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Applied and Environmental Microbiology, November 2001, p. 5032-5036, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5032-5036.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Regulation of the Packaging of Bacillus
thuringiensis 
-Endotoxins into
Inclusions
Lily
Chang,
Rian
Grant, and
Arthur
Aronson*
Department of Biological Sciences,
Purdue University, West Lafayette, Indiana 47907
Received 5 April 2001/Accepted 14 August 2001
 |
ABSTRACT |
During sporulation, many Bacillus
thuringiensis subspecies synthesize several related
-endotoxins which are packaged into bipyramidal intracellular
inclusions. These inclusions are solubilized in the alkaline, reducing
conditions of the midguts of susceptible insect larvae and are
converted by proteolysis to active toxins. The toxins insert into the
membranes of cells lining the midgut and form cation-selective
channels, which results in lethality. There are three -endotoxins,
Cry1Ab3, Cry1Ca1, and Cry1Da1, present in the inclusions
produced by a B. thuringiensis subsp.
aizawai cell. While the ratio of the steady-state
mRNAs for these three protoxins has been shown to differ
(cry1Ab3/cry1Ca1/cry1Da1 mRNA ratio, 4:2:1), the
half-lives of the cry1Da1 and cry1Ab3 mRNAs were found to be similar, indicating that there were differences in the
transcription rates. The relative contents of these -endotoxins in
purified inclusions from B. thuringiensis
subsp. aizawai have been measured previously, and an even
greater relative deficiency of the Cry1Da1 protoxin (ratio, 20:12:1)
was found. In order to account for this deficiency, other steps which
could be involved in inclusion formation, such as translation and
packaging, were examined. The three cry genes have the same
dual overlapping promoters, but the ribosome binding sequence
for the cry1Da1 gene was not the consensus sequence.
Translation was enhanced about fourfold by changing to the consensus
sequence. In addition, the relative amount of Cry1Da1 protoxin
in inclusions was twofold lower when cells were
sporulated in Luria-Bertani (LB) medium than when cells were
sporulated in a glucose-yeast extract medium. This difference was
attributable to packaging since the relative amounts of Cry1Da1 antigen in cells sporulating in the two media were the same. Some factor(s) required for packaging of the Cry1Da1 protoxin in inclusions is apparently limiting in LB medium. Differences in the initial transcription rates, translation efficiencies, and packaging all contribute to the -endotoxin composition of an inclusion.
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INTRODUCTION |
A large number of Bacillus
thuringiensis subspecies contain several
plasmid-encoded -endotoxin genes (cry genes) that lead to
production of intracellular inclusions, each of which is comprised of
related -endotoxins (2, 24). These proteins are
synthesized as protoxins, and the members of a major class, designated
Cry1 (molecular mass, about 130 kDa), contain 15 to 17 cysteine
residues in their carboxyl halves (18). Apparently, all of
these cysteines form intermolecular disulfide bonds when they are
packaged in inclusions (11). For example, B. thuringiensis subsp. aizawai HD133 contains
the cry1Ca1 and cry1Da1 genes in close proximity on a 120-MDa plasmid plus the cry1Ab3 gene on a 45-MDa
plasmid (6). The protoxins encoded by these three
cry genes have very similar sequences in their carboxyl
halves, the portions of the protoxins involved in cross-linking. They
differ from each other in specific regions in their amino halves and
thus have unique but overlapping specificities for target insects
(18).
When an inclusion is ingested by a susceptible insect larva (certain
Lepidoptera in this case), it is solubilized in the alkaline, reducing
conditions of the larval midgut. Trypsin-like enzymes remove the
carboxyl halves of the protoxins, releasing ca. 60-kDa toxins which
have a well-conserved three-domain structure (24). The
toxins bind reversibly but with high affinity to receptors on cells
lining the larval midgut. This binding is rather specific and is
attributable to certain toxin-specific sequences in loops connecting
the 
sheets in domains II and III (24). Subsequently, there is an irreversible binding step involving a close association of
most of the toxin with the membrane and insertion of certain amphipathic 
-helices in domain I (7). The toxin
molecules aggregate (either before or after insertion) and form
cation-selective channels that result in osmotic lysis of the cells and
larval death (20).
