biotechnology Information at the National Library of
Medicine(32) .
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.
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.
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).
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
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).
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.
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 (
)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.
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).
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.
; 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.
and BM1P1 genes, we sought to confirm this conclusion by
determining the effect of anti-Bm3R1 antiserum
(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).
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
.
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
.
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.
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) .
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. (
)
-D-galactopyranoside; PAGE,
polyacrylamide gel electrophoresis; DTT, dithiothreitol.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.