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Recent developments in the biotechnology of Bacillus thuringiensis
Biotechnology Advances 18 (2000) 143–145
Meeting report
Recent developments in the biotechnology of Bacillus thuringiensis The 3rd Pacific Rim Conference on the Biotechnology of Bacillus thuringiensis was held at Huazhong Agricultural University, Wuhan, China, October 5–9, 1999 attracting 114 participants from 18 countries. The 104 papers that were presented at this meeting were compiled as a proceedings volume entitled ‘Biotechnology of Bacillus thuringiensis’ (Ziniu Yu, Ming Sun, and Ziduo Liu, Editors) and published by Science Press, Beijing. Some of the highlights of this meeting are summarized in this report. 1. Screening and identification of strains and pesticidal crystal protein genes Even though a large number of B. thuringiensis strains have been isolated, and 28 groups of insecticidal crystal protein genes have been cloned, evaluation of B. thuringiensis strains is ongoing. Some insect pests are highly sensitive to B. thuringiensis, but most are not. Thus, it is necessary to continue to isolate and characterize new and more effective strains of B. thuringiensis. Several labs reported the isolation of highly toxic B. thuringiensis strains effective against Noctuida larvae including Spodoptera exigua and S. littoralis. B. thuringiensis strains with toxicity against lepidopteran larvae such as Tribolium castaneum, Oryzaephlius surinamensis, and Trichoplusia ni; dipteran larvae such as Aedes aegypti; coleopteran larvae such as scarabaeid beetles, Anomala exleta, A. corpulenta, and A. cuprea, were reported to be isolated and analyzed. Thuringiensin- and Zwittermicin-producing strains and its synergistic activity were characterized. Some labs provided detailed reports of their extensive B. thuringiensis culture collections. Various PCR techniques have been used to identify the crystal protein genes in highly toxic strains or to screen specific genes in B. thuringiensis collections. The genes that attract the most interest are cryl type genes, such as those highly toxic to the most agriculturally important insects including diamondback moth, cotton worm, and Spodoptera larvae. Efforts to identify cry1I, cry8, cry9 genes and coleopteran-specific genes are also very active. 2. Characterization of insecticidal crystal proteins and their genes Bacillus thuringiensis subsp. wuhanensis was analyzed and shown to produce novel Cry1Ab, Cry1Ac, and Cry1D crystal proteins whose toxicity against silkworm, diamondback moth, and common cutworm is different from that of the typical Cry proteins. The first binary toxin was reported in B. thuringiensis subsp. thompsoni. Previously, only the Cry34 0734-9750/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S0 7 3 4 - 9 7 5 0 ( 0 0 ) 0 0 0 2 9 -X
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Meeting Report/Biotechnology Advances 18 (2000) 143–145
protein was found to be toxic, but not Cry40. Further, helper protein genes p19 and p20 were isolated and their function was analyzed. It was found that the helper protein genes can increase the expression of crystal protein genes and thereby increase the total toxicity of the target strains, as well as facilitate the crystallization of Cry1Ac protein. An interesting finding is that the crystal inclusion body of B. thuringiensis strain CTC consists entirely of a cell surface-like protein, a B. anthracis-like S-layer protein. This raises the question, that, if a cell structural component forms an inclusion body during sporulation, whether the strain can be recognized as B. thuringiensis. Finally, the toxicity against animal parasitic nematodes, and the related crystal proteins in some strains were characterized.
3. Genetics and gene functioning To remain competitive, improved B. thuringiensis insecticide strains are being constructed to produce novel combinations of toxins, additional toxic proteins, and a higher level of toxin production. These modifications should increase the efficacy and enlarge the insect spectrum of existing B. thuringiensis toxins. In addition to the more than 170 crystal protein genes cloned and sequenced, genetic elements, helper protein genes p19, p20, and p21, the use of strong promoters, mRNA stabilizing sequences, and various 3⬘ stem-loop structures have been used to increase the production of toxin proteins from 1.25- to 10-fold. In addition, the transcriptional regulation mechanism of the cry1 gene was reported. In the future, manipulation of this sequence may also be used to overproduce B. thuringiensis toxins. The construction of several unique delivery systems was reported. In one instance, a plasmid replication origin from B. thuringiensis subsp. tenebrionis was isolated and used to construct a genetically stable plasmid vector. A vector based on transposon Tn4430 was also constructed, and a novel mobile insertion cassette MIC231 from Bacillus cereus was identified. The transcriptional regulation mechanism of the cry1 gene upstream sequence was reported. Some small plasmids from B. thuringiensis were compared from different strains, and these might also someday serve as vectors for cry genes.
