This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ito, T.
Right arrow Articles by Asano, S.-i.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ito, T.
Right arrow Articles by Asano, S.-i.
Agricola
Right arrow Articles by Ito, T.
Right arrow Articles by Asano, S.-i.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, August 2006, p. 5673-5676, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.01894-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Cloning and Expression of Two Crystal Protein Genes, cry30Ba1 and cry44Aa1, Obtained from a Highly Mosquitocidal Strain, Bacillus thuringiensis subsp. entomocidus INA288

Takeshi Ito, Tomonori Ikeya, Ken Sahara, Hisanori Bando, and Shin-ichiro Asano

Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

Received 10 August 2005/ Accepted 12 June 2006


arrow
ABSTRACT
 
Two novel crystal protein genes, cry30Ba and cry44Aa, were cloned from Bacillus thuringiensis subsp. entomocidus INA288 and expressed in an acrystalliferous strain. Cry44Aa crystals were highly toxic to second-instar Culex pipiens pallens (50% mortality concentration [LC50] = 6 ng/ml) and Aedes aegypti (LC50 = 12 ng/ml); however, Cry30Ba crystals were not toxic.


arrow
INTRODUCTION
 
Bacillus thuringiensis subsp. israelensis produces crystal proteins that have been successfully used for controlling the mosquito population (2). Although no resistance to B. thuringiensis subsp. israelensis toxins in the field has been reported, weak resistance has been observed in the laboratory in populations that are under artificial selection pressure due to high concentrations of B. thuringiensis subsp. israelensis toxins (3). Therefore, it is possible that field populations of insects resistant to B. thuringiensis subsp. israelensis toxins may emerge if large concentrations of B. thuringiensis subsp. israelensis toxins are used for an extended period. Hence, several screening programs have been set up that aim to isolate new strains producing novel mosquitocidal crystal proteins that could replace or be used in combination with B. thuringiensis subsp. israelensis. Bacillus thuringiensis subsp. entomocidus INA288 has been isolated from Indonesian soil, and it produces large cuboidal crystals (5). Although known mosquitocidal cry genes, such as cry2A, cry4A, cry4B, and cry11A, were not detected by PCR, this strain showed a toxicity comparable to that of B. thuringiensis subsp. israelensis against second-instar Aedes aegypti. This indicated the presence of a novel mosquitocidal cry gene(s).

In order to clone the novel mosquitocidal cry gene(s), PCR was performed using a forward primer, 288-5 (5'-ACAAATTATAAAGATTGGCT-3'), and two reverse primers, 288-31 (5'-ATCCCCTCCTGTATGACCAGGTCC-3') and 288-32 (5'-GAGTAATTGGCAGAAATTC-3'), which were designed on the basis of common DNA sequences of known mosquitocidal crystal proteins. The 1.6-kb and 1.8-kb PCR products were amplified by using 288-31 and 288-32 as reverse primers, respectively. We designated 1.6-kb and 1.8-kb PCR products as 288A and 288B, respectively. 288A and 288B were labeled and used as probes to hybridize with the total DNA obtained from B. thuringiensis subsp. entomocidus INA288. An XbaI fragment of about 8 kb was hybridized with the 288A probe. The 7- to 9-kb XbaI fragments were purified and cloned into the XbaI site of pUC119, and Escherichia coli DH5{alpha} was transformed using these plasmids. Colony hybridization was performed for screening against recombinant E. coli DH5{alpha} colonies, and the plasmid was extracted from the positive clone. The positive clone contained an XbaI DNA insert of about 8 kb. Sequencing analysis revealed the presence of two open reading frames (ORFs) oriented in the same direction (Fig. 1). The first ORF corresponded to a polypeptide of 686 amino acids (aa) with a deduced molecular mass of 77.5 kDa. This protein was further classified as Cry44Aa1. The second ORF, located downstream of cry44Aa1, encoded a polypeptide of 541 aa with a deduced molecular mass of 64.1 kDa and was designated as ORF2-44A. A nucleotide sequence similar to the –35 and –10 regions of the {sigma}35-dependent promoter was found upstream of cry44Aa. A palindromic sequence that could form an mRNA hairpin loop with a {Delta}G of –20.53 kcal/mol and a shorter sequence that could act as a {rho}-factor-independent transcriptional terminator were found downstream of the orf2-44A gene. Nucleotide sequences capable of acting as ribosome binding sites were also found just upstream of cry44Aa and orf2-44A (data not shown). These results indicate that cry44Aa and orf2-44A form an operon.


Figure 1
View larger version (6K):
[in this window]
[in a new window]
  		FIG. 1. Organization of the genes in the cry30Ba and cry44Aa operons. The positions and directions of the genes are indicated by open arrows. The eight regions conserved among Cry proteins are indicated by black rectangles. The arrowhead and asterisks indicate the putative promoter region and the transcriptional terminator, respectively. Black diamonds indicate tentative ribosome binding sites.

