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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1993, p. 815-821
0099-2240/93/030815-07$02.00/0 Copyright © 1993, American Society
for Microbiology
High-Level cryIVD and cytA Gene Expression in Bacillus
thuringiensis Does Not Require the 20-Kilodalton Protein, and the
Coexpressed Gene Products Are
Synergistic in Their Toxicity to Mosquitoes CHENG CHANG,1 YONG-MAN
yU,2 SHU-MEI DAI,2 SARA K. LAW,2
AND SARJEET S. GILL1' 2*
Department of Entomology2 and Interdepartmental Graduate Program in
Environmental Toxicology, 1 University of California, Riverside,
California 92521
Received 31 August 1992/Accepted 8 December 1992
Interactions among the 20-kDa protein gene and the cyt4 and cryIVD
genes located in a 9.4-kb HindIll fragment were studied. A series
of plasmids containing a combination of these different genes was
constructed by using the Escherichia colil/Bacilus thuringiensis
shuttle vector pHT3101. The plasmids were then used to transform an
acrystalliferous strain, cryB, derived from B. thuringiensis subsp.
kurstaki. The results from sodium dodecyl sulfate-polyacrylamide
gel electrophoresis and immunoblot analyses suggest that although
the 20-kDa protein is required for the efficient CytA protein
production in E. coli, it is not required in B. thuringiensis. With
or without the truncated 20-kDa protein gene, the CytA and/or
Cry1VD proteins are
produced and form parasporal inclusions in B. thuringiensis cells.
However, more-efficient expression is obtained when a second
protein, probably acting as a chaperonin, is present. In addition,
the time course
studies show that the CytA and CrylVD proteins are coordinately
produced. Both the crude B. thuringiensis culture and purified
inclusions from each recombinant B. thuringiensis strain are toxic
to Culex quinquefas- ciatus larvae. The parasporal inclusions
formed in B. thuringiensis cells are mosquitocidal, with CytA
synergizing CryIVD toxicity.
Bacillus thuringiensis subsp. israelensis and B. thurin- giensis
subsp. morrisoni (PG-14) both produce spherical parasporal
inclusions that are highly toxic to dipteran larvae, such as
mosquitoes and black flies (18, 27). The parasporal inclusions from
both subspecies produce 27-, 72-, 125-, and 135-kDa polypeptides,
the CytA, CryIVD, CryIVB, and CryIVA proteins, respectively (12-15,
19). The B. thurin- giensis subsp. morrisoni parasporal inclusions
also contain a
144-kDa protein not found in B. thuringiensis subsp. israe- lensis
(26). The genes encoding the CytA, CryIVA, CryIVB, and CryIVD
proteins have been cloned and expressed in both Escherichia coli
and B. thuringiensis (1, 4, 8, 9, 13, 24, 33, 35). All proteins
common to both subspecies are mos-
quitocidal, with the Cry proteins having the greatest insec-
ticidal activity (2, 4, 8, 10, 33). The CytA protein, in addition
to its mosquitocidal activity (34), is also hemolytic and cytolytic
(17, 31). The insecticidal activity of B. thuringiensis subsp.
israe-
lensis is a complex interaction of the four inclusion body
proteins, CryIVA, CryIVB, CryIVD, and CytA (15). Among these
proteins, CytA exclusion from inclusion bodies has relatively
little effect on insecticidal activity (9). Although each protein
is mosquitocidal, none is as active as the intact parasporal
inclusion. The higher insecticidal activity of the intact
parasporal inclusions is primarily due to the synergis- tic
interaction between the proteins present in these inclu- sions (2,
5, 8, 20, 38, 39).
In addition to the synergistic interaction between CytA and CryIVD,
these proteins are encoded by the same
HindIII fragment which also encodes a 20-kDa protein. In
* Corresponding author.