4. Mechanism of action and insect resistance The mechanism of binding of the B. thuringiensis Cry1Ac toxin to Lymantria dispar APN was reported. It was found that the binding of Cry1Ac to gypsy moth APN is a sequential two-step binding mechanism. The first step is a fast-on, fast-off binding of domain III to the GalNAc moiety on the APN receptor. This step determines the on-rate of the overall reaction and must precede the second step, which is a slow-on, slow-off binding of domain III to amino acid residues of the APN receptor. Several basic and hydrophobic residues are known to be important in binding to the APN receptor, and hydrophobic residues play a critical role in the off-rate. Substitution of domain III of the Cry1Ac protein confers toxicity against the lepidopteran Trichoplusia ni to the coleopteran-specific Cry3A protein. The function of Cry4A, and the role of interdomain salt bridges in the pore-forming ability of this protein were analyzed. The pore formation activity of Cry toxins in membranes isolated from differ-
Meeting Report/Biotechnology Advances 18 (2000) 143–145
145
ent insect orders was compared. A comparison of the binding properties of Cry1A and Cry9Ca proteins to the midgut brush border membranes of Culex pipiens and Bombyx mori was reported. Finally, insect resistance of Spodoptera exigua and Plutella xylostella to B. thuringiensis was reported. 5. Transgenic plants The properties and the application of transgenic cotton, canola and rapeseed, poplar, coffee, rice, and tropical maize, all expressing B. thuringiensis toxins, were reported. This work used a synthetic cry1C gene, a partially modified cry1Ac gene, an arrowhead proteinase inhibitor gene, a synthetic cry1Ac gene, and a fully modified cry1B gene. For transgenic cotton in Thailand, the efficacy ranged from 0 to 60%. The potential for using transgenic rice in China to protect rice from insect attacks was shown. The development of insect resistance to transgenic plants expressing B. thuringiensis toxins is a concern and is currently being monitored and analyzed in Australia. 6. Production and application Production strategies for B. thuringiensis toxins have been developed in China using various types of media including organic wastewater. Fermentation strategies for the production of recombinant B. thuringiensis were also studied. The application and efficacy of B. thuringiensis products in China, Canada, Germany, Egypt, Brazil, and Malaysia were reported. Finally, human health surveillance monitoring in Canada was reported. Next meeting The 4th Pacific Rim Conference on the Biotechnology of Bacillus thuringiensis will be held in Sydney, Australia, in 2001.
Ming Sun and Ziniu Yu Department of Microbial Science and Technology Huazhong Agricultural University Wuhan, Hubei 430070 P.R. China
Meeting report
Recent developments in the biotechnology of Bacillus thuringiensis The 3rd Pacific Rim Conference on the Biotechnology of Bacillus thuringiensis was held at Huazhong Agricultural University, Wuhan, China, October 5–9, 1999 attracting 114 participants from 18 countries. The 104 papers that were presented at this meeting were compiled as a proceedings volume entitled ‘Biotechnology of Bacillus thuringiensis’ (Ziniu Yu, Ming Sun, and Ziduo Liu, Editors) and published by Science Press, Beijing. Some of the highlights of this meeting are summarized in this report. 1. Screening and identification of strains and pesticidal crystal protein genes Even though a large number of B. thuringiensis strains have been isolated, and 28 groups of insecticidal crystal protein genes have been cloned, evaluation of B. thuringiensis strains is ongoing. Some insect pests are highly sensitive to B. thuringiensis, but most are not. Thus, it is necessary to continue to isolate and characterize new and more effective strains of B. thuringiensis. Several labs reported the isolation of highly toxic B. thuringiensis strains effective against Noctuida larvae including Spodoptera exigua and S. littoralis. B. thuringiensis strains with toxicity against lepidopteran larvae such as Tribolium castaneum, Oryzaephlius surinamensis, and Trichoplusia ni; dipteran larvae such as Aedes aegypti; coleopteran larvae such as scarabaeid beetles, Anomala exleta, A. corpulenta, and A. cuprea, were reported to be isolated and analyzed. Thuringiensin- and Zwittermicin-producing strains and its synergistic activity were characterized. Some labs provided detailed reports of their extensive B. thuringiensis culture collections. Various PCR techniques have been used to identify the crystal protein genes in highly toxic strains or to screen specific genes in B. thuringiensis collections. The genes that attract the most interest are cryl type genes, such as those highly toxic to the most agriculturally important insects including diamondback moth, cotton worm, and Spodoptera larvae. Efforts to identify cry1I, cry8, cry9 genes and coleopteran-specific genes are also very active. 2. Characterization of insecticidal crystal proteins and their genes Bacillus thuringiensis subsp. wuhanensis was analyzed and shown to produce novel Cry1Ab, Cry1Ac, and Cry1D crystal proteins whose toxicity against silkworm, diamondback moth, and common cutworm is different from that of the typical Cry proteins. The first binary toxin was reported in B. thuringiensis subsp. thompsoni. Previously, only the Cry34 0734-9750/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S0 7 3 4 - 9 7 5 0 ( 0 0 ) 0 0 0 2 9 -X
144
Meeting Report/Biotechnology Advances 18 (2000) 143–145
protein was found to be toxic, but not Cry40. Further, helper protein genes p19 and p20 were isolated and their function was analyzed. It was found that the helper protein genes can increase the expression of crystal protein genes and thereby increase the total toxicity of the target strains, as well as facilitate the crystallization of Cry1Ac protein. An interesting finding is that the crystal inclusion body of B. thuringiensis strain CTC consists entirely of a cell surface-like protein, a B. anthracis-like S-layer protein. This raises the question, that, if a cell structural component forms an inclusion body during sporulation, whether the strain can be recognized as B. thuringiensis. Finally, the toxicity against animal parasitic nematodes, and the related crystal proteins in some strains were characterized.
3. Genetics and gene functioning To remain competitive, improved B. thuringiensis insecticide strains are being constructed to produce novel combinations of toxins, additional toxic proteins, and a higher level of toxin production. These modifications should increase the efficacy and enlarge the insect spectrum of existing B. thuringiensis toxins. In addition to the more than 170 crystal protein genes cloned and sequenced, genetic elements, helper protein genes p19, p20, and p21, the use of strong promoters, mRNA stabilizing sequences, and various 3⬘ stem-loop structures have been used to increase the production of toxin proteins from 1.25- to 10-fold. In addition, the transcriptional regulation mechanism of the cry1 gene was reported. In the future, manipulation of this sequence may also be used to overproduce B. thuringiensis toxins. The construction of several unique delivery systems was reported. In one instance, a plasmid replication origin from B. thuringiensis subsp. tenebrionis was isolated and used to construct a genetically stable plasmid vector. A vector based on transposon Tn4430 was also constructed, and a novel mobile