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Applied and Environmental Microbiology, March 2001, p. 1090-1096, Vol. 67, No. 3
Facultad de Ciencias Exactas y Naturales,
Departamento de Química Biológica, Ciudad Universitaria,
Universidad de Buenos Aires, 1428 Buenos Aires,
Argentina,1 and Unité des
Bacteries Entomopathogenes, Institut Pasteur, 75724 Paris Cedex 15, France2
Received 25 July 2000/Accepted 10 November 2000
In the same way that cry genes, coding for larvicidal
delta endotoxins, constitute a large and diverse gene family, the
cyt genes for hemolytic toxins seem to compose another set
of highly related genes in Bacillus thuringiensis. Although
the occurrence of Cyt hemolytic factors in B. thuringiensis
has been typically associated with mosquitocidal strains, we have
recently shown that cyt genes are also present in strains
with different pathotypes; this is the case for the
morrisoni subspecies, which includes strains biologically
active against dipteran, lepidopteran, and coleopteran larvae. In
addition, while one Cyt type of protein has been described in all of
the mosquitocidal strains studied so far, the present study confirms
that at least two Cyt toxins coexist in the more toxic antidipteran
strains, such as B. thuringiensis subsp.
israelensis and subsp. morrisoni PG14, and that
this could also be the case for many others. In fact, PCR screening and
Western blot analysis of 50 B. thuringiensis strains
revealed that cyt2-related genes are present in all strains
with known antidipteran activity, as well as in some others with
different or unknown host ranges. Partial DNA sequences for several of
these genes were determined, and protein sequence alignments revealed a
high degree of conservation of the structural domains. These findings
point to an important biological role for Cyt toxins in the final in
vivo toxic activity of many B. thuringiensis strains.
Bacillus thuringiensis
constitutes a large family of strains found in different habitats
(2, 6) and highly specialized as insect pathogens. The
main insecticidal factors displayed by these bacilli are the parasporal
crystalline inclusions synthesized during the sporulation process
(1). Other insecticidal factors also contribute to the
final biological effects: vegetative insecticidal proteins, proteases,
chitinases, exotoxins, and lipases have all been described (17,
30).
The delta endotoxins known so far fall into two categories, Cry and
Cyt, that do not share significant sequence homology, although both
types of toxins seem to work through pore formation that leads to cell
lysis and irreversible damage of the insect midgut (24, 25,
26). While Cry toxins act via specific receptor recognition and
binding (20), no specific receptors have been described
for Cyt toxins, although they show specificity of action in vivo
(13, 27).
More than 100 genes coding for Cry proteins have been isolated to date;
they constitute a biodiverse family with different insect and noninsect
targets including nematodes and protozoans (8, 19, 37).
Antidipteran B. thuringiensis strains commonly feature the
presence of Cyt proteins with cytolytic and hemolytic activities.
Cyt1Aa, according to the new nomenclature (8), is a major
component of the toxic crystal of B. thuringiensis subsp.
israelensis (16) and was the first cytolytic
toxin to be isolated and thoroughly characterized (4).
Since then, others have been detected in different antidipteran
B. thuringiensis strains: some related to Cyt1, like the one
reported for the medellin subspecies (38), and
some that have been classified into other groups based on immunological
criteria (34, 44).
We have recently detected the presence of a second cyt gene
in B. thuringiensis subsp. israelensis, named
cyt2Ba1, which is functional and expresses a 27- to 28-kDa
polypeptide (21); its predicted protein product is 67%
similar to Cyt2Aa1, the 29-kDa cytolytic toxin from B. thuringiensis subsp. kyushuensis (28).
According to these results, the B. thuringiensis subsp.
israelensis crystal would comprise three types of Cry toxins
(Cry4A, Cry4B, and Cry11A), two types of Cyt toxins (Cyt1A and Cyt2B), and other components yet to be defined, all working altogether to give
the final biological activity (12).
PCR amplification and Southern blot analysis confirmed the presence of
homologues of cyt2Ba from B. thuringiensis subsp.
israelensis in other mosquitocidal strains and,
interestingly, also in the anticoleopteran subspecies
morrisoni serovar Tenebrionis and others belonging to the
morrisoni subspecies.
