From the Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907
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ABSTRACT |
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During sporulation, Bacillus
thuringiensis produces inclusions comprised of different amounts
of several related protoxins, each with a unique specificity profile
for insect larvae. A major class of these genes designated
cry1 have virtually identical dual overlapping promoters,
but the upstream sequences differ. A gel retardation assay was used to
purify a potential regulatory protein which bound with different
affinities to these sequences in three cry1 genes. It was
identified as the E2 subunit of pyruvate dehydrogenase. There was
specific competition for binding by homologous gene sequences but not
by pUC nor Bacillus subtilis DNA; calf thymus DNA competed
at higher concentrations. The B. thuringiensis gene
encoding E2 was cloned, and the purified glutathione
S-transferase-E2 fusion protein footprinted to a consensus
binding sequence within an inverted repeat and to a potential bend
region, both sites 200-300 base pairs upstream of the promoters.
Mutations of these sites in the cry1A gene resulted in
decreased binding of the E2 protein and altered kinetics of expression
of a fusion of this regulatory region with the lacZ gene.
Recruitment of the E2 subunit as a transcription factor could couple
the change in post exponential catabolism to the initiation of protoxin synthesis.
Most Bacillus thuringiensis subspecies contain
multiple, plasmid-encoded protoxin genes that are very actively
transcribed primarily during sporulation (1, 2). There is extensive synthesis of the related protoxins which are often packaged into the
same inclusion. Each of these protoxins has a somewhat different specificity (7), and there may be synergism among some (8, 9).
Many but not all of these genes contain very similar overlapping
promoters (2, 3) recognized in the mother cell during sporulation by
Given the similarity of the overlapping promoter regions for three of
these cry1 genes, the sequences upstream for ~1
kbp2 were examined and found
to differ substantially
(10).3,4
These regions appear to be important for regulation because expression of cry1-lacZ fusion plasmids in B. thuringiensis
was enhanced by their presence.1 Employing a gel
retardation assay, a novel DNA binding protein was identified and
purified, and its gene was cloned. The binding sites in the upstream
regions of two of these cry genes were determined by
footprinting. The effects of mutations in these sites indicated that
this protein is likely to have a role in regulating the expression of
this class of protoxin genes.
Cell Growth--
B. thuringiensis subsp.
kurstaki HD1, strain 80-21 (11), subsp. aizawai
HD133, and a plasmid-cured (with mitomycin C) acrystalliferous derivative of B. thuringiensis subsp. kurstaki
HD1 designated Mit94 were grown in G-Tris medium at
30 °C (12) in a New Brunswick incubator shaker. This medium contains
0.2% glucose as the principal carbon source, which was replaced with
0.1% potassium gluconate for the Isolation of Regions Upstream of the Promoters--
A region of
280 bp upstream of the cryIAb gene was prepared
by PCR using oligonucleotides 5'-AATAGGATCCTTCCTATATTTACTTTGCCC-3', containing a BamHI site, and
5'-GGTTTGAATTCCGTTAACTTATTTTAAAGT-3', containing an EcoRI site.
The region upstream of the promoters of the cry1C gene was
isolated as a 656-bp BglII/HindIII fragment from
a 7-kbp EcoRI fragment containing this gene, including 2.5 kbp upstream of the promoters (13). This fragment was isolated from low
melting agarose with GELase (Epicentre Technologies) and cloned into
the HindIII and BamHI sites of pUC18. For gel
retardation, this clone was digested with EcoRI and
HindIII, and the 656-bp fragment was reisolated as described above.
The cryID gene (14) was cloned as a 3.8 kbp KpnI
fragment from B. thuringiensis subsp. aizawai
HD133, strain 5 (11), into the Escherichia coli/B.
thuringiensis shuttle vector pHT3101 (15). A 2.2-kbp
NdeI fragment embracing most of the coding region was deleted from this plasmid, creating p
The E2 binding site containing a potential bend region was mutated from
5'-CTCAATTTGTATATGTAAAATAGGAAAAGTG to
5'-CTCAGTCTGTCTATGTAGAACAGGACAAGTG (bold letters indicate changes including the creation of an
MspHI site for screening) employing oligonucleotide
5'-CACTTCTCCTGTTGTACATAGACAGACTGAG. The inverted repeat (IR; see Fig.
