Biochemical and Multiplex PCR Analysis of Toxic Crystal Proteins to Determine Genes in Bacillus thuringiensis Mutants
The Egyptian Bacillus thuringiensis isolate (M5) produce crystal proteins that is toxic against insects was irradiated with UV light to induce mutants. Upon testing 10 of the resulting mutants for their toxicity against cotton leafworm larvae, the three mutants 62, 64 and 85 proved to be the most toxic ones. Upon testing these mutants along with their parental isolate by SDS-PAGE analysis of spores-crystals proteins as well as vegetative cells proteins, new induced bands appeared in the three mutants by UV radiation and also they showed disappearance of some other bands as compared with the wild type isolate. Multiplex PCR technique, with five sets of specific primers, was used to detect the three types of cryI genes cryIAa, cryIAb and cryIAc. Results showed that these three genes exist, as distinctive bands, in the wild type isolate (M5) as well as in mutants 62 and 85, while the mutant 64 had two distinctive bands of cryIAb and cryIAc genes, and a faint band of cryI Aa gene. Finally, these results revealed that mutant 62 is considered as the promising mutant since it is UV resistant, highly toxic against Spodoptera littoralis and active against a wide range of Lepidopteran insects.
<p>[1] Chattopdhyay, A.; N. B. Bhatnagar and R. Bhatnagar "Bacterial
insecticidal toxins" Criti. Rev. Microbiol. 30:33-54, 2004.
[2] Beegle, C. C. and T. Yamamoto "History of Bacillus thuringiensis
Berliner research and development" Can. Entomol. 124:587-616, 1992.
[3] Feitelson, J. S. "The Bacillus thuringiensis family tree. In: Advanced
Engineered Pesticides. Kim L. (ed.). Marcel Dekker, New York, 1993,
pp. 63-71.
[4] Laemmli, U. K. "Cleavage of structural proteins during the assembly of
the head bacteriophage T4". Nature. 277:680- 685. 1970.
[5] Donovan, W. P.; J. M. Gonzalez; M. P. Gilbert and C. Dankocsik"
Isolation and characterization of EG2158, a new strain of Bacillus
thuringiensis toxic to coleopteran larvae, and nucleotide sequence of the
toxic gene" Mol. Gen. Genet. 214:366-372, 1988.
[6] Gill, S. H.; L. Kiehne; J. L. and T. Yamamoto "Cloning of a novel
cryIC-type gene from a strain of Bacillus thuringiensis subsp. gallaiae"
Appl. Environ. Microbiol. 59:1131-1137, 1995.
[7] Liang, H. ; Y. Liu ; J. Zhu ; P. Guan ; S.Li ; S.wang ; A. Zheng ; H. Liu
and P. Li " Characterization of cry2-type genes of Bacillus thuringiensis
strains from soil isolated of Sihuan Basin, China" Brazillian J.
Microbiol. 42: 140-146, 2011.
[8] Kati, H; K. Sezen and Z. Demirabag "Characterization of a highly
pathogenic Bacillus thuringiensis strain isolated from common
cockchafer, Melolontha melolontha" Folia Microbiol. 52:147-152, 2007.
[9] Carlabrese, D. M. and K. W. Nickerson “A comparison of protein
crystal subunit sizes in Bacillus thuringiensis” Can. J. Microbiol.
26:1006-1010, 1980.
[10] Jarrett, P. "Potency factors in the delta-endotoxin of Bacillus
thuringiensis var. aizawi and the significance of plasmids in their
control" J. Appli. Bacteriol. 58: 437-448, 1985.
[11] Baum, J. A.; C. Chu; M. Rupar; G. R. Brown; W. P. Donovan; J. E.
Huesing; O. Ilagan; T. M. Malvar and M. Pleau "Binary toxins from
Bacillus thuringiensis active against the western corn rootworm,
Diabrotica virgifera virgifera LeConte" Appli. Environ. Microbiol.
70:4889-4898, 2004.
[12] Milne, R.; D. Murphy and P. G. Fast "Bacillus thuringiensis- endotoxin:
An improved technique for the separation of crystals from spores" J.
Invert. Pathol. 29:230-231, 1977.
[13] Lynch, M. J. and P. Baumann" Immunological comparisons of the
crystal protein from strains of Bacillus thuringiensis" J. Invert. Pathol.
46:47-57. 1985.
[14] Bourque, S.; J. R. Valero; J. Mercier; M. C. Lavoie and C. Levesquer
"Multiplex polymerase chain reaction for detection and differentiation
of the microbial insecticide Bacillus thunrngiensis" Appli. Environ.
Microbiol. 59: 523- 527, 1993.