We report a broader screening for cyt2 genes and
Cyt2-related proteins. Partial DNA sequences corresponding to the
central portions of the predicted protein products were determined for several of these genes and aligned with other known Cyt toxins.
The positive correlation found between the coexistence of Cyt1-Cyt2
proteins and high mosquitocidal activity, in addition to the high
conservation found among the sequences determined so far, point to an
important biological role for Cyt proteins in the overall toxicity of
the crystals. There is growing evidence that Cyt and Cry proteins
interact in specific, synergistic combinations in the insect gut to
exert their final biological effects (7, 33, 42). Our
results indicate that cyt genes coding for hemolytic toxins
are widely distributed among a range of B. thuringiensis subspecies and constitute another family of highly related genes.
Strains, plasmids, and media.
The B. thuringiensis strains used in this work are listed in Tables
1 and 2.
Escherichia coli DH5
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.3.1090-1096.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Bacillus thuringiensis cyt Genes for
Hemolytic Endotoxins Constitute a Gene Family
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
(D. Hanahan) was used for plasmid
propagation (22). B. thuringiensis subsp.
kyushuensis, B. thuringiensis subsp.
morrisoni serovar Tenebrionis, B. thuringiensis subsp. kurstaki, B. thuringiensis subsp.
fukuokaensis, B. thuringiensis subsp.
darmstadiensis, B. thuringiensis subsp.
morrisoni HD518, B. thuringiensis subsp.
morrisoni HD12, and B. thuringiensis subsp. israelensis 4Q2-72 were obtained from the Bacillus Genetic
Stock Center (Ohio State University, Columbus). All other strains are from the IEBC collection held by the Laboratoire des Bactéries and Champignons Entomopathogènes (Pasteur Institute, Paris,
France). Strains were maintained in Schaeffer's sporulation agar
medium (36) and grown in Luria-Bertani medium
(32) for DNA isolation. Liquid cultures were grown with
aeration (shaking) at 37°C (E. coli) or 30°C (B. thuringiensis). Ampicillin was added to autoclaved media at 100 µg/ml.
TABLE 1.
cyt2-positive strains: host ranges and the
presence of IS240
TABLE 2.
cyt2-negative strains: host ranges and the
presence of IS240-related sequences
DNA manipulations. Restriction enzymes and T4 DNA ligase (Gibco-BRL) were used as recommended by the manufacturers. DNA fragments were purified from gels with a Gene Clean kit (Bio 101). Plasmids from E. coli were prepared as described by Birnboim and Doly (3). Plasmid DNA was isolated from B. thuringiensis strains as described previously (5) and further purified by using Qiagen columns (Diagen GmbH; Qiagen, Inc.).
DNA sequencing. cyt2B-like genes were amplified by using the upper and lower primers described below. PCR products were cloned into pGEM-T Easy Vector (Promega) and sequenced.
PCR reaction conditions. A total of 20 to 50 ng of purified plasmid DNA was added to the PCR mixtures (2.2 mM concentrations of deoxynucleoside triphosphates, 2 mM MgCl2, 0.5 U of Taq polymerase [Promega], and 100 ng of PCR primers) in a final volume of 50 µl. The oligonucleotide primers were as follows: upper, 5'-AATACATTTCAAGGAGCTA-3', and lower, 5'-TTTCATTTTAACTTCATATC-3'. Amplification was performed in a thermal cycler (M. J. Research Minicycler PTC100) by using a single denaturation step (3 min at 94°C), followed by a 35-cycle program, with each cycle consisting of the following: denaturation at 94°C for 45 s, annealing at 45°C for 45 s, and extension at 72°C for 1 min. A final extension step (72°C for 5 min) was also included. Next, 20-µl samples from each PCR reaction were electrophoresed on 1.5% agarose gels in 0.5× Tris-borate-EDTA buffer at 100 V for 30 to 35 min and stained with ethidium bromide.
Computer analysis. DNA sequences were analyzed with the National Center for Biotechnology Information's BLAST WWW server and with the MegAlign program (Macintosh, v3.03; DNASTAR, Inc.).