E. coli, CytA levels are increased because of posttransla- tional
stabilization by this 20-kDa protein (1, 32). It is not known,
however, whether this 20-kDa protein plays any role in CytA or
CryIVD levels in B. thuingiensis. To investigate the role of the
20-kDa protein, a recombinant system in B. thuringiensis (4) was
used. We report here that the 20-kDa protein is not required for
the production of high levels of either CytA or CryIVD in B.
thurngiensis. However, the presence of an additional protein
increases the CytA and CryIVD levels in an acrystalliferous strain
of B. thuringien- sis. Mosquitocidal activity evaluations also
demonstrate that CytA synergizes the insecticidal activity of
CryIVD to Culex quinquefasciatus.
MATERIALS AND METHODS
Bacterial strains, plasmids, and general methods. Bacterial strains
used in this study were E. coli JM101, JM109, and XL-1 (Stratagene,
La Jolla, Calif.), BMH 71-18 mutS (Promega, Madison, Wis.), and the
acrystalliferous strain cryB, derived from B. thuringiensis subsp.
kurstaki HD1, obtained from A. Aronson, Department of Biology,
Purdue University. The plasmid pMl, which encodes the B. thur-
ingiensis subsp. morrisoni (PG-14) CytA, CryIVD, and 20-kDa
proteins, was obtained from B. Federici and S. Sivasubramanian of
our department (14). The E. coliIB. thuringiensis shuttle vector
pHT3101 (22) was obtained from D. Lereclus, Institut Pasteur,
Paris, France. Site-directed mutagenesis was performed with
pSELECT-1 by using the Altered Sites in vitro mutagenesis system
(Promega) follow- ing the manufacturer's instructions while all
other cloning was performed with pBluescriptIl SK+ (Stratagene).
Stan- dard protocols were used for restriction enzyme
digestion,
815
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ligation, and E. coli transformation (29). B. thuringiensis cryB
cells were transformed by electroporation essentially as described
previously (22).
Construction of the pCG plasmid series. The construction of pCG1
has been described previously (4). For pCG2 construction, pM1 was
restricted with EcoRI. The 5.7-kb fragment, which contains cytA and
a truncated cryIVD gene, was separated by agarose gel
electrophoresis and purified by Geneclean (Bio 101, La Jolla,
Calif.). The purified fragment was ligated into the unique EcoRI
site of pHT3101 and then used for transformation in E. coli JM101.
Transformants, which were selected on Luria-Bertani agar containing
ampi- cillin (50 ,ug/ml), 20 mM isopropyl-p-D-thiogalactopyrano-
side (IPTG), and 80 mg of 5-bromo-4-chloro-3-indolyl-p3-D-
galactopyranoside (X-Gal) per ml were isolated, and the sizes and
orientations of the EcoRI inserts were determined. To construct
pCG4, the cryIVD EcoRI site was deleted by
site-directed mutagenesis with the Altered Sites in vitro
mutagenesis system. Briefly, the 5.3-kb BamHI-PstI frag- ment from
pCG1 which contains the cryIVD gene was inserted into the
BamHI-PstI sites of pSELECT-1. The cryIVD internal EcoRI site was
then deleted by primer- directed mutagenesis. The mutant,
identified by an EcoRI site deletion, was then restricted with
EcoRI. The 4.4-kb EcoRI fragment which contains cryIVID, a
truncated cytA gene, and a truncated 20-kDa protein gene was then
inserted into the unique EcoRI site of pHT3101, resulting in pCG4.
For pCG5 construction, the 4.9-kb pMl BamHI-ClaI frag- ment was
subcloned into pBluescriptII SK+. The pCG2 2.0-kb SacI-BamHI
fragment was then inserted into SacI- BamHI sites, giving pCGB12.
The 6.9-kb pCGB12 KpnI- SacI fragment which contains the cryIID,
cytA, and 20-kDa protein genes was then inserted into the shuttle
vector pHT3101, resulting in the plasmid pCG5. The 4.4-kb pM1
EcoRV-PstI fragment was isolated and
ligated with a SacI linker, and the resulting fragment was inserted
into the unique SacI-PstI sites of pHT3101, result- ing in the
construction of pCG6. To obtain pCG8, the 6.9-kb SacI-Clal fragment
from pCG5 was ligated with the 7.2-kb SacI-ClaI fragment of pCG1.