Labeled 288B was hybridized with a BamHI fragment of approximately 20 kb, and a lambda library of BamHI fragments of B. thuringiensis subsp. entomocidus INA288 DNA was constructed. The phage DNA was extracted from a positive lambda clone (lambda 288B) and was digested with various restriction enzymes. Restriction enzyme-digested DNA fragments were electrophoresed and Southern blotted to screen for an insert of a suitable size. The 7-kb BamHI-HindIII fragments and 8-kb HindIII fragments, which hybridized with the 288B probe, were cloned into pUC119 to yield pUC288B1 and pUC288B2. A partial nucleotide sequence of pUC288B1 and pUC288B2 in the region containing the cry gene was determined. Two ORFs that were oriented in the same direction were detected. The first ORF corresponded to a polypeptide of 683 aa with a deduced molecular mass of 77.4 kDa. This protein was further classified as Cry30Ba1. The second ORF, located downstream of cry30Ba1 and coding for a polypeptide of 545 aa with a deduced molecular mass of 62.0 kDa, was designated ORF2-30B. There is no known Cry protein that shows high homology to Cry44Aa; the protein that showed maximum homology was Cry19Ba from Bacillus thuringiensis subsp. higo (32% identity). Therefore, this protein belongs to a novel class of {delta} endotoxins. Cry30Ba was similar to Cry30Aa from Bacillus thuringiensis subsp. medellin (58% identity). Both Cry44Aa and Cry30Ba contained the five conserved blocks (block 1 to block 5) that are present in almost all Cry proteins. On the other hand, ORF2-44A and ORF2-30B contained three conserved blocks (block 6 to block 8) that were present in the carboxyl-terminal half of 130-kDa-type crystal proteins, and the two ORFs showed high homology to those carboxyl-terminal halves (Fig. 1). Thus, cry44Aa/orf2-44A and cry30Ba/orf2-30B have a gene structure similar to that of the 130-kDa-type crystal protein genes. A similar gene structure, in which a typical 130-kDa protein cry gene was split into two separate ORFs, has been observed in cry39A (7), cry40A (GenBank accession no. AB074414), cry40B (GenBank accession no. AB112346), cry10A (13), and cry19A (12). The cause of the 130-kDa-type Cry protein’s division into two segments has not been elucidated. At present, this gene configuration has been detected only in cry genes from mosquitocidal strains.

In order to test the insecticidal activities of Cry44A and Cry30B, expression plasmids were constructed and introduced into an acrystalliferous Bacillus thuringiensis subsp. kurstaki HD-1 mutant (Bt51) (15). When the cry44Aa and cry30Ba were expressed without their orf2 genes, the resulting Bt51 transformants did not produce any detectable crystalline inclusion bodies (data not shown). Similar results have been reported previously (7, 12). As mentioned above, ORF2-44A and ORF2-30B correspond to the carboxyl-terminal half of a typical 130-kDa Cry protein. The 130-kDa Cry proteins with truncated carboxyl-terminal portions do not form crystalline inclusion bodies (1, 11, 14), and the carboxyl-terminal half of the 130-kDa Cry protein is considered to be involved in crystallization and/or stabilization but not in toxicity (10). Therefore, ORF2-44A and ORF2-30B were considered to have similar functions. When cry44Aa and cry30Ba were expressed with their orf2 genes, recombinants Bt51-pHY44A and Bt51-pHY30B produced crystals that were detectable under a light microscope. Bt51 transformants and B. thuringiensis subsp. entomocidus INA288 were induced to sporulate and autolyze after 72 h of incubation. After being harvested and washed, spores and crystals were observed using a scanning electron microscope as described by Iizuka et al. (6). In comparison with wild-type INA288, which produced large cuboidal crystals (Fig. 2A), recombinants Bt51-pHY44A and Bt51-pHY30B produced smaller, amorphous crystals (Fig. 2B and C). These crystals were purified on discontinuous sucrose gradients as described by Nishimoto et al. (9) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 3). The molecular masses of major polypeptides in the crystals purified from wild-type INA288 were 74, 70, 65, 60, 43, 24, and 14 kDa. Two major polypeptides of 73 and 65 kDa were detected in crystals obtained from Bt51-pHY44A. The sequences of the first five N-terminal amino acids of 73- and 65-kDa polypeptides were identical to those of Cry44A and ORF2-44A, respectively. In the crystals obtained from Bt51-pHY30B, major polypeptides of 73 and 66 kDa were detected. The sequences of the first five N-terminal amino acids of 73- and 66-kDa polypeptides were identical to those of Cry30B and ORF2-30B, respectively.