Nucleotide sequence accession numbers. The nucleotide sequence data reported here have been submitted to the GenBank-EMBL database and were assigned accession numbers as follows (gene, number, B. thuringiensis subspecies, strain designation [where available]): cyt2Ba2, AF020789, morrisoni, PG14; cyt2Ba3, AF022884, fukuokaensis; cyt2Ba4, AF022885, morrisoni, HD12; cyt2Ba5, AF022886, morrisoni, HD518; cyt2B-, pending, medellin, 163-130; and cyt2Ba6, AF034926, morrisoni (serovar Tenebrionis).
Protein analysis. B. thuringiensis strains were grown in Schaeffer's liquid sporulation medium (36) until lysis. Spore-crystal mixtures from 10-ml samples were harvested by centrifugation at 12,000 × g and then washed once in 1 M NaCl-2 mM phenylmethylsulfonyl fluoride-10 mM EDTA. Pellets were resuspended in sample buffer (32) supplemented with phenylmethylsulfonyl fluoride and EDTA as described earlier, boiled for 10 min, and subjected to sodium dodecyl sulfate (SDS)-15% polyacrylamide gel electrophoresis (PAGE). Protein concentrations were determined by the Bradford assay (Bio-Rad) on samples solubilized as previously described (10). Proteins were electrotransferred to nitrocellulose membranes and detected immunologically by the following method: the membrane was treated with 3% low-fat milk in a 1× Tris-buffered saline (TBS) solution at room temperature with gentle shaking for 1 h. Incubations with anti-Cyt2Aa1 (kindly provided by David Ellar, University of Cambridge) or anti-Cyt2Ba recombinant protein from B. thuringiensis subsp. israelensis were performed at room temperature for 1 h and then overnight at 4°C. Anti-Cyt2Aa1 was used at a 1:500 dilution, and anti-Cyt2Ba was added at a 1:1,000 dilution in a solution containing 3% low-fat milk.
Membranes were washed with gentle shaking at room temperature with three changes of 1× TBS for 10 min before being incubated with the secondary antibody with gentle shaking for 1 h. The Gibco-BRL detection system (biotinylated second antibody, streptavidin-alkaline phosphatase, and nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate toluidinium) was used as recommended by the manufacturer. Secondary antibody bound to the filter was visualized after three washes in 1× TBS for 5 min, one wash with NP-40 at 0.05%, and then one final wash in 1× TBS for 5 min.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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PCR analysis. A pair of oligonucleotide primers was designed from two highly conserved regions shared by the cyt2 genes from B. thuringiensis subsp. israelensis and B. thuringiensis subsp. kyushuensis (21). This primer set was used in PCR amplification experiments in order to search for the presence of this gene in a wider range of B. thuringiensis strains. As shown in Table 1, amplification products of the expected size (469 bp) were observed in all of the strains with known antidipteran activities. Other strains showing this fragment were either nonmosquitocidal (i.e., B. thuringiensis subsp. morrisoni serovar Tenebrionis and strain HD518) or have been described to have both antidipteran and antilepidopteran activities (HD12). Finally, the PCR-positive B. thuringiensis subsp. andalousiensis and subsp. ostriniae have not yet been fully characterized regarding their host ranges.
On the other hand, the PCR-negative group shown in Table 2 is composed mostly of nonmosquitocidal strains and the strain B. thuringiensis subsp. fukuokaensis T03C001 which, interestingly, was also found to be negative for IS240 by hybridization standards. These findings suggest that the known correlation between a mosquitocidal pathotype and the presence of cyt genes may be particularly strong for cyt2-related genes. The morrisoni subspecies seems to be a special case, since all of the strains belonging to this group show cyt2 genes, although not all are known to display mosquitocidal activities.Sequence alignments. PCR amplification products from most of the positive strains were cloned and sequenced. DNA sequence comparisons made with the BLASTN and MegAlign programs revealed a high degree of homology (>90%). Interestingly, this high homology was also observed in the nonmosquitocidal strains B. thuringiensis subsp. morrisoni serovar Tenebrionis and HD518.
Sequence alignments of the central portions of the predicted protein products (154 amino acids from residues 62 to 216) representing 58% of the whole proteins, confirmed as shown in Fig. 1, that predicted helices and 
sheets are highly conserved throughout the different gene versions
compared to the previously described Cyt2 toxins (see Discussion).