To construct pCG10, the 4.9-kb pCG6 SpeI-BamHI fragment, which
lacks the B. thuringiensis origin of replication but retains the E.
coli replication origin, was isolated. This fragment was then
ligated with the 1.2-kb SpeI-BamHI fragment of pCGE1, which
contains the 3' cryIVD fragment but lacks the 20-kDa protein gene,
resulting in the formation of pCG9; the BamHI site in pCGE1 was
introduced by the polymerase chain reaction. For expression in B.
thunngiensis, the 3.6-kb pHT3101 BamHI fragment containing the B.
thunngiensis replication origin was inserted into the unique pCG9
BamHI site; this new construct, pCG10, contains only the cryIVD
gene. The plasmid pCG12 was constructed by replacing the
6.9-kb SacI-ClaI fragment of pCG5, which contains all three genes,
with the 4.0-kb SacI-ClaI fragment of pCG6, which contains cryIVD
and the gene encoding the 20-kDa protein. For construction of
pCG13, the 2.9-kb SacI-EcoRV frag- ment of pCG5, which contains
cytA, was isolated from the agarose gel, and XbaI 10-mer linkers
were used to modify the blunt end. After restriction, the SacI-XbaI
fragment was cloned into the corresponding restriction sites of
pHT3101. For construction of pCG17, the 0.93-kb pCGB12
HaeIII-
ClaI fragment, containing the 20-kDa protein gene and a 32-bp 3'
end of cryIVD, was used to replace the 3.98-kb fragment containing
the 20-kDa protein gene and cryIVD. The resulting construct was
then cloned into pHT3101 to
obtain pCG13, which contains cytA, 32 bp of cryIVD, and the 20-kDa
protein gene.
cyt4 and cryIVD gene expression. B. thunngiensis subsp. kurstaki
recombinant CG strains containing the pCG plas- mids were analyzed
for the CytA and CryIVD proteins. These B. thuningiensis subsp.
kurstaki transformants were grown on nutrient agar plates
containing 50 p,g of erythro- mycin per ml for 3 days at 30°C. The
bacterial cultures were isolated by washing the culture plates with
deionized water. Crude culture aliquots were boiled in sample
treatment buffer and analyzed by discontinuous sodium dodecyl
sulfate polyacrylamide gel electrophoresis, (SDS-PAGE) with 4.5%
acrylamide (pH 6.8) and 10% acrylamide (pH 8.8) as the stacking and
separating gels, respectively (21). The gels were then stained with
0.1% Coomassie blue R-250. Alternatively, the proteins resolved by
SDS-PAGE were transferred to nitrocellulose for 16 h at a constant
current of 250 mA, and the nitrocellulose was then probed with
rabbit antibody developed against either the purified whole
parasporal inclu- sion or the purified 72-kDa toxin of B.
thuringiensis subsp. israelensis by using methods described
previously (16). Goat anti-rabbit immunoglobulin G-alkaline
phosphatase was used as the second antibody, and chromogenic
development was then achieved with nitroblue tetrazolium chloride
(1 mg/ml in H20) and 5-bromo-4-chloro-3-indolyl phosphate (5 mg/ml
in dimethylformamide). For time course studies, cultures were
terminated at 12,
24, 36, 48, and 72 h. The plate bacterial cultures were isolated
with 10 mM EDTA and sedimented by centrifuga- tion at 15,000 x g
for 10 min. After the protein concentration was estimated (23), the
cultures were analyzed by discontin- uous SDS-PAGE and
immunoblotting as described above.
Purification of parasporal inclusions. B. thunngiensis subsp.
kurstaki CG strains were cultured on nutrient agar plates
containing 50 ,ug of erythromycin per ml for 5 days at 30°C to
ensure sporulation and complete autolysis. The spore-parasporal
inclusion mixture was thoroughly washed with 1 M NaCl-10 mM EDTA
and sedimented by centrifu- gation at 15,000 x g for 10 min. The
pellet was resuspended in water, sonicated, loaded onto a
continuous 40 to 70% Renografin density gradient, and centrifuged
at 15,000 rpm for 30 min in an SW28 rotor as described previously
(40). The parasporal inclusions were then subjected to a second
centrifugation. The purified parasporal inclusions were washed
three times with distilled water, the protein concen- tration was
measured (23), and the inclusions were stored at 40C.