[15] Ceron, J.; L. Cvarrubias; R. Quintero; A. Ortiz; M. Ortiz; E. Aranda; L.
Lina and A. Bravo "PCR analysis of the cryI insecticidal crystal family
genes from Bacillus thuringiensis" Appli. Environ. Microbiol. 60:353-
356, 1994.
[16] Ben-Dov, E.; A. Zaritsky; E. Dahan; Z. Barak; R. Siani; R.Manasherob;
A. Khamraev; E. Trttskaya; A. Dubitsky; N.Berezina and Y. Margalith "
Extended screening by PCR for seven cry-group genes from fieldcollected
strains of Bacillus thuringiensis. Appli. Environ. Microbial"
63:4883-4890, 1994.
[17] Von-Tersch, M. A. and T. M. Gonzalez "Bacillus thuringiensis cry ET1
toxin gene and protein toxic to Lepidopteran insects, patent No:
5356623 supplemented for registration in (10-8-1994), 1994.
[18] Hames, B. D. "One-dimensional polyacrylamide gel electrophoresis. In:
Gel electrophoresis of proteins. A practical approach. Hames B.D. and
Rickwood D. (eds.). Oxford University Press, New York, 1995, pp. 1-
147.
[19] Saxena, D.; E. Ben-Dov; R. Manasherob; Z. Barak; S. Boussiba and A.
Zaritsky "A UV tolerant mutant of Bacillus thuringiensis subsp.
Kurstaki producing melanin" Curr. Microbiol. 44:25-30, 2002.
[20] Ruan, L.; Z. Yu; B. Fang; W. He; Y. Wang and P. Shen "Melanin
pigment formation and increased UV resistance in Bacillus
thuringiensis following high temperature induction" System. Appl.
Microbiol. 27:286-289, 2004.
[21] Kumar, S. ; U. Tuteja ; K. Sarika ; D. Kumar Singh and O. Kumar
"Rapid Multiplex PCR assay for the simultaneous detection of the
Brucella Genus, B. abotus, B. melitensis, and B. suis." J. Microbiol.
Biotechnol. 21(1):89-92, 2011.</p>
<p>[1] Chattopdhyay, A.; N. B. Bhatnagar and R. Bhatnagar "Bacterial
insecticidal toxins" Criti. Rev. Microbiol. 30:33-54, 2004.
[2] Beegle, C. C. and T. Yamamoto "History of Bacillus thuringiensis
Berliner research and development" Can. Entomol. 124:587-616, 1992.
[3] Feitelson, J. S. "The Bacillus thuringiensis family tree. In: Advanced
Engineered Pesticides. Kim L. (ed.). Marcel Dekker, New York, 1993,
pp. 63-71.
[4] Laemmli, U. K. "Cleavage of structural proteins during the assembly of
the head bacteriophage T4". Nature. 277:680- 685. 1970.
[5] Donovan, W. P.; J. M. Gonzalez; M. P. Gilbert and C. Dankocsik"
Isolation and characterization of EG2158, a new strain of Bacillus
thuringiensis toxic to coleopteran larvae, and nucleotide sequence of the
toxic gene" Mol. Gen. Genet. 214:366-372, 1988.
[6] Gill, S. H.; L. Kiehne; J. L. and T. Yamamoto "Cloning of a novel
cryIC-type gene from a strain of Bacillus thuringiensis subsp. gallaiae"
Appl. Environ. Microbiol. 59:1131-1137, 1995.
[7] Liang, H. ; Y. Liu ; J. Zhu ; P. Guan ; S.Li ; S.wang ; A. Zheng ; H. Liu
and P. Li " Characterization of cry2-type genes of Bacillus thuringiensis
strains from soil isolated of Sihuan Basin, China" Brazillian J.
Microbiol. 42: 140-146, 2011.
[8] Kati, H; K. Sezen and Z. Demirabag "Characterization of a highly
pathogenic Bacillus thuringiensis strain isolated from common
cockchafer, Melolontha melolontha" Folia Microbiol. 52:147-152, 2007.
[9] Carlabrese, D. M. and K. W. Nickerson “A comparison of protein
crystal subunit sizes in Bacillus thuringiensis” Can. J. Microbiol.
26:1006-1010, 1980.
[10] Jarrett, P. "Potency factors in the delta-endotoxin of Bacillus
thuringiensis var. aizawi and the significance of plasmids in their
control" J. Appli. Bacteriol. 58: 437-448, 1985.
[11] Baum, J. A.; C. Chu; M. Rupar; G. R. Brown; W. P. Donovan; J. E.