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Expression of cyt2B-related genes.
Western blot
experiments were performed on spore-crystal extracts from
cyt2-positive and some negative strains. Three groups could
be defined according to their immunoreactivity with an antiserum raised
against the B. thuringiensis subsp. israelensis
Cyt2Ba1 recombinant protein (see Materials and Methods) (Fig.
2): group 1 includes strains that showed
one reactive band of ca. 26 to 27 kDa (B. thuringiensis
subsp. israelensis 1884, B. thuringiensis subsp.
israelensis 4Q2-72, and B. thuringiensis subspp.
canadensis, tohokuensis, and thompsoni
12007 and B51 [AAT021]); group 2 includes strains that showed one
reactive band of ca. 33 kDa (B. thuringiensis subspp.
higo, ostriniae, and morrisoni HD12
and B. thuringiensis subsp. morrisoni serovar
Tenebrionis); and group 3, which includes strains B. thuringiensis subsp. morrisoni PG14 and B. thuringiensis subsp. morrisoni HD518, which showed both
bands with similar intensities.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Fifty B. thuringiensis strains belonging to various serotypes and host ranges were screened for the presence of cyt2-related genes using PCR amplification. This analysis revealed a wide distribution for these genes coding for cytolytic factors, especially but not only, among strains that display antidipteran activities.
The presence of a cytolytic factor has been always associated with the antidipteran host ranges. In fact, Cyt1 toxins are the first ones described in the most active B. thuringiensis subspecies, israelensis and morrisoni PG14 (15). Although the exact role of these factors in the final biological activity of the crystals has not been fully defined (7, 10, 23) it is clear that they are able to synergize with some Cry toxins (33, 43). It has been recently shown that Cyt1 can lower resistance to Cry toxins in a number of target insects, as well as enhance the activity of B. sphaericus strains (40, 41). Positive interactions have been observed for Cry4-Cyt1 combinations (42, 43), whereas antagonistic effects were found with the Cry1Ac-Cyt1Aa combination (9). Also, sensitivity to Cyt1 was reported in Cry3-resistant B. thuringiensis subsp. morrisoni serovar Tenebrionis (18). This could be consistent with the fact that while cyt genes have been found to naturally occur in cry3- and cry4-bearing strains, it has not been detected in strains displaying exclusively antilepidopteran activities due to Cry1 toxins (reference 21 and this work).
The presence of insertion sequences in B. thuringiensis is very broad (see reference 31 for a review), and many of them are structurally associated with cry genes (37). Although IS240 insertion sequences have invariably been found in antidipteran strains, IS240-related sequences have been found to occur in a wide range of B. thuringiensis strains (35). These sequences have been found in the 5' region of the cyt1Ab1 gene from B. thuringiensis subsp. medellin (38) and upstream of the cry11B gene from B. thuringiensis subsp. jegathesan (11). Similar observations were made for a plasmid in B. thuringiensis subsp. fukuokaensis (14).
The strong correlation observed between the presence of cyt2 genes, IS240 sequences, and antidipteran activity in the best-studied mosquitocidal strains (Tables 1 and 2) could reflect structural associations that might have promoted cyt dispersion among them. Therefore, the presence of cyt2-related genes could be considered a strong indicator of antidipteran host range, even when these genes may be present in other pathotypes as well.
The application of primers based on both cyt2 and IS240 sequences to PCR screening programs could help in the detection of new antidipteran strains with high predictability. According to this approach, B. thuringiensis subsp. ostriniae and subsp. andalousiensis should prove to be dipteran active once their biological activities are characterized.
Figure 4 integrates the cyt2
gene family into the Cyt branch of the B. thuringiensis
toxin dendrogram (8). Although this tree has been
constructed based on partial sequences for many cyt2 genes
and therefore may change once the full sequences are determined, it
reflects the high degree of relatedness of the Cyt family of proteins.