Insect bioassays. The larval mosquitocidal activity of each B.
thuringiensis CG strain was determined. For determina- tion of
crude bacterial culture toxicity, the cultures were serially
diluted and added to distilled water (total volume, 10 ml)
containing 10 fourth-instar C. quinquefasciatus larvae. The 24-h
mortality was determined by counting the number of surviving
larvae. Bioassays were performed in triplicate. The mosquitocidal
activity of purified inclusions from the
B. thuringiensis subsp. kurstaki CG series was also assessed.
Briefly, 0.1 ml of parasporal inclusion dilutions was added to 99.9
ml of distilled water containing 20 fourth-instar C.
quinquefasciatus larvae. To determine a 50% lethal concen- tration
(LC50), 10 different inclusion concentrations (1 to 1,000 ng/ml)
were used, with at least four replicates per concentration. The
24-h mortality was determined, and LC50 and 95% lethal
concentration (LC95) values were calculated by probit analyses
(28). Controls for the bioassays utilized mosquito larvae reared
similarly but not exposed to either
816 CHANG ET AL.
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cytA ciyIVD
E
FIG. 1. Construction of the pCG plasmid series and determination of
cryIVD and cytA gene expression in B. thuningiensis (Bt) and the
mosquitocidal activity of the gene products obtained. B, BamHI; C,
ClaI; E, EcoRI; EV, EcoRV; H, HindIII; P, PstI; Sa, SacI; X, XbaI.
+ + +, + +, and + indicate that 100, >50, or <50% mortality,
respectively, was observed in the mosquito larva bioassay with
crude bacterial cultures.
the crude bacterial cultures or the purified parasporal inclu-
sions.
RESULTS
To determine the interactions between the CytA, CryIVD, and the
20-kDa proteins or their genes, several DNA frag- ments derived
from the 9.4-kb HindIII fragment ofpMl were introduced into the
shuttle vector pHT3101 for expression in B. thuringiensis (Fig. 1).
The CytA and CryIVD protein levels were detected by SDS-PAGE
analysis (Fig. 2). Immu- noblot analysis confirmed the identity of
the CytA and CryIVD proteins (data not shown). In all experiments,
the 20-kDa protein was not observed in the acrystalliferous strain
cryB.
Role of the 20-kDa protein in cyt4 gene expression. The 20-kDa
protein is reportedly required for efficient CytA production in E.
coli (1, 32). The plasmids pCG2 and pCG13 were therefore
constructed to determine whether the 20-kDa protein is required for
cytA gene expression in the strain cryB derived from B.
thunngiensis subsp. kurstaki. Our results show that CG2 and CG13,
cryB strains transformed with pCG2 and pCG13, respectively,
expressed significant amounts of the 27-kDa CytA protein at levels
readily detect- able by SDS-PAGE (Fig. 2). High CytA levels similar
to those in CG2 were also observed in CG17 (data not shown).
However, the CytA protein in CG13 is synthesized in lower
kDa
72
27
1 2 3 4 58a 7 9 10 81112131415161718
FIG. 2. SDS-PAGE analysis of cryIVD and cytA gene expression in B.
thuningiensis subsp. kurstaki (cryB). Lanes 1 to 10 were
loaded
with 10 pLg of total protein from each B. thuningiensis crude
culture
(3 days at 30'C). Lanes 11 to 18 were loaded with 5 p.g of purified
inclusions from each indicated B. thuringiensis strain. Lanes: 1
and
11, CG1; 2 and 12, CG2; 3 and 13, CG4; 4 and 14, CG5; 5 and
15,
CG6; 6 and 16, CG8; 7 and 17, CG10; 8 and 18, CG12; 9, CG13;
10,
cryB; S, Standard proteins. Molecular size markers are indicated
on
the right.