4) was mutated from 5'-CCTGCAATTCATCTTGAATTGTAAATGC to
5'-CCTGCAGTTAAGCCTGAATTGTAAATGC with the introduction of a PstI site for screening.
The cry1Ab upstream region as a 310-bp HindIII
fragment (see Fig. 4) was cloned into pGEM 11(+) for production of
single-stranded template in E. coli CJ236
(dut Purification of the Binding Protein--
Strain Mit9 was grown
in 2 liters of G-Tris medium until about 70% of the cells contained
phase bright endospores. B. subtilis JH642 was grown in 500 ml of nutrient sporulation medium until >50% contained phase bright
endospores (the maximum is about 70% versus >90% for
Mit9). Cells were harvested by centrifugation and washed once with 20 ml of 100 mM KCl, 5 mM EDTA, pH 8.0. The pellets were suspended in 1/5 volume of buffer A (50 mM
Tris, pH 7.4, 1 mM EDTA, 100 µg/ml phenylmethylsulfonyl
fluoride), washed twice with this buffer, resuspended in 1/50 the
original volume of buffer A plus 1 mM dithiothreitol, and
lysed in a French press at 9000 p.s.i. The lysate was centrifuged
at 4500 × g for 10 min at 4 °C. The supernatant was
withdrawn and centrifuged in an Eppendorf microcentrifuge for 10 min at
4 °C. This supernatant was then heated to 45 °C for 10 min to
inactivate DNases while leaving binding activity unaltered.
The heated extract was fractionated by the addition of solid
(NH4)2SO4 to final concentrations
of 25, 40, 50, 60, 80, and 100% of saturation. The protein precipitate
after each step was collected by centrifugation at 8000 × g for 20 min at 4 °C, dissolved in 0.5 × TBE (0.04 M Tris, 0.04 M sodium borate, 2 mM
EDTA, pH 8.0), and dialyzed overnight at 4 °C against two changes of
2 liters each of this buffer. Each fraction was then tested for binding
activity as described below.
The active 50-60% (NH4)2SO4
fraction from Mit9 was further fractionated by passage over a
heparin-agarose column (17). One column volume was loaded in 0.5 × TBE at 4 °C, and the effluent was passed back over the column ten
times. After washing with 4 column volumes of 0.5 × TBE, the
column was eluted with a KCl gradient from 0 to 0.4 M in
0.5 × TBE. One-ml fractions were collected, concentrated 4-fold
by lyophilization, and dialyzed against 0.5 × TBE. Fractions were
assayed for binding activity as described below.
Purification was also performed by excising and eluting the retarded
DNA band from polyacrylamide gels. Elution was performed in a Little
Blue Tank elutrap system (ISCO) using 0.05 × TBE in the sample
wells and 0.5 × TBE in the electrophoresis chamber. The
DNA-protein complex concentrated in this way was dissociated by
addition of KCl to 0.4 M for 8 h at 27 °C. The DNA
was then digested for 1 h at 37 °C with 20 units of DNase I,
and the protein was fractionated in 10% SDS-PAGE (16). The gel was
transferred to polyvinylidene difluoride and stained (18). Protein
bands were excised, and the sequence of the first 25 amino acids was determined in an automated sequenator (Purdue Center for Macromolecular Structure).