It has been known for some time that both the overall toxicity and the
specificity profile of a particular B. thuringiensis subspecies for target insects vary
substantially depending on the medium used for growth and sporulation
(14). The medium-dependent differences, especially the
specificity profile differences, imply that there is regulation of the
amount of each of the protoxins produced. In support of this
possibility, differences in the steady-state levels of mRNAs for
the cry1Ab3, cry1Ca1, and cry1Da1 genes in B. thuringiensis subsp. aizawai
HD133 were determined by measuring each level relative to the gene
content (4). While the gene content did differ, probably
due to differences in plasmid copy number, the steady-state mRNA
levels relative to the gene content were about 1.00:0.50:0.25.
In a subsequent study, the -endotoxin contents of purified
inclusions from this organism were compared by quantifying unique peptides produced by trypsin digestion of the proteins solubilized from
purified inclusions (22). A ratio of 1.00:0.60:0.05 was obtained, which was substantially different from the steady-state mRNA ratio, especially in terms of the relatively low recovery of
the Cry1Da1 protoxin. One difference in these experiments, aside from
the fact that -endotoxin-specific peptides were measured instead of steady-state mRNAs, was the media used for growth
and sporulation.
In order to resolve the apparent discrepancy, the relative
contributions of transcription, translation, and packaging to the -endotoxin content of an inclusion were determined. Differences in
the initial transcription rates of the genes were established by
measuring mRNA half-lives. A lower rate of translation of the cry1Da1 mRNA due to a suboptimal ribosome binding
sequence was also found. In addition, a medium-dependent difference in
packaging of the Cry1Da1 -endotoxin in inclusions contributed
to the relatively low level of this protoxin. The latter finding was
unexpected and indicates that a cellular component(s) involved in
packaging can be limiting, which influences the protoxin content of an
inclusion and thus the toxicity profile of a B. thuringiensis subspecies.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
B.
thuringiensis subsp. aizawai HD133 and HD68
were obtained from the H. Dulmage culture collection maintained by L. Nakamura at the USDA Northern Regional Research Center, Peoria, Ill.
Strain #5 is a derivative of HD133 spontaneously cured of the 45-MDa plasmid containing the cry1Ab3 gene (6). Strain
80-21 is a derivative of B. thuringiensis
subsp. kurstaki HD1 spontaneously cured of a 44-MDa plasmid
containing the cry1Ab3 gene (3). These strains
were transformed by electroporation with various lacZ fusion
plasmids as described below. Cells were grown and sporulated either in
Luria-Bertani (LB) medium or in a glucose-yeast extract medium (G-tris
medium) (5). Liquid cultures comprising 10 to 15% of the
flask volume were grown at 30°C in a New Brunswick shaker at 250 rpm.
Escherichia coli DH5, which was used for plasmid
construction, was grown at 37°C in LB medium containing ampicillin
(50 µg/ml) when necessary.
Plasmid construction.
A 940-bp oligonucleotide from the
upstream region of the cry1Da1 gene extending through the
first five codons was synthesized by PCR by using a plasmid containing
a 1.6-kb fragment of the cry1Da1 gene as the template
(21, 23). The primers were
5'-GCTATGATCTAGATTACGAATTCGAGCTCG-3' from the T7
primer region and the multiple cloning site of pUC18 contained in
vector pHT3101 (21) and
5'-CATTGGTCTAGATTATTTATTTCCATAAACTATCCCCTA-3' starting in the cry1Da1 coding region and extending
through the ribosome binding site (boldface type indicates
XbaI sites). The PCR conditions used have been described
previously (25). Both primers contained XbaI
sites, so the PCR product was digested with XbaI and ligated
into pUC18. The orientation was established by exploiting a
BglII site in the cloned fragment. The cry1Da1 fragment was then excised with HindIII and BamHI
for ligation into a double-digested B. thuringiensis-E. coli shuttle vector, pHT304-18Z
(1), to form a transcriptional fusion with lacZ (15). The resulting 10.7-kb plasmid contained both the
cry1Da1 and Bacillus subtilis spoVG ribosome
binding sites separated by 31 bp. The latter site was not optimal for
translation in B. subtilis (9). In
addition, changing the cry1Da1 ribosome binding sequence from AAGGGGAT (Fig. 1) to an
optimal B. subtilis sequence, AAGGAGGT (9), by PCR performed with the second
oligonucleotide described above but with the opitmal ribosome binding
sequence resulted in enhanced expression of -galactosidase
(see Results). It is likely, therefore, that this ribosome binding site
rather than the spoVG site was used for translation in the
lacZ fusions.
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FIG. 1.
Sequence of the cry1Da1 upstream region,
including the overlapping promoters and the initiation of translation.