In fact, analysis of the predicted amino acid sequences shows that the
different Cyt2 versions show a high degree of conservation in
-helices and -sheets known to be involved in the formation of the
lytic pore and in the structural integrity of the toxin molecule. The
conservation of specific motifs (highlighted in Fig. 1, in the loops
between and domains) that have been associated with cytolytic
activity, interaction with the insect gut membrane, or pore formation
indicate an important biological role for these toxins (29,
39).
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Further work on the biological activity of isolated Cyt2 toxins alone
and in combination with other Cyt and Cry toxins, as well as the
construction of cyt2
mutants, is under way and
will help in our understanding of the mechanisms operating in the
toxicity of the native crystals. This knowledge may have great impact
on the application of toxin combinations to insect resistance
management programs.
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ACKNOWLEDGMENTS |
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This work was supported by research grants from the Conservation, Food, and Health Foundation (United States) and CONICET (Argentine National Research and Technology Council).
We are grateful to Carmen Sanchez Rivas in whose laboratory this work was performed and for helpful discussion.
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FOOTNOTES |
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* Corresponding author. Mailing address: Virrey Loreto 1865, 3rd, 1426 Buenos Aires, Argentina. Phone: 54-11-47833244. Fax: 54-11-45763342. E-mail: crubi{at}qb.fcen.uba.ar.
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REFERENCES |
|---|
|
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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|---|
| 1. |
Agaisse, H., and D. Lereclus.
1995.
How does Bacillus thuringiensis produce so much insecticidal crystal protein?
J. Bacteriol.
177:6027-6032 |
| 2. | Bernhard, K., P. Jarrett, M. Meadows, J. Butt, D. J. Ellis, G. M. Roberts, S. Pauli, P. Rodgers, and H. D. Buerges. 1997. Natural isolates of Bacillus thuringiensis: worldwide distribution, characterization, and activity against insect pests. J. Invertbrate Pathol. 70:59-68. |
| 3. |
Birnboim, H., and J. Doly.
1979.
A rapid alkaline extraction procedure for screening recombinant plasmid DNA.
Nucleic Acids Res.
7:1513-1523 |
| 4. | Bourgouin, C., A. Delécluse, and G. Rapoport. 1986. Characterization of the genes encoding the haemolytic toxin and the mosquitocidal delta-endotoxin of Bacillus thuringiensis israelensis. Mol. Gen. Genet. 205:390-397[CrossRef][Medline]. |
| 5. |
Bourgouin, C.,
A. Klier, and G. Rapoport.
1986.
Characterization of the genes encoding the haemolytic toxin and the mosquitocidal  -endotoxins of Bacillus thuringiensis var. israelensis.
Mol. Gen. Genet.
205:390-397.
|
| 6. | Chaufaux, J., Marchal, N. Gilois, I. Jehanno, and C. Buisson. 1997. Investigation of natural strains of Bacillus thuringiensis in different biotopes throughout the world. Can. J. Microbiol. 43:337-343. |
| 7. |
Crickmore, N.,
E. J. Bone,
J. A. Williams, and D. J. Ellar.
1995.
Contribution of the individual components of the -endotoxin crystal to the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis.
FEMS Microbiol. Lett.
131:249-254[CrossRef].
|
| 8. | Crickmore, N., D. R. Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D. H. Dean. 1998. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62:808-813. |
| 9. |
Del Rincón Castro, M. C.,
J. Barajas-Huerta, and J. Ibarra.
1999.
Antagonism between Cry1Ac1 and Cyt1A1 toxins of Bacillus thuringiensis.
Appl. Environ. Microbiol.
65:2049-2053 |
| 10. |
Delécluse, A.,
J.-F. Charles,
A. Klier, and G. Rapoport.
1991.
Deletion by in vivo recombination shows that the 28-kilodalton cytolytic polypeptide from Bacillus thuringiensis subsp. israelensis is not essential for mosquitocidal activity.
J. Bacteriol.
173:3374-3381 |
| 11. | Delécluse, A., M.-L. Rosso, and A. Ragni. 1995. Cloning and expression of a novel toxin gene from Bacillus thuringiensis subsp. jegathesan encoding a highly mosquitocidal protein. Appl. Environ. Microbiol. 61:4230-4235[Abstract]. |
| 12. | Delécluse, A., V. Juárez-Pérez, and C. Berry. 2000. Vector-active toxins: structure and diversity, p. 101-125. In J.-F. Charles, A. Delécluse, and X. Nielsen-Le Roux (ed.), Entomopathogenic bacteria: from laboratory to field application. Kluwer Academic Publishers, Dordrecht, The Netherlands. |
| 13. |
Drobniewski, F. A.,
B. H. Kowles, and D. J. Ellar.
1987.