DNA Labeling--
DNA was treated with calf intestinal alkaline
phosphatase (CIP; 2 units per mg of DNA) at 37 °C for 45 min,
extracted with phenol and precipitated with ethanol. The DNA was then
dissolved in 10 mM Tris, 20 mM
Gel Retardation Assays--
Retardation assays (19) were
performed in 3.5% native polyacrylamide gels (acrylamide:bisacrylamide
ratio of 60:1) as per Fried and Crothers (20) and Garner and Revzin
(21). After investigating several buffers, it was found that the best
retardation was observed when protein and 32P-labeled DNA
were mixed in 20 µl of 0.5 × TBE and incubated at 16 °C for
20 min. Five µl of bromphenol blue-xylene cyanol in 5 M
sucrose was added to the samples, which were loaded onto vertical gels
prepared in 0.5 × TBE and subjected to electrophoresis at 4 °C
and 10 V/cm until the bromphenol blue dye front was about 3/4 of the
way down the gel (approximately 2.5 h for a 15-cm gel). The gel
was dried at 80 °C under vacuum and autoradiographed.
Cloning the pdhC Gene from B. thuringiensis--
The
pdhC gene encoding the E2 subunit of pyruvate dehydrogenase
(PDH) from B. thuringiensis was cloned as a fusion with the glutathione S-transferase gene in the pGEX-KG expression
vector (22). PCR oligonucleotides:
5'-TAGGAGGTCGGGATCCGTGGCATTTGAATT-3' containing a
BamHI site and 5'-ATAGGGAAATCTCGAGCTACCATAACATTA-3' containing a XhoI site were based on the sequences of
the B. subtilis pdhC gene (23) and used to clone a 1350-bp
region of DNA from B. thuringiensis corresponding to its
pdhC gene. This gene was cloned in-frame as a
BamHIXhoI fragment into the pGEX-KG vector to
produce plasmid pCB117. It was sequenced and had a deduced open reading
frame encoding a polypeptide of the expected size.
A fusion protein consisting of B. thuringiensis PDH-E2 fused
to glutathione S-transferase was produced after
transformation of pCB117 into E. coli TG1 and induction with
isopropyl-1-thio-
To obtain a less extensive E2 fusion protein, this gene was also cloned
into the His6 expression vector, pQE30 (Qiagen). The BamHI/XhoI fragment was cloned into pUC18 and
then excised as a BamHI/SphI fragment for cloning
in-phase in pQE30. E. coli DH5 DNase I Footprinting--
Fifty ng of purified, end-labeled DNA
was incubated with varying amounts of the E2 protein and 5 µg of
poly(dI·dC) in 20 µl of 0.5 × TBE. The binding reaction was
carried out for 20 min at 16 °C, after which 0.05 units of DNase I
(Boehringer Mannheim) in 12.5 mM MgCl2 was
added and the tubes incubated at 25 °C for 90 s. The reaction
was stopped by the addition of 5 µl of 30 mM EDTA and
extraction of the mixture with phenol-chloroform. The DNA was
precipitated with 2 volumes of ethanol and dissolved in Sequenase stop
buffer (United States Biochemical). After heating at 90 °C for 5 min, samples were loaded onto a 6% polyacrylamide gradient gel
containing 8 M urea in 90 mM Tris, 89 mM borate, 2.5 mM EDTA, pH 8.3 (16) and
electrophoresed at 1000 V and 75 mA. Gels were then dried and autoradiographed.
DNA sequence was obtained according to the standard method for
double-stranded DNA sequence analysis (16) with the addition of
Mn2+ to the reaction mix to allow sequence determination
near the primer (25).
Binding Activity in Sporulating Cells--
The 50-60%
(NH4)2SO4 fraction of a crude
extract from sporulating but not from growing cells of B. thuringiensis strain Mit9 retarded a 280-bp fragment from the
cry1A upstream region, a 656-bp fragment from
cry1C, and an ~1.6-kbp fragment from cry1D
(Fig. 1). In all cases, at least a
1,000-fold excess of poly(dI·dC) was present. The further addition of
a small excess of homologous unlabeled DNA competed for binding,
whereas the same concentration of pUC18 did not (Fig.
2A). Even a 100-fold excess of
pUC18 DNA (either linear or as 0.3 + 2.4-kbp PvuII
fragments) or sonicated B. subtilis DNA did not
compete.4 There was some inhibition of retardation by a
50-fold excess of sonicated calf thymus DNA (Fig. 2B).