The upstream oligonucleotide used to amplify this region was
complementary to the T7 primer region and extended into the multiple
cloning site of pUC18 which was present in the cloned
cry1Da1 fragment used for PCR (see Materials and Methods).
The downstream oligonucleotide extended from within the
cry1Da1 coding region through the ribosome binding site. The
ribosome binding sequence is overlined and was changed to AAGGAGGT
by modifying the oligonucleotide primer as described in Materials
and Methods. The positions of the overlapping promoter regions ( 10
and 35) for the  E and K forms of RNA
polymerase are indicated. The GenBank accession number of the sequence
is AF337948.
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|
The plasmids were isolated from transformed
E. coli DH5
,
and constructs were confirmed by restriction enzyme digestion profiles
and by sequencing. Plasmids were introduced into various
B. thuringiensis strains by electroporation, with plating
on G-tris medium containing
erythromycin (25 µg/ml) for the
pHT304-18Z constructs. Each resistant
colony was then streaked onto the
same medium onto which 0.1 ml
of a 10% (wt/vol) solution of
methylumbelliferyl-
-
D-galactoside
(Sigma) in 50%
dimethyl formamide had been spread to confirm expression
of the
lacZ gene (
12). The transformed cells were
grown in G-tris
medium containing the appropriate antibiotic at 30°C.
Duplicate
samples were removed at hourly intervals starting at the end
of
growth and were frozen at
70°C. Assays for
-galactosidase
activity
were performed as previously described (
12), and
the results
are reported below in Miller units per unit of optical
density
at 600 nm (OD
600).
Immunoblotting.
Twenty milliliters of cells was harvested at
two stages of sporulation, when about 30% of the cells
contained phase-dull endospores and when about 70 to 80% of the cells
contained phase-white to bright endospores. The values were somewhat
lower for LB medium, in which the sporulation efficiency was only 60 to
70%, compared to >95% for G-tris medium. The cells were harvested by
centrifugation at 10,000 × g in a Sorvall SS4 rotor,
washed, and lysed as previously described (12). The amount
of cell extract protein loaded onto sodium dodecyl
sulfate-polyacrylamide gels was adjusted to compensate for the
difference in sporulation frequency.
The remainder of each culture was incubated for a total of 40 h
until primarily free spores and inclusions were present. These
spores
and inclusions were harvested and washed, and the inclusions
were
purified with Renografin-76 (66% diatrizoate meglumine-10%
diatrizoate sodium) (Solvay) gradients as previously described
(
6). Inclusion proteins were solubilized by incubating
suspensions
in 30 to 50 µl of 0.03 M
Na
2CO
3-1%
-mercaptoethanol (pH 9.8)
for 20 min at 37°C. Following centrifugation for 5 min in an Eppendorf
microcentrifuge, the pellets were reextracted with 0.5 volume
of
the same buffer, and the supernatants were
pooled.
The inclusion and cell extracts were dialyzed for 20 h at 4°C
versus 2,000 volumes of 0.03 M NaHCO
3. The dialyzed
preparations
were then treated with tosylsulfonyl phenylalanyl
chloromethyl
ketone (TPCK)-trypsin (Sigma) at a ratio of 1:50
for 90 min at
37°C; then the same amount of trypsin was added and the
preparations
were incubated for an additional 90 min. The
specificity of each
of the Cry antibodies was for the toxin rather than
for the protoxin,
and thus the trypsin treatment prior to
electrophoresis was necessary.
The preparations were then dialyzed as
described above by using
50-cut dialysis tubing (Spectrum). Aliquots
were removed for protein
determinations (BCA reagent; Pierce Chemical
Co.), and the remaining
portions of the preparations were frozen at
70°C.
Portions of each preparation (25 to 50 µg of extract protein and 5 to
10 µg of inclusion protein) were fractionated on sodium
dodecyl
sulfate-10% polyacrylamide gel electrophoresis gels and
transferred
to polyvinylidene difluoride membranes (Immobilon
P; Millipore) in a
Hoffer semidry apparatus. The additional treatments
used have been
described previously (
12). The developed blots
were
scanned, and major bands were quantified with NIH Image software.
Experiments were repeated with the same extracts (sometimes the
protein
concentrations were varied) at least two additional times
with
different cell and inclusion preparations. The average densities
of
defined stained areas from two experiments were determined
(the
standard deviations were ±10%), and the relative values for
the three
toxin antigens were
determined.
A monoclonal antibody specific for the Cry1Ab3 toxin (
23)
and a polyclonal rabbit antibody specific for the Cry1Ca1 toxin
have
been described previously (
4). An antibody reacting with
both the Cry1Ca1 and Cry1Da1
-endotoxins was kindly provided
by D. Dean. An affinity column was prepared by coupling purified
Cry1Ca1
toxin (
12) to React-Gel (1,1'carbonyldiimidazole
cross-linked
to 6% agarose; Pierce) as described previously
(
17). The effluent
from the mixed antibody was collected
and passed through a second
affinity column. As discussed below, this
second effluent did
not react with the Cry1Ca1 toxin but did react with
a mixture
of Cry1Ca1 and Cry1Da1 toxins (see Fig.
3).
Determination of mRNA stability.
Cells (300 ml) were
grown at 30°C in G-tris medium in 2-liter flasks. At the two stages
of sporulation described above, 30-ml portions of cells were
transferred to tubes containing 20 ml of frozen, crushed G-tris medium.
The cells were harvested immediately by centrifugation in a Sorvall
SS34 rotor at 12,000 × g for 10 min. The pellets were
washed once with 15 ml of 0.05 M sodium acetate-0.1 M NaCl-0.001 M
EDTA (pH 5.5). The cells were suspended in 1 ml of this buffer plus 0.2 ml of a 30% bentonite suspension in deionized water and lysed by
passage through a French press at 9,000 lb/in2. The lysed
cells were collected directly in an equal volume of phenol-chloroform
(24:1). RNA was purified as described previously (8). A
solution of rifampin (10 mg per ml in 50% ethanol) was added to the
remaining culture to obtain a final concentration of 100 µg/ml.
Thirty-milliliter samples were removed after 10, 20, and 30 min and
used for RNA extraction as described above. The cry1Ab3 and
cry1Da1 mRNAs in each sample were quantified following electrophoresis, transfer, and hybridization with gene-specific 32P-labeled oligonucleotides (4). The
hybridizing bands on X-ray film were quantified with a phosphorimager,
and decay curves were plotted with Kaleidagraph.
| |
RESULTS |
mRNA stability.
Differences in the steady-state amounts of
the three cry mRNAs reported previously (4)
could have been due to differences in rates of transcription and/or
turnover. The stabilities of the cry1Ab3 and
cry1Da1 mRNAs in B. thuringiensis subsp. aizawai HD133 were
measured (Fig. 2). The half-lives
were 14 to 18 min, and there were no significant differences. This
range is comparable to that reported for cry1Aa mRNA
(28). Therefore, the smaller amount of steady-state
Cry1Da1 mRNA cannot be attributed to a less stable mRNA and
must be due to differences in the initial transcription rates.
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FIG. 2.
(A) Autoradiograms of Northern gels of RNAs from
sporulating cells sampled after the addition of rifampin at the times
indicated (see Materials and Methods). Hybridization was to a
32P-labeled cry1Da1 (Cry1D) or
32P-labeled cry1Ab3 (Cry1Ab) probe. (B)
X-ray film was quantified with a phosphorimager, and the
percentages of the initial values were plotted on a log scale versus
time in order to calculate mRNA half-lives of cry1Ab3
mRNA ( ) and cry1Da1 mRNA ( ). Standard errors
are indicated by error bars.
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Alteration of the ribosome binding sequence improved
expression.
The two- or fourfold differences in transcription were
not as great as the differences in the relative amounts of the two protoxins in inclusions produced in LB medium (ratio, 1:20)
(22). Control at the level of translation was a
possibility since the ribosome binding sequence for the
cry1Da1 gene is AAGGGGAT (Fig. 1) rather than the
optimal B. subtilis consensus sequence, AAGGAGGT (9), which is present in the cry1Ab3 and
cry1Ac1 genes. When the cry1Da1 sequence was
changed to the latter sequence in a cry1Da1-lacZ fusion
plasmid, the -galactosidase specific activity increased from 300 to
1,400 Miller units per OD600 unit in strain 80-21 and from
450 to 1,800 Miller units per OD600 unit in strain #5.
Relative accumulation of the protoxins in sporulating cells and
inclusions.
Antibodies specific to each of the three toxins were
prepared either in rabbits (Cry1Ca1 and Cry1Da1) or as a monoclonal
antibody (Cry1Ab3). The specificity of each antibody is shown in
Fig. 3. Since purified Cry1Da1 toxin was
not available, the specificity of the absorbed Cry1Da1 antibody (see
Materials and Methods) was determined by its reaction with toxins from
strain #5 inclusions. This strain contains only the cry1Ca1
and cry1Da1 genes and thus reacts with the Cry1Ca1 and
Cry1Da1 antibodies but not with the Cry1Ab3 antibody. The Cry1Da1
antibody did not react with purified Cry1Ca1 toxin (Fig. 3) or with the
Cry1Ab3 toxin (data not shown). In a given immunoblot, the
reactions with all three antibodies increased as the amounts of the
toxins increased.
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FIG. 3.
Immunoblots obtained with 200 ng of purified Cry1Ab3
toxin (lanes 6, 9, and 10), 500 ng of purified Cry1Ca1 toxin (lanes 1, 4, 5, and 8), and a mixture of the Cry1Ca1 and Cry1Da1 toxins (500 ng)
from purified inclusions from strain #5 (lanes 2, 3, and 7). Lanes 1 and 2 were treated with Cry1Da antibody, lanes 3 to 6 were
treated with Cry1Ca1 antibody, and lanes 7 to 10 were treated with
Cry1Ab3 antibody.
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|
The relative amounts of the toxins in extracts of sporulating
B. thuringiensis subsp.
aizawai
HD133 and in purified inclusions
produced in LB or G-tris medium were
determined (Fig.
4 and
5).
The ratio of the three antigens in extracts of
sporulating cells
in both LB medium and G-tris medium (Fig.
4) was
1.0:2.1:0.8.
These values are averages based on two separate
experiments, and
the standard deviations were ±10%. Samples were also
taken when
more than 70% of the cells contained phase-white
endospores, and
similar results were obtained. There did not appear to
be medium-dependent
translational impairment of the
cry1Da1
gene, nor is it likely
that the Cry1Da1 protoxin was less stable in
cells sporulating
in LB medium.
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FIG. 4.
Immunoblots of trypsin-treated extracts from
B. thuringiensis subsp. aizawai
HD133 grown in G-tris medium (G) (25 µg of protein) or LB medium (LB)
(30 µg of protein) until 30% of the cells in G-tris medium or ca.
25% of the cells in LB medium contained phase-gray endospores. Samples
were prepared as described in Materials and Methods. Lanes 1 and 2 were
treated with the Cry1D antibody, lanes 3 and 4 were treated with
the Cry1Ca1 antibody, and lanes 5 and 6 were treated with the Cry1Ab3
antibody. The blots were scanned and quantitated as described in
Materials and Methods.
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FIG. 5.
Immunoblots of trypsin-treated extracts from purified
inclusions prepared from B. thuringiensis
subsp. aizawai HD133 grown and sporulated in G-tris medium
(G) or LB medium (LB). Preparations were treated with the Cry1Ab3,
Cry1Ca1, and Cry1Da1 antibodies as described in Materials and Methods.
Lanes 1 to 4 were treated with Cry1Da1 antibody, lanes 5 to 8 were
treated with Cry1Ca1 antibody, and lanes 9 and 10 were treated with
Cry1Ab3 antibody. Lanes 1, 2, 5, 6, and 9 contained inclusions from
G-tris medium; lanes 1 and 2 were duplicates, as were lanes 5 and 6. Lanes 3, 4, 7, 8, and 10 contained inclusions from LB medium; lanes 3 and 4 were duplicates, as were lanes 7 and 8.
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|
Inclusions from the two media, however, did differ in terms of
the relative content of the Cry1Da1 toxin (Fig.
5). The
Cry1Da1/Cry1Ca1/Cry1Ab3
ratios for G-tris medium inclusions and LB
medium inclusions were
2.0:3.3:1.4 and 1.0:3.3:1.4, respectively. These
values are averages
based on two separate experiments, and the standard
deviations
were ±10%. There was twofold less Cry1Da1 toxin in
inclusions
from LB medium than in inclusions from G-tris
medium.
B. thuringiensis subsp.
aizawai
HD68 is the original source of the cloned
cry1Da1 gene
(
19). The relative content of this
toxin in inclusions
formed by this subspecies in LB medium was
also twofold less than the
relative content in inclusions formed
in G-tris medium (R. Grant,
unpublished
data).
| |
DISCUSSION |
The results presented here account for the relatively low content
of Cry1Da1 -endotoxin in inclusions. In a previous study (4), the steady-state ratio of the cry1D,
cry1C, and cry1Ab mRNAs was reported to be 1:2:4.