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APPL. ENVIRON. MICROBIOL.
amounts than in strains CG2 and CG17, and in addition, the CG13
CytA protein is proteolytically cleaved to a 24-kDa protein. Thus,
the 27-kDa protein is less stable in CG13 than in CG2 and CG17.
After 5 days of culture on nutrient agar plates at 30°C, strain CG2
formed large ovoid parasporal inclusions during sporulation. These
ovoid inclusions were readily observed under phase-contrast
microscopy and in quantities sufficient to form a distinct band
when they were purified by Renografin gradients.
Since the EcoRI fragment used to construct the pCG2 contains a
portion of the cryIVD gene and its promoter, immunoblot analyses
were performed to detect the presence of the CryIVD (72-kDa)
protein by using antibodies raised against the intact B.
thuringiensis subsp. israelensis paras- poral inclusions. The
truncated cryIVD gene in this con- struct does not have a
translation stop codon, and the cryIVD and P-galactosidase genes
are in frame; therefore, the fusion protein formed includes part of
the ,B-galactosi- dase protein. A 74-kDa CryIVD-13-galactosidase
fusion pro- tein containing a C-terminal deletion of 77 amino acids
from the CryIVD protein but containing 98 amino acid residues from
the P-galactosidase was produced and confirmed by immunoblot
analysis (data not shown). Although strain CG13 can form
inclusions, spore formation in this strain is aberrant and the
bacterial cells do not autolyze after 6 days of culture at 30°C.
The bacterial strain CG13 produces the 27-kDa protein and a 24-kDa
proteolytic product (Fig. 2). However, because of the lack of
autolysis in the CG13 strain, parasporal inclusions are difficult
to purify from this strain. Consequently, strain CG17 was
constructed and produced CytA levels comparable to the CytA levels
in CG2. Our observations show that the 20-kDa gene product is not
required for efficient CytA protein production in B. thurin-
giensis, in contrast to that observed in E. coli.
B. thuringiensis CryIYD production in the absence of the 20-kDa
protein. We previously obtained high cryIVD gene expression in B.
thuringiensis by using the shuttle vector pHT3101 (4). However,
this construct contained, in addition to cryl'D, a truncated cytA
gene and the 20-kDa protein gene. Therefore, to determine whether
the 20-kDa protein is involved in the CryIVD protein production,
the plasmids pCG4, pCG6, and pCG1O were constructed (Fig. 1).
Plasmid pCG4 contains cryIVD, a truncated cytA gene, and a trun-
cated 20-kDa protein gene. The plasmid pCG6 has only cryIVD and a
20-kDa protein gene, while pCG10 has only cryIVD but lacks the
invert repeats (10). These plasmids were then used for
transformation of the strain cryB derived from B. thunngiensis
subsp. kurstaki. The results of SDS-PAGE (Fig. 2) and immunoblot
anal-
yses show that the cryIVD gene is expressed in all three bacterial
strains, i.e., CG4, CG6, and CG10; but bacterial strain CG1O
produces lower CryIVD levels than do strains CG4 and CG6 (Fig. 3
and 4). During SDS-PAGE and immunoblotting analyses, most of the
CryIVD products were observed in the pellet fraction following
centrifugation at 15,000 x g for 10 min, demonstrating that the
CrylVD protein is mostly in the inclusion and that only a small
portion is released into the supernatant after cell lysis. Time
course studies of CG6 and CG10 strains showed that the CryIVD
protein production can be observed within 24 h (Fig. 5, lanes 4 and
9).
IS231-like transposase role in CryIVD and CytA protein production.