The specificity of this binding was confirmed by footprinting (Figs. 4
and 6) as well as by the effectiveness of a B. thuringiensis protein fraction as compared with that from B. subtilis
(Fig. 2C). There was greater retardation of the
cry1A fragment by equivalent amounts of E2 in the 50-60%
ammonium sulfate fraction from B. thuringiensis Mit9 than
that from B. subtilis JH642 (Fig. 2C). A
comparable preparation from E. coli did not retard.
Retardation of 1 pmol of the cry1Ab fragment was complete
when incubated with about 2 µg of the 50-60% ammonium sulfate
fraction from B. thuringiensis. Complete retardation of the
cry1C and cry1D DNAs required 10-20-fold more
protein, indicating lower affinities and/or more binding sites. There
were several degrees of retardation of these DNAs (arrows on
the right panel of Fig. 1) in contrast to the
cry1A DNA. The presence of multiple retarded complexes may
be because of several binding sites with different affinities for the protein.
Characterization of the Binding Protein--
The binding protein
was isolated by electroelution of an excised, retarded band followed by
dialysis and digestion with DNase I. This procedure resulted in
recovery of a single protein of ~60 kDa (Fig.
3). The sequence of the first 25 residues
of this band was 92% identical to that of dihydrolipoamide
acetyltransferase, the E2 subunit of PDH from B. subtilis
and Bacillus stearothermophilus (Table
I). The E2 subunit is a 48-kDa protein
containing lipoic acid (which decreases its mobility in SDS-PAGE).
Because sufficient quantities of E2 could not be renatured after
elution from the retarded complex, the 50-60%
(NH4)2SO4 fraction was
further purified by elution from a heparin-agarose column with a KCl
gradient. The greatest binding activity (based on retardation of
cry1A DNA) was in the 0.2-0.3 M KCl fraction
that contained the E2 protein as >50% of the total based on staining
and confirmed by immunoblotting with antiserum against the B. subtilis PDH complex. The E1 Footprinting of the Binding Sites--
The purified glutathione
S-transferase fusion protein protected three regions in the
280-bp cry1Ab DNA (Fig. 4,
A and B and Fig.
5). The most distal of these and the
inverted repeat share a common sequence. The third region was within a
stretch of intrinsically bent DNA (29) with a 10-bp spacing between
each of the protected regions. There were also several hypersensitive
sites on both strands (arrows). A major protected site in
the cry1C DNA was within an inverted repeat (Fig.
6) almost identical in sequence to that
in the cry1Ab DNA. There were also a multiplicity of
hypersensitive sites in this DNA but no detectable bend region. The
footprint with the heparin-agarose fraction was the same as that
obtained with the purified fusion protein.
Function of the Upstream Region in cry1 Gene
Transcription--
The binding site within the bend region and the IR
were mutated as described under "Experimental Procedures." Both
were found to have lower affinities for purified His6-E2
protein than the wild type fragment (Fig.
7). The apparent Kd
value for the wild type, assuming that it was the monomer of the
His6-E2 adduct that bound, was 4-6 nM. E2 is a
multimer in the PDH complex (30), however, and it is likely that a
multimeric form of this protein is required for binding to DNA. As
mentioned above, only His6-E2 extracted from the crude
membrane fraction with 4-6 M urea in buffer was active.
The extent of gel retardation by His6-E2 purified from the
E. coli clone was greater than that of E2 purified from
B. thuringiensis (Figs. 1 and 7), implying different
aggregation states perhaps because of concentration effects or the
presence or absence of lipoamide.
Cells containing lacZ fusions with either the
cry1A wild type or mutant (bend and IR) upstream regions
were sampled during sporulation, and the kinetics of Regulation of protoxin genes is of interest not only because of
their insecticidal properties but from the perspective of how a cell
recruits regulatory elements for a group of structural genes that have
very likely become part of the genetic repertoire of this
Bacillus relatively late in evolution. One aspect of the regulation is the presence of dual overlapping promoters, which ensures
a constant rate of transcription of these cry genes during an extended period of sporulation. In addition, each of these genes is
independently regulated, so factors other than the promoters must be
involved. The sequences upstream of the promoters for the
differentially regulated cry1A, cry1C, and cry1D
genes differ substantially (although some features are shared; see
below) so we began a search for DNA binding proteins.