Figure 2
View larger version (75K):
[in this window]
[in a new window]
  		FIG. 2. Scanning electron microscopy showing spores and crystalline inclusions produced by the B. thuringiensis parental strain and transformants. Magnification, x10,000.


Figure 3
View larger version (54K):
[in this window]
[in a new window]
  		FIG. 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crystal proteins produced by the B. thuringiensis parental strain and transformants. Lane 1, molecular mass markers.

Purified crystals obtained from Bt51 recombinants including Bt51-pHY/IAaP-IVA, which produced Cry4A crystals (4), and crystals obtained from B. thuringiensis subsp. entomocidus INA288 were tested against first-instar larvae of Anopheles stephensi and second-instar larvae of Culex pipiens pallens and Aedes aegypti (Table 1). Five larvae were transferred to each well of a 24-well titer plate that contained 950 µl of tap water per well. Fifty microliters of the serial dilutions of purified crystal proteins was added. Larval mortality was recorded after 24 h of incubation, and 50% mortality concentrations (LC50s) were calculated by probit analysis. Thirty larvae were tested against each dilution, and the bioassays were performed more than three times. The Cry44A crystals showed strong toxicity against C. pipiens (LC50 = 0.006 µg/ml) and A. aegypti (LC50 = 0.012 µg/ml), and the Cry44A crystals were approximately 49 times and 22 times more toxic than Cry4A crystals against C. pipiens and A. aegypti, respectively. In addition, the Cry44A crystals were slightly toxic against A. stephensi (LC50 = 1.265 µg/ml). Crystals from B. thuringiensis subsp. entomocidus INA288 showed similar tendencies in mosquitocidal activity. This suggests that Cry44Aa plays a major role in the mosquitocidal activity of B. thuringiensis subsp. entomocidus INA288 crystals. The investigation of the activation processes of Cry44Aa protoxins and binding properties of Cry44Aa toxins for brush border membranes prepared from mosquito larvae is in progress in order to gain an understanding of the mechanism of action of mosquitocidal crystal protein Cry44A. In contrast to Cry44A, the Cry30B crystals at a 100-µg/ml concentration were not toxic against any mosquito species tested in this study. Cry29Aa from B. thuringiensis subsp. medellin strain 161-131 is nontoxic to C. pipiens, A. stephensi, and A. aegypti. However, Cry29A shows a fourfold synergistic activity with Cry11Bb against A. aegypti (8). Therefore, Cry30Ba may also possess a similar synergistic property. Even if there is a synergy, it should be subtle and/or masked by the presence of other proteins in the native INA288 crystal since the native INA288 crystals, which contain both Cry44A and Cry30B, are four- or fivefold less toxic than pure Cry44A crystals. An additional bioassay in combination with other Cry proteins such as Cry44Aa is required.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Mosquitocidal activities of purified crystals from B. thuringiensis strains

In this paper, we reported the cloning and expression of two novel crystal protein genes, cry44Aa/orf2-44A and cry30Ba/orf2-30B, from highly mosquitocidal B. thuringiensis subsp. entomocidus INA288. The cry44Aa/orf2-44A gene is highly toxic to C. pipiens and A. aegypti and appears to be a promising alternative to B. thuringiensis subsp. israelensis or may be used in combination with B. thuringiensis subsp. israelensis toxins. It is essential to perform additional bioassays with these Cry toxins against the resistant mosquito colonies selected with B. thuringiensis subsp. israelensis toxins. These studies will serve as evidence that novel cry genes from INA288 such as cry44Aa/orf2-44A may be useful in managing resistance and/or as a component of synthetic combinations of mosquitocidal toxins.


arrow
Nucleotide sequence accession numbers.
 
The nucleotide sequence data are available in the EMBL, GenBank, and DDBJ nucleotide sequence databases under accession numbers AB161456 (cry44Aa1 and orf2-44A) and AB125059 (cry30Ba1 and orf2-30B).


arrow
ACKNOWLEDGMENTS
 
We are grateful to Sugyo Hastowo and Bibiana W. Lay of Universitas Katolik Indonesia Atma Jaya for isolating and identifying B. thuringiensis subsp. entomocidus INA288.

This work was supported in part by the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (T. Ito).