An open reading frame encoding an IS231-like transposase downstream
of the 20-kDa protein gene was reported in B. thunngiensis subsp.
israelensis (1). The potential role of the transposase-like
fragment in B. thuiin-
- 7 2 1 t D E~7
S1 2 05X03L6 7i=00 FIG. 3. SDS-PAGE analysis of the 20-kDa protein
influence on
cryIVD gene expression. Each B. thuringiensis strain was incubated
at 30°C for 3 days, and the lysates were then centrifuge at 15,000
x g for 10 min. The pellets were brought to the original volume
with H20. Samples (10 p1) from either the supernatant or pellet
fractions were used for electrophoresis. Lanes 1 to 4 were loaded
with samples from the pellets, and lanes 5 to 8 were loaded samples
from the supernatant fractions. Lanes: 1 and 5, CG4; 2 and 6, CG6;
3 and 7, CG10; 4 and 8, cryB; S, standard proteins. The protein
band in lane 8 is near the dye front and has a molecular size of
<20 kDa. The 72-kDa marker is indicated on the right.
giensis subsp. mornisoni PG-14 was evaluated by using the B.
thuringiensis expression system because in some crude bacterial
cultures increased CryIVD levels were observed. Two plasmids, pCG5
and pCG8, were constructed, with the latter having the DNA encoding
the putative transposase fragment; SDS-PAGE and immunoblot analyses
showed that there was no difference in CryIVD and CytA levels in
these two constructs (Fig. 2, lanes 4 and 6). Temporal expression
of the cryIVD and cytA genes. Expres-
sion of the cryIVD and cytA genes was monitored over a 72-h period.
Low-level expression was observed within 24 h, with high levels
apparent within 48 h (Fig. 6). CryIVD and CytA protein levels did
not increase further after a 96-h incuba- tion. The time course
studies showed that CryIVD and CytA are synthesized simultaneously.
However, since each pro- tein is synthesized independently (Fig. 2
and 4), it appears that their genes are independently
regulated.
Mosquitocidal activity and inclusion shape. All B. thunn- giensis
subsp. kurstaki strains transformed with the pCG series form
parasporal inclusions during sporulation. Under light microscopy,
the inclusions formed by CG4 and CG6 were similar to those observed
for CG1 (4). However, the inclusions formed by CG2, CG8, and CG17
are large and ovoid in shape. Thus, high CytA levels result in an
inclusion formation whose shape is different from that observed in
wild-type B. thuringiensis subsp. israelensis. Both the crude
bacterial cultures and the purified inclu-
sions from these strains are toxic to C. quinquefasciatus larvae
(Fig. 1; Table 1). Parasporal inclusions obtained from CG strains
(CG1, CG4, and CG6), which produced only the CryIVD protein, all
had similar LC50 values (Table 1).
818 CHANG ET AL.
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kDa Q-7
1 234 56 788
FIG. 4. Immunoblot analysis of the 20-kDa protein influence on
cryBIVD gene expression. B. thuringiensis strains were incubated at
30'C for 3 days, and the lysates were then centrifuge at 15,000 x g
for 10 min. The pellets were brought to the original volume with
H20. Supernatants (5 p.1) and pellet fractions (2 p.l) were used.
Rabbit antiserum developed against B. thuringiensis subsp. israe-
lensis CrylVD protein was used for detection. Lanes 1 to 4 were
loaded with samples from the pellets and lanes 5-8 were loaded with
samples from the supernatant fractions. Lanes: 1 and 5, CG4; 2 and
6, CG6; 3 and 7, CG10; 4 and 8, cryB; S, standard proteins. The
prominent 30- to 34-kDa bands in lanes 5 and 6 are CryIVD
proteolytic products (5). Molecular size markers are indicated on
the right.
Strains containing both CytA and CryIVD have the highest
mosquitocidal activity, while strains containing only CytA are the
least mosquitocidal (Table 1). A densitometer scan of the proteins
produced in the CG8 strain (Fig. 2) showed that the CytA and CryIVD
proteins were produced in a ratio of 7:3. Since the CytA protein
has little mosquitocidal activity, the LC50 value that is observed
for pCG8 parasporal inclu- sions therefore results from ca. 11 ng
of the CrylVD protein per ml.