Extracts of sporulating but not vegetative cells of B. thuringiensis subsp. kurstaki contained a protein that
bound to regions of DNA upstream of the cry1Ab,
cry1C, and cry1D gene promoters. The major
binding protein in the heparin-purified fraction from B. thuringiensis was identified as the E2 subunit of PDH. The purified GST-E2 or His6-E2 fusion proteins footprinted to
specific sites in the cry1A and cry1C upstream
regions. The presence of three close binding sites in the
cry1A but not the cry1C sequence may account for
the higher affinity for the E2 oligomer by the former (Fig. 1) and thus
a basis for the differential regulation of these cry 1 genes.
The evidence for specific binding of this novel DNA binding protein is
1) the presence of a consensus binding sequence (5'-cAAGAT/gG/tAA) in
two of the three sites in the cry1A sequence and within the inverted repeat in the cry1C sequence. There was also
binding to an intrinsically bent region in the cry1A DNA.
The binding site(s) in the cry1D DNA have not been mapped.
2) There was optimal competitive binding by the homologous DNA and very
little or no competition by nonspecific DNAs such as poly(dI·dC),
pUC18, or B. subtilis DNAs. Sonicated calf thymus did
compete at higher concentrations (Fig. 2B), probably because
of the complexity of sequences in this DNA including potential bend
regions. 3) There was no binding to a digest of pUC18, which included a
fragment similar in size (about 0.3 kbp) to that of the
cry1A upstream fragment. 4) There was higher affinity
binding by the E2 protein from B. thuringiensis as compared
with that from B. subtilis. While the deduced sequences of
the B. thuringiensis and B. subtilis E2 proteins
are >80% identical, there is considerably less homology in and around
the so-called hinge region.4 This region of E2 links the
lipoyl domain to the E1 and E3 binding sites (30), and it is a
potential DNA binding region (37).
The stoichiometry and patterns of DNA retardation indicated extensive
cooperativity in the binding, probably involving conformational changes
to allow some form of the E2 protein to bind. The icosohedral core of
PDH is comprised of 60 E2 subunits (30), so it is likely that some
multimeric form is involved in binding as indicated by the requirement
of a urea extraction in the purification protocol of
His6-E2 from E. coli. The role of lipoamide,
which is a component of E2 in the PDH complex, is not known. Whatever
conformational changes of the DNA occurred could be reversed by
treatment with protease K, demonstrating that the continued presence of
E2 was essential. In many respects, the binding was similar to that of the Lrp protein (31), which may serve as a useful paradigm.
The importance of these E2 binding sites was indicated by the effect of
mutations on the binding of His6-E2 and on the expression of lacZ fusions (Figs. 7 and 8). Both the rates and final
amounts were reduced, but the former may be particularly important.
These parasporal inclusions have a crystalline array (32) so that the
deposition of the disulfide cross-linked protoxins (33) must be an
orderly process, very likely dependent upon chaperones and other
factors (2). A decrease in the rate of protoxin accumulation such as
that resulting from mutations in the upstream binding sites for the E2
protein (Fig. 8) could substantially alter the relative amount of the
Cry1A protoxin in the inclusion.
The consensus binding sequence is also present close to the start site
of transcription of the pdhC gene in B. subtilis
(23) as well as near the origin of replication (34, 35). The latter was
identified as a region of the chromosomal DNA bound to the membrane
primarily by a protein of 60 kDa, which was subsequently identified as
E2.5 The E2 protein in
Neurospora crassa (designated MRP3) appears to be a
mitochondrial ribosomal protein (36) so its recruitment for other
functions is not without precedence.
A role for the E2 subunit in regulation implies a connection between
catabolism and protoxin synthesis. It has been known for some time that
the protoxin yield per spore or per mg of dry weight varies
considerably with the subspecies studied and with the media (38).