Nonspecific ionic effects on the cytolytic properties of Bacillus thuringiensis -endotoxin.
Curr. Microbiol.
15:295-299[CrossRef].
|
| 14. | Dunn, M. G., and D. J. Ellar. 1997. Identification of two sequence elements associated with the gene encoding the 24-kDa crystalline component in Bacillus thuringiensis subspecies fukuokaensis: an example of transposable element archaelogy. Plasmid 37:205-215[CrossRef][Medline]. |
| 15. |
Earp, D. J., and D. J. Ellar.
1987.
Bacillus thuringiensis var. morrisoni strain PG14: nucleotide sequence of a gene encoding a 27 kDa crystal protein.
Nucleic Acids Res.
15:3619 |
| 16. | Earp, D. J., E. S. Ward, and D. J. Ellar. 1987. Investigation of possible homologies between crystal proteins of three mosquitocidal strains of Bacillus thuringiensis. FEMS Microbiol. Lett. 42:195-199. |
| 17. |
Estruch, J. J.,
G. W. Warren,
M. A. Mullinis,
G. J. Nye,
J. A. Craig, and M. G. Koziel.
1996.
Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects.
Proc. Natl. Acad. Sci. USA
93:5389-5394 |
| 18. |
Federici, B. A., and L. S. Bauer.
1999.
Cyt1Aa protein of Bacillus thuringiensis is toxic to the cottonwood leaf beetle, Chrysomela scripta, and suppresses high levels of resistance to Cry3A.
Appl. Environ. Microbiol.
64:4368-4371 |
| 19. | Feitelson, J. S. 1993. The Bacillus thuringiensis family tree, p. 63-71. In L. Kim (ed.), Advanced engineered pesticides. Marcel Dekker, Inc, New York, N.Y. |
| 20. | Gill, S. S., E. A. Cowles, and P. P. Pietrantonio. 1992. The mode of action of Bacillus thuringiensis endotoxins. Annu. Rev. Entomol. 37:615-636[CrossRef][Medline]. |
| 21. | Guerchicoff, A., R. Ugalde, and C. Rubinstein. 1997. Identification and characterization of a previously undescribed cyt gene in Bacillus thuringiensis subsp. israelensis. Appl. Environ. Microbiol. 63:2716-2721[Abstract]. |
| 22. | Hanahan, D. 1983. Studies on the transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580[Medline]. |
| 23. | Held, G. A., Y.-S. Huang, and C. Y. Kawanishi. 1986. Effect of removal of the cytolytic factor of Bacillus thuringiensis subsp israelensis on mosquito toxicity. Biochem. Biophys. Res. Commun. 141:937-941[CrossRef][Medline]. |
| 24. |
Knowles, B. H., and D. J. Ellar.
1987.
Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis -endotoxins with different specificity.
Biochim. Biophys. Acta
924:509-518.
|
| 25. |
Knowles, B. H., and D. J. Ellar.
1987.
Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis -endotoxins with different specificity.
Biochim. Biophys. Acta
924:509-518.
|
| 26. |
Knowles, B. H.,
M. R. Blatt,
M. Tester,
J. M. Horsnell,
J. Carroll,
G. Menestrina, and D. J. Ellar.
1989.
A cytolytic -endotoxin from Bacillus thuringiensis var. israelensis forms cation-selective channels in planar lipid bilayers.
FEBS Lett.
244:259-262[CrossRef][Medline].
|
| 27. |
Koni, P. A., and D. J. Ellar.
1994.
Biochemical characterization of Bacillus thuringiensis cytolytic delta-endotoxins.
Microbiology
140:1869-1880 |
| 28. | Koni, P. A., and D. J. Ellar. 1993. Cloning and characterization of a novel Bacillus thuringiensis cytolytic delta-endotoxin. J. Mol. Biol. 229:319-327[CrossRef][Medline]. |
| 29. |
Li, J.,
P. A. Koni, and D. J. Ellar.
1996.