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. Phone and fax: 81-011-706-2487. E-mail: ito-t{at}abs.agr.hokudai.ac.jp. Back


arrow
REFERENCES
 
    1
  1. Adang, M. J., M. J. Staver, T. A. Rocheleau, J. Leighton, R. F. Barker, and D. V. Thompson. 1985. Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta. Gene 36:289-300.[CrossRef][Medline]
    2. 2
  2. Becker, N. 2000. Bacterial control of vector-mosquitoes and black flies, p. 383-398. In J.-F. Charles, A. Delécluse, and C. Nielsen-LeRoux (ed.), Entomopathogenic bacteria: from laboratory to field application. Kluwer, Dordrecht, The Netherlands.
    3. 3
  3. Georghiou, G. P., and M. C. Wirth. 1997. Influence of exposure to single versus multiple toxins of Bacillus thuringiensis subsp. israelensis on development of resistance in the mosquito Culex quinquefasciatus (Diptera: Culicidae). Appl. Environ. Microbiol. 63:1095-1101.[Abstract]
    4. 4
  4. Hashimoto, N., J. Sasaki, S. Asano, H. Bando, and T. Iizuka. 1996. Bacillus thuringiensis ICP gene expression under the control of cryIA(a) gene promoter. J. Seric. Sci. Jpn. 65:185-191.
    5. 5
  5. Hastowo, S., B. W. Lay, and M. Ohba. 1992. Naturally occurring Bacillus thuringiensis in Indonesia. J. Appl. Bacteriol. 73:108-113.
    6. 6
  6. Iizuka, T., M. Ishino, and T. Nakajima. 1982. Comparative morphology of parasporal crystals and characterizations of plasmid DNA from various subspecies of entomopathogenic bacteria, Bacillus thuringiensis. J. Fac. Agric. Hokkaido Univ. 13:423-431.
    7. 7
  7. Ito, T., K. Sahara, H. Bando, and S. Asano. 2002. Cloning and expression of novel crystal protein genes cry39A and 39orf2 from B. thuringiensis subsp.aizawai Bun1-14 encoding mosquitocidal proteins. J. Insect Biotechnol. Sericol. 71:123-128.
    8. 8
  8. Juárez-Pérez, V., M. Porcar, S. Orduz, and A. Delécluse. 2003. Cry29A and Cry30A: two novel {delta}-endotoxins isolated from Bacillus thuringiensis serovar medellin. Syst. Appl. Microbiol. 26:502-504.[CrossRef][Medline]
    9. 9
  9. Nishimoto, T., H. Yoshisue, K. Ihara, H. Sakai, and T. Komano. 1994. Functional analysis of block 5, one of the highly conserved amino acid sequences in the 130-kDa CryIVA protein produced by Bacillus thuringiensis subsp.israelensis. FEBS Lett. 348:249-254.[CrossRef][Medline]
    10. 10
  10. Park, H. W., D. K. Bideshi, and B. A. Federici. 2000. Molecular genetic manipulation of truncated Cry1C protein synthesis in Bacillus thuringiensis to improve stability and yield. Appl. Environ. Microbiol. 66:4449-4455.[Abstract/Free Full Text]
    11. 11
  11. Rang, C., M. Bes, V. Lullien-Pellerin, D. Wu, B. A. Federici, and R. Frutos. 1996. Influence of the 20-kDa protein from Bacillus thuringiensis ssp. israelensis on the rate of production of truncated Cry1C proteins. FEMS Microbiol. Lett. 141:261-264.[CrossRef][Medline]
    12. 12
  12. Rosso, M. L., and A. Delécluse. 1997. Contribution of the 65-kilodalton protein encoded by the cloned gene cry19A to the mosquitocidal activity of Bacillus thuringiensis subsp. jegathesan. Appl. Environ. Microbiol. 63:4449-4455.[Abstract]
    13. 13
  13. Thorne, L., F. Garduno, T. Thompson, D. Decker, M. Zounes, M. Wild, A. M. Walfield, and T. J. Pollock. 1986. Structural similarity between the lepidoptera- and diptera-specific insecticidal endotoxin genes of Bacillus thuringiensis subsp. "kurstaki" and "israelensis." J. Bacteriol. 166:801-811.[Abstract/Free Full Text]
    14. 14
  14. Wabiko, H., G. A. Held, and L. A. Bulla, Jr. 1985. Only part of the protoxin gene of Bacillus thuringiensis subsp. berliner 1715 is necessary for insecticidal activity. Appl. Environ. Microbiol. 49:706-708.[Abstract/Free Full Text]
    15. 15
  15. Yamamoto, T., A. Ehmann, J. M. Gonzalez, and B. C. Carlton. 1988. Expression of three genes coding for 135-kDa entomocidal proteins in Bacillus thuringiensis kurstaki. Curr. Microbiol. 17:5-12.[CrossRef]

Applied and Environmental Microbiology, August 2006, p. 5673-5676, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.01894-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ito, T.
Right arrow Articles by Asano, S.-i.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ito, T.
Right arrow Articles by Asano, S.-i.
Agricola
Right arrow Articles by Ito, T.
Right arrow Articles by Asano, S.-i.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»