DISCUSSION
Adams et al. (1) reported that the 20-kDa protein is essential for
efficient CytA production in E. coli. No CytA is detected in the
absence of the 20-kDa protein, and CytA accumulation in E. coli is
due to its stabilization by the 20-kDa protein (32). Stabilization
apparently occurs because the CytA is protected from proteolysis in
E. coli. In the CG13 strain, which contains only cytA4, CytA
inclusions were formed. Some of the CytA protein produced in CG13
is, however,, processed to a 24-kDa peptide. Similarly, in the CG2
strain, which contains cytA and a 3' truncated cryIVD but lacks the
20-kDa protein gene,, large CytA oval inclu- sions are detected and
high CytA levels are observed. Therefore, the 20-kDa protein is not
essential for CytA protein production in B. thuringiensis. However,
the pres- ence of a second protein, either the 20-kDa protein or
CrylVD, enhances the stability of CytA in an acrystallifer- ous B.
thwingiensis subsp. kurstaki strain,, cryB. Armstrong et al. (3)
reported that the 24-kDa peptide is the
- 72 kDa
FIG. 5. Results of time course studies showing the 20-kDa pro- tein
influence on cryIVD gene expression by immunoblot analyses. Rabbit
antiserum developed against the B. thuringiensis subsp. israelensis
CryIVD protein was used for detection. Lanes: 1, 250 ng of pure B.
thuringiensis subsp. israelensis CryIVD protein; 2 to 6, cryIVD
gene expression in strain CG6 at 6, 12, 24, 48, and 72 h,
respectively; 7 to 11, cryIVD gene expression in strain CGlO at 6,
12, 24, 48, and 72 h, respectively; 12, cryB. One microgram of
total protein was loaded in each lane. S, standard proteins. The
promi- nent 30- to 34-kDa bands are CryIVD proteolytic products.
The 72-kDa marker is indicated on the right.
active form of the CytA toxin. Further, Ward et al. (34) showed
that the residues Arg-25 and Arg-30 are important for the CytA
inclusion formation in E. coli. These two residues are removed when
the 27-kDa CytA protein is proteolytically cleaved to a 24-kDa
protein (17). Conse- quently, the 24-kDa protein produced in CG13
is probably not readily packaged into the inclusion body, and
hence, the soluble 24-kDa protein could be cytotoxic to the
bacterial cell (11); thus, CytA levels are lower. However, if the
CytA protein is continuously packaged, this protein synthesis
continues, resulting in high cytA gene expression as ob- served in
CG2. Strain CG13 does not autolyze; after 6 days of incubation at
30°C, the inclusion body is still maintained in the cell. This lack
of autolysis could be due to the cleaved CytA protein cytotoxicity,
or, alternatively, the presence of either CryIVD or the 20-kDa
protein is required for autolysis because all strains that
contained either gene encoding these proteins autolyzed after
sporulation. CryIVD levels are low in the CG1O strain, which lacks
the
20-kDa gene and the inverted repeats normally present 3' to the
cryIVD coding region. In B. thuringiensis, the presence of inverted
repeats following a structural gene increases the stability of the
mRNA (10, 25, 33, 37). The high CryIVD levels in CG4, which
contains only a truncated 20-kDa gene and cryIVD, are comparable to
those observed in the wild- type bacterial strain and support the
possible role of the invert repeats. Alternatively, the absence of
an additional protein, such as the 20-kDa protein, contributes to
the low CryIVD yield in CG10. Hence, in CG4 either the 20-kDa
protein is not required for CryIVD production or the 20-kDa
truncated gene product maintains its activity even though ca. 50%
of the 20-kDa gene was deleted, although the mainte- nance of its
biological function is unlikely.
Therefore, it appears that in acrystalliferous B. thuring-
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$ 1 2 3 4 5 6 7 8 9 101112
FIG. 6. Temporal expression of the cytA and cryID genes. Lanes 1 to
6 were probed with rabbit antiserum developed against the intact B.
thuringiensis subsp. israelensis parasporal inclusion, while lanes
7 to 12 were probed with rabbit antiserum developed against the
CryIVD protein of B. thuringiensis subsp. israelensis. Lanes: 1 and
7, 24-h crude culture from strain CG6; 2 and 8, 48-h culture from
strain CG6; 3 and 9, 96-h culture from strain CG6; 4 and 10, 24-h
culture from strain CG8; 5 and 11, 48-h culture from strain CG8; 6
and 12, 96-h culture from strain CG8. In each well, 2 ,ul of crude
culture was loaded. S, standard proteins. Molecular size markers
are indicated on the right.