Structure of the mosquitocidal -endotoxin CytB from Bacillus thuringiensis sp. kyushuensis and implications for membrane pore formation.
J. Mol. Biol.
257:129-152[CrossRef][Medline].
|
| 30. | Lövgren, A., M.-Y. Shang, A. Engström, G. Dalhammmar, and R. Landén. 1990. Molecular characterization of immune inhibitor A, a secreted virulence protease from Bacillus thuringiensis. Mol. Microbiol. 4:2137-2146[CrossRef][Medline]. |
| 31. | Mahillon, J., R. Rezsöhazy, B. Hallet, and J. Delcour. 1994. IS231 and other Bacillus thuringiensis transposable elements: a review. Genetica 93:13-26[CrossRef][Medline]. |
| 32. | Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 33. | Poncet, S., A. Delécluse, A. Kilier, and G. Rapoport. 1995. Evaluation of synergistic interactions between the CryIVA, CryIVB and CryIVD toxic components of Bacillus thuringiensis subsp. israelensis crystals. J. Invertebrate Pathol. 66:131-135. |
| 34. | Ragni, A., I. Thiéry, and A. Delécluse. 1996. Characterization of six highly mosquitocidal Bacillus thuringiensis strains that do not belong to H-14 serotype. Curr. Microbiol. 32:48-54[CrossRef][Medline]. |
| 35. | Rosso, M.-L., and A. Delécluse. 1997. Distribution of the insertion sequence IS240 among Bacillus thuringiensis strains. Curr. Microbiol. 34:348-353[CrossRef][Medline]. |
| 36. | Schaeffer, P., J. Millet, and J. Aubert. 1965. Catabolic repression of bacterial sporulation. Proc. Natl. Acad. Sci. USA 554:701-711. |
| 37. | Schnepf, E., N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, R. D. Zeigler, and H. D. Dean. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Appl. Environ. Microbiol. 62:775-806. |
| 38. | Thiery, I., A. Delecluse, M. C. Tamayo, and S. Orduz. 1996. Identification of a gene for Cyt1A-like hemolysin from Bacillus thuringiensis subsp. medellin and expression in a crystal-negative Bacillus thuringiensis strain. Appl. Environ. Microbiol. 63:468-473[Abstract]. |
| 39. | Ward, E. S., D. J. Ellar, and C. N. Chilcott. 1988. Single amino acid changes in the Bacillus thuringiensis var. israelensis delta-endotoxin affect the toxicity and expression of the protein. J. Mol. Biol. 202:527-535[CrossRef][Medline]. |
| 40. |
Wirth, M. C.,
B. A. Federici, and W. E. Walton.
2000.
Cyt1A from Bacillus thuringiensis synergizes activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae).
Appl. Environ. Microbiol.
66:1093-1097 |
| 41. | Wirth, M. C., W. E. Walton, and B. A. Federici. 2000. Cyt1A from Bacillus thuringiensis restores toxicity of Bacillus sphaericus against resistant Culex quinquefasciatus (Diptera: Culicidae). J. Med. Entomol. 37:401-407[Medline]. |
| 42. | Wu, D., and F. N. Chang. 1985. Synergism in mosquitocidal activity of 26 and 65 kDa proteins from Bacillus thuringiensis subsp. israelensis crystal. FEBS Lett. 190:232-236[CrossRef]. |
| 43. | Wu, D., J. J. Johnson, and B. A. Federici. 1994. Synergism of mosquitocidal toxicity between CytA and CryIVD proteins using inclusions produced from cloned genes of Bacillus thuringiensis. Mol. Microbiol. 13:965-972[Medline]. |
| 44. |
Yu, Y.,
M. Ohba, and S. S. Gill.
1991.
Characterization of mosquitocidal activity of Bacillus thuringiensis subsp. fukuokaensis crystal proteins.
Appl. Environ. Microbiol.
57:1075-1081 |
Applied and Environmental Microbiology, March 2001, p. 1090-1096, Vol. 67, No. 3
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.3.1090-1096.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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