The ratios were about the same for cells that sporulated in G-tris
medium and cells that sporulated in LB medium. These steady-state
values are attributable to differences in the rates of transcription
since the half-lives of the cry1Ab3 and cry1Da1
mRNAs were the same (14 to 18 min) (Fig. 2). The half-life of
cry1Aa mRNA was reported to be about 12 min
(28), which is much greater than the half-lives of most
mRNAs in vegetative cells (2 to 3 min). The stability of these
cry mRNAs is in part due to a large stem-loop structure
at the 3' end (28).
The cry1D, cry1C, and cry1Ab -endotoxin genes
in B. thuringiensis subsp.
aizawai HD133 have very similar dual overlapping promoters
(10), and yet the steady-state amounts of the three mRNAs differ. The differences may be due to sequences upstream of
the promoters which differ for ca. 1 kb in the three genes. There is
some evidence that this region is involved in transcriptional regulation (27).
A second factor contributing to the relatively small amount of the
Cry1Da1 protoxin is the fact that the ribosome binding sequence differs
from the B. subtilis consensus sequence
(9), which results in fourfold-lower translation of a
cry1Da1-lacZ fusion. Transcription and translation thus
account for ca. 10- to 15-fold less Cry1Da1 than Cry1Ab3 toxin. A third
factor which augments this difference in inclusions is a
medium-dependent limitation in packaging. Overall, a 20- to 30-fold
difference in the amounts of these two protoxins in inclusions formed
in LB medium would be expected, which is very similar to the ratio of
these protoxins in purified inclusions (22).
Many B. thuringiensis subspecies produce a
mixture of related Cry1 protoxins which are packaged together in a
crystalline inclusion. The sequences of the carboxyl halves of these
protoxins are well conserved and include 15 to 19 cysteines.
Apparently, all of these cysteines participate in intermolecular
disulfide bond formation (11). Such interactions among
-endotoxins to form a regular crystalline array probably require
other factors. For example, there is a stabilizing protein, P20, which
enhances accumulation of a cytolytic toxin produced by
B. thuringiensis subsp.
israelensis (26, 29). There may be a
functionally similar protein encoded by an open reading frame in the
operon which also contains a cry2 -endotoxin gene
(13). While these examples suggest some specificity for
-endotoxins, packaging or stabilizing factors which are probably not
specific for the -endotoxins may also have functions. Crystalline
inclusions are produced by Bacillus cereus transformants
containing clones of only a cry gene (8), implying that plasmid-encoded packaging functions are not essential.
Other cellular components involved in such packaging are not
known but could include one or more enzymes involved in disulfide interchange reactions, such as protein disulfide isomerase
(16) and perhaps some of the chaperone(s). The
smaller amount of the Cry1Da1 -endotoxin in inclusions
formed in LB medium than in inclusions formed in G-tris medium
indicates that there is a limitation in one or more of these packaging
factors in LB medium. There also appeared to be a limitation when the
Cry1Ac1 -endotoxin was synthesized at a very high and
apparently excessive rate due to promoter-up mutations
(25). The excess protoxin was rapidly turned over, which
resulted in smaller inclusions. There appears to be a close coupling
between -endotoxin synthesis and packaging in inclusions. The
Cry1Da1 -endotoxin which is produced in relatively small amounts may
not bind efficiently to one of the limiting factors; i.e., there may be
some specificity of the packaging factors for the various
-endotoxins.
However, it is puzzling that the Cry1Da1 -endotoxin is produced at
all given the limited capacity of sporulating cells to synthesize
-endotoxins (3). In addition to its relatively small
quantity, it is not very active against the array of insects that have
been tested, nor is its specificity profile unique (18). It could function synergistically with other toxins and thus may be
required only in small amounts. This would allow the presence in
inclusions of larger amounts of the more active Cry1Ab3 and Cry1Ca1 protoxins.
| |
ACKNOWLEDGMENTS |
Lan Wu assisted in some of the experiments. We thank D. Dean,
Ohio State University, for providing the Cry1Ca1/Cry1Da1 antibody.
Lily Chang was a visiting scientist supported by the Ministry of
Agriculture, People's Republic of China.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, Lilly Hall, Purdue University, West Lafayette, IN 47907. Phone: (765) 494-4992. Fax: (765) 494-0876. E-mail:
aaronson{at}bilbo.bio.purdue.edu.
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Applied and Environmental Microbiology, November 2001, p. 5032-5036, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5032-5036.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