During growth on glucose, bacilli excrete acetic acid, pyruvic acid,
and acetoin (39) which are catabolized during sporulation (40). The
pyruvate is utilized rapidly, and PDH is then no longer required for
catabolism. At about this time, the E2 subunit is found in the soluble
fraction of sporulating cells4 (35). The source of the
soluble E2 could be dissociation of the PDH complex and/or
transcription from its own promoter (23), which does function at the
end of growth.4 The E2 consensus binding sequence in this
promoter region is thus all the more intriguing.
The presence of soluble E2 would signal the end of growth on sugars,
and its recruitment for regulating the plasmid-encoded cry
genes would provide a mechanism for selectively enhancing their
transcription in postexponential cells. The amount of soluble E2 in
sporulating cells would depend upon prior growth conditions and perhaps
autoinduction of the pdhC gene. Such factors could integrate
cell growth with the subsequent transcription of the cry genes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
E and
K forms of RNA polymerase (2, 4).
Dual promoters ensure transcription of these particular genes
(cry1) throughout much of
sporulation1 (5), but they
are differentially transcribed (6). Control of expression of the
cry genes is necessary not only to ensure a balance of
transcription with mother-cell spore genes which utilize the same forms
of RNA polymerase but for regulating the relative amounts of the
various protoxins and their assembly into an inclusion (2).
EXPERIMENTAL PROCEDURES
-galactosidase assays (see below).
Bacillus subtilis JH642 was grown in nutrient sporulation
medium at 37 °C. Growth was monitored by A600
in a Perkin Elmer Model 35 spectrophotometer and sporulation in the
phase microscope.
ID from which the
cryID upstream region including the promoters was isolated
as a 1.6-kbp KpnI1-NdeI fragment.
, ung
). Mutagenesis followed
the procedure of Kunkel (16), and plasmids produced in E. coli DH5
were screened initially for the presence of an
additional MspHI or PstI site in the insert and
then sequenced to confirm the changes. The mutagenized
HindIII fragment was isolated from the pGEM clone as
described above, and the fragment inserted into the single
HindIII site in a lacZ fusion vector containing the cry1A promoter region (see below). The orientation was
established on the basis of the sizes of HpaI restriction fragments.
-mercaptoethanol, 10 mM MgCl2, pH 7.5, and incubated with l0 units of T4 polynucleotide kinase and 300 µCi of
[
-32P]ATP (150 mCi ml
1) per µg of DNA
for 90 min at 37 °C in a total volume of 25 µl. To remove the
label from one end, the 32P-DNA was digested with
BamHI for footprinting the transcribed strand and with
EcoRI for the nontranscribed strand. The labeled DNA was
purified by excision from a low-melting agarose gel and digestion with GELase.
-D-galactopyranoside (22). The fusion
protein that was purified by elution from a glutathione-agarose column
(24) reacted with anti-PDH-E2 antibody from Staphylococcus
aureus, kindly provided by Dr. H. Hemila (23). The glutathione
S-transferase could not be removed by thrombin digestion
without disrupting the B. thuringiensis PDH-E2 protein, which contains an internal thrombin cleavage site.
containing this clone was
grown in LB-ampicillin (25 µg ml
1) and expression was
induced by addition of 1 mM
isopropyl-1-thio-
-D-galactopyranoside for 3 h.
Cells were lysed as per the Qiagen manual. Although there was
substantial His6-E2 in the soluble fraction, the E2 protein solubilized from the pellet with 6 M urea in 0.05 M Na2HPO4, 0.3 M NaCl,
pH 8.0 was most active in gel retardation. The latter was fractionated
on a Ni2+-agarose column using a step gradient of 0.1-0.3
M imidazole in the above buffer. After dialysis for 18 h at 4 °C against 4000 volumes of 0.5 × TBE, fractions were
assayed for gel retardation activity, and the most active fraction was
stored at
80 °C.