Huesing; O. Ilagan; T. M. Malvar and M. Pleau "Binary toxins from
Bacillus thuringiensis active against the western corn rootworm,
Diabrotica virgifera virgifera LeConte" Appli. Environ. Microbiol.
70:4889-4898, 2004.
[12] Milne, R.; D. Murphy and P. G. Fast "Bacillus thuringiensis- endotoxin:
An improved technique for the separation of crystals from spores" J.
Invert. Pathol. 29:230-231, 1977.
[13] Lynch, M. J. and P. Baumann" Immunological comparisons of the
crystal protein from strains of Bacillus thuringiensis" J. Invert. Pathol.
46:47-57. 1985.
[14] Bourque, S.; J. R. Valero; J. Mercier; M. C. Lavoie and C. Levesquer
"Multiplex polymerase chain reaction for detection and differentiation
of the microbial insecticide Bacillus thunrngiensis" Appli. Environ.
Microbiol. 59: 523- 527, 1993.
[15] Ceron, J.; L. Cvarrubias; R. Quintero; A. Ortiz; M. Ortiz; E. Aranda; L.
Lina and A. Bravo "PCR analysis of the cryI insecticidal crystal family
genes from Bacillus thuringiensis" Appli. Environ. Microbiol. 60:353-
356, 1994.
[16] Ben-Dov, E.; A. Zaritsky; E. Dahan; Z. Barak; R. Siani; R.Manasherob;
A. Khamraev; E. Trttskaya; A. Dubitsky; N.Berezina and Y. Margalith "
Extended screening by PCR for seven cry-group genes from fieldcollected
strains of Bacillus thuringiensis. Appli. Environ. Microbial"
63:4883-4890, 1994.
[17] Von-Tersch, M. A. and T. M. Gonzalez "Bacillus thuringiensis cry ET1
toxin gene and protein toxic to Lepidopteran insects, patent No:
5356623 supplemented for registration in (10-8-1994), 1994.
[18] Hames, B. D. "One-dimensional polyacrylamide gel electrophoresis. In:
Gel electrophoresis of proteins. A practical approach. Hames B.D. and
Rickwood D. (eds.). Oxford University Press, New York, 1995, pp. 1-
147.
[19] Saxena, D.; E. Ben-Dov; R. Manasherob; Z. Barak; S. Boussiba and A.
Zaritsky "A UV tolerant mutant of Bacillus thuringiensis subsp.
Kurstaki producing melanin" Curr. Microbiol. 44:25-30, 2002.
[20] Ruan, L.; Z. Yu; B. Fang; W. He; Y. Wang and P. Shen "Melanin
pigment formation and increased UV resistance in Bacillus
thuringiensis following high temperature induction" System. Appl.
Microbiol. 27:286-289, 2004.
[21] Kumar, S. ; U. Tuteja ; K. Sarika ; D. Kumar Singh and O. Kumar
"Rapid Multiplex PCR assay for the simultaneous detection of the
Brucella Genus, B. abotus, B. melitensis, and B. suis." J. Microbiol.
Biotechnol. 21(1):89-92, 2011.</p>
@article{"International Journal of Biological, Life and Agricultural Sciences:61864", author = "Fatma N. Talkhan and H. H. Abo-Assy and K. A. Soliman and Marwa M. Azzam and A. Z. E. Abdelsalam and A. S. Abdel-Razek", title = "Biochemical and Multiplex PCR Analysis of Toxic Crystal Proteins to Determine Genes in Bacillus thuringiensis Mutants", abstract = "The Egyptian Bacillus thuringiensis isolate (M5) produce crystal proteins that is toxic against insects was irradiated with UV light to induce mutants. Upon testing 10 of the resulting mutants for their toxicity against cotton leafworm larvae, the three mutants 62, 64 and 85 proved to be the most toxic ones. Upon testing these mutants along with their parental isolate by SDS-PAGE analysis of spores-crystals proteins as well as vegetative cells proteins, new induced bands appeared in the three mutants by UV radiation and also they showed disappearance of some other bands as compared with the wild type isolate. Multiplex PCR technique, with five sets of specific primers, was used to detect the three types of cryI genes cryIAa, cryIAb and cryIAc. Results showed that these three genes exist, as distinctive bands, in the wild type isolate (M5) as well as in mutants 62 and 85, while the mutant 64 had two distinctive bands of cryIAb and cryIAc genes, and a faint band of cryI Aa gene. Finally, these results revealed that mutant 62 is considered as the promising mutant since it is UV resistant, highly toxic against Spodoptera littoralis and active against a wide range of Lepidopteran insects.
", keywords = "Bacillus thuringiensis, biological control, cry1
genes, multiplex PC, SDS- PAGE analysis.", volume = "7", number = "7", pages = "704-6", }