iensis subsp. kurstaki, the presence of an additional protein,
whether it is CryIVD or the 20-kDa protein, results in the
stabilization of CytA. Similarly, higher CryIVD levels are observed
in the presence of a 20-kDa protein or truncated 20-kDa and CytA
proteins. In the absence of a second protein, the protein levels
observed are lower. Likewise, the presence of an additional
protein, the ORF2 gene product, also appears to be critical for
high-level stable expression of CryIIA (7, 36). The role of a
second protein in high-level cry and cyt gene
expression is uncertain. Potentially, these proteins, even
TABLE 1. Mosquitocidal toxicity of the CytA and CryIVD purified
parasporal inclusions to fourth-instar
C quinquefasciatus larvae
Strain Mosquitocidal toxicity' LC5o (ng/ml) LC95 (ng/ml)
CG1 39.7 (33.9-46.5) 184 (140-266) CG2 301 (227-443) 3,980
(1,960-12,600) CG4 37.3 (31.2-44.2) 150 (115-216) CG6 36.5
(29.4-44.5) 204 (151-308) CG8 25.6 (21.6-30.2) 91.9 (71.3-132) CG13
> 1,000 NDb
a Values in parentheses represent the fiducial limits at LC50 and
LC95 levels.
b ND, not determined.
small ones as observed in CG4, can apparently protect the CytA,
CryIVD, and CryIIA proteins from protease cleavage prior to
parasporal inclusion body formation. Alternatively, this additional
protein could provide the matrix or scaffold- ing for CytA, CryIIA,
and CryIVD packaging and parasporal inclusion formation. Time
course studies show that both CytA and CryIVD are
detected by 24 h of incubation at 30°C. The two toxin genes
encoding these proteins are thus expressed at almost the same time;
however, it is not known whether these two genes are coordinately
regulated. Further, CytA levels higher than CryIVD levels are
observed. The relative amounts of these two proteins are similar to
those found in the intact inclusion body of B. thuringiensis subsp.
israelen- sis. A large ovoid inclusion is formed by bacterial
strains
producing CytA. This shape differs from that found in B.
thuringiensis subsp. israelensis, suggesting that the large
parasporal inclusion in B. thuringiensis subsp. israelensis is an
aggregate of the CryIVA, CryIVB, and CytA proteins, while CryIVD is
packaged separately (4). Inclusions from each B. thuringiensis
strain except CG13 are highly toxic to C. quinquefasciatus larvae.
A synergistic effect was ob- served for B. thuringiensis strains
which produced both CytA and CryIVD. CytA, which has low
mosquitocidal activity, synergizes CryIVD toxicity by about four-
to five- fold (30). This study demonstrates that if both CytA and
CryIVD are present in the same inclusion a synergistic interaction
is observed. These results are in agreement with published reports
which state that purified CytA synergizes CryIVD mosquitocidal
activity (5, 20, 38, 39). The mecha- nism by which CytA synergizes
CryIVD mosquitocidal activity is unknown. Potentially, the cell
membrane aggre- gates formed by CytA (6) could facilitate increased
CryIVD interaction with target cell membranes or, alternatively,
expedite translocation or transportation of an active CryIVD moiety
to its target. However, the mosquitocidal activity of CytA and
CryIVD
together, with an LC50 of 26 ng/ml to C. quinquefasciatus, is an
order of magnitude lower than that observed with intact parasporal
inclusions from B. thuringiensis subsp. israelen- sis, with an LC50
of 3.8 ng/ml (4). Consequently, the other CryIV proteins also
contribute significantly to the intact B. thuringiensis subsp.
israelensis parasporal inclusion toxicity to Culex species.
ACKNOWLEDGMENTS
This work was supported in part by grants from NIH ES03298, the
University of California System-wide Biotechnology Research and
Education Program, and the University of California Mosquito
Control Research Program.
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