-Galactosidase Activity--
A cryIAb
promoter-lacZ fusion was constructed in an E. coli/Bacillus shuttle vector.1 A
HindIII-digested, PCR-cloned 310-bp upstream fragment from the cryIAb gene (containing the region from
218 to
528;
see Fig. 5) was inserted in both orientations into a single
HindIII site upstream of the protoxin promoters. This
fragment containing the mutated bend or IR regions was also inserted
into this vector, and the plasmids were electroporated (26) into strain
80-21. Cells were grown in G-Tris medium (12) containing 0.l% yeast extract and 0.1% potassium gluconate plus 5 µg/ml chloramphenicol. Differences in induction of
-galactosidase (but not growth) were more evident when potassium gluconate rather than glucose was the
primary carbon source. Samples of 0.2 ml were taken at various times
during growth and sporulation, sonicated for 10 s, and 30-50 µl
were assayed for
-galactosidase activity (27) as modified by
Giacomini et al. (28). Growth was monitored by
A600 in the Perkin Elmer spectrophotometer and
sporulation followed in the phase microscope, measuring the percent
phase dull, phase white, and phase bright endospores in each sample.
RESULTS
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Fig. 1.
Gel retardation assays of cry1Ab
(1 pmol) in the left panel and of
cry1C (0.2 pmol; right panel,
lanes1-4) and cry1D (0.1 pmol; right
panel, lanes 5-8) DNA isolated and labeled as described
under "Experimental Procedures." Retardation was with the
heated, 50-60% ammonium sulfate fraction from strain Mit9. In the
left panel, lane 1 contains no protein,
lanes 2-8 contain 3.6, 1.8, 0.9, 0.72, 0.54, 0.36, and 0.18 µg, respectively. In the right panel, lane 1 is
cry1C DNA with no protein; lanes 2-4 contain
0.5, 1.3, and 4.0 µg of protein, respectively. Lane 5 is
cry1D DNA with no protein; lanes 6-8 contain
0.5, 1.3, and 4.0 µg of protein, respectively. Arrows
indicate various retardation states as discussed in the text.
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Fig. 2.
Gel retardation assays of the competition by
homologous and heterologous DNAs (A and
B) or by different sources of the binding protein
(C). A, lane 1, 100 ng of
32P-cry1C fragment; lane 2, plus 8 µg of total protein from a crude extract of strain Mit9; lane
3, 4 µg of the 50-60% ammonium sulfate fraction from Mit9;
lane 4, as in lane 3 plus 200 ng of pUC18; and
lane 5, as in lane 3 plus 200 ng of the
cry1C fragment. B, lane 1, 2 ng
32P-cry1A fragment; lane 2, plus 2 µl of the Mit9 heparin-agarose fraction containing 1.5 µg of E2
antigen; lanes 3 and 4, as in lane 2 plus 40 or 100 ng of sonicated calf thymus DNA, respectively;
lanes 5 and 6, as in lane 2 plus 10 or
20 ng of cry1A fragment, respectively. Panel C,
lane 1, 2 ng of 32P-cry1A fragment;
lanes 2 and 4, as in lane 1 plus
either 2.5 or 5 µl of the 50-60% ammonium sulfate fraction from
Mit9 containing 2 or 4 µg of E2 antigen, respectively; lanes
3 and 5, as in lane 1 plus 3 or 6 µl of
the 50-60% ammonium sulfate fraction from B. subtilis
containing 2 or 4 µg of E2 antigen, respectively.
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Fig. 3.
10% SDS-PAGE stained gel of the DNA binding
protein. Lane Mr, molecular mass standards
as indicated (in thousands); lane 2, bovine serum albumen
(68 kDa); lane 3, 50-60% ammonium sulfate fraction from
strain Mit9; lane 4, 50 to >200-kDa proteins eluted from a
gel of proteins as in lane 3; lane 5, 20-50 kDa
proteins eluted from a gel as in lane 3; lane 6,
protein from a retarded band that had been eluted and digested with
DNase I as described under "Experimental Procedures." The protein
fraction in lane 4 but not lane 5 retarded
cry1A DNA. The band labeled 60 kDa in lane 6 also
retarded and was transferred to polyvinylidene difluoride for
sequencing. The DNase I band was identified by sequencing.
Comparisons of the amino acid sequences of the N-terminal 25 residues
of PDH E2 proteins from several Gram (+) bacteria with that from
Bacillus thuringiensis
and E1
subunits of PDH eluted
at a lower salt concentration, and this fraction retarded poorly
(41).
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Fig. 4.
Footprints of the transcribed (panel
A) and nontranscribed (panel B) strands of
the cry1A upstream region DNA (50 ng). The E2-GST
fusion protein was added in amounts of 60 ng (lane 2A) and
150 ng (lane 3A) or 10, 50, and 200 ng in lanes
2B, 3B, and 4B, respectively.
Arrowheads indicate DNase hypersensitive sites.
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Fig. 5.
Sequence of the upstream region of the
cry1Ab gene (M. Geiser, personal
communication and A. Aronson, unpublished results. Boxed
sequences indicate those protected in the DNase footprints (Fig.
4). The boxed inverted repeat (convergent overlying
arrows) is based on footprinting results with both the transcribed
and nontranscribed strands. Start sites of transcription from the BtI
( E) and BtII (
K) promoters are indicated
by arrows; the ribosome binding site is
overlined. Lowercase letters indicate
oligonucleotide primers used to construct a 310-bp HindIII
PCR fragment of the upstream region. Letters in bold are the
HindIII sites.
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Fig. 6.
Footprint of the nontranscribed strand of the
cry1C upstream DNA (50 ng). Lane 1, no
protein; lane 2, 1 µg of the heparin-agarose purified
fraction; lanes 3-5, 60, 150, and 500 ng, respectively, of
the E2-GST purified fusion protein. Protected sequences are indicated
as are the DNase I hypersensitive sites (arrowheads). The
major protected region is part of an inverted repeat that is very
similar in sequence to the one in the cry1Ab sequence (Fig.
5).
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Fig. 7.
Gel retardation of 0.5 pmol of
cry1A wild type (lanes 1-3), mutated
IR (lanes 4-7), and mutated bend region (lanes
8-10) DNAs. Lanes 1, 4, and
8, no protein; lanes 2 and 5, 1.0 µg
of His6-E2; lanes 3, 6 and
9, 2.0 µg of His6-E2; lanes 7 and
10, 3.0 µg of His6-E2.
-galactosidase
synthesis was determined (Fig. 8).
Addition of the upstream DNA resulted in an enhancement of
-galactosidase synthesis. Both the initial rate and the final
activity were reduced in strains containing the mutated upstream
regions.
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Fig. 8.
Kinetics of
-galactosidase synthesis (Miller units) in B. thuringiensis strain 80-21 containing a plasmid with only
the cry promoters fused to the lacZ
gene (
); with the promoters plus 310bp cry1A
upstream region (
); the 310 bp with the bend region mutated
(
); and the 310 bp with the IR mutated (
). Sampling in
duplicate was begun 1 h after the end of growth, and the values
for each time point were averaged (± 3%).
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Dr. H. Hemila for providing antibodies to the PDH complex and to the E2 protein as well as a clone of the B. subtilis pdhC gene. Dr. Lan Wu provided considerable technical assistance.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant GM 34035.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF039908.
Supported by a Predoctoral Fellowship from the National Science
Foundation. To whom correspondence should be addressed. Tel.: 765-494-7061; Fax: 765-494-0876; E-mail:
twalter{at}bilbo.bio.purdue.edu.
1 M. Sedlak, T. Walter, and A. Aronson, manuscipt in preparation.
3 M. Geiser, personal communication.
4 A. Aronson, unpublished results.
5 W. Firshein, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: kbp, kilobase pair(s); IR, inverted repeat; bp, base pair(s); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; PDH, pyruvate dehydrogenase; GST, glutathione S-transferase.
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REFERENCES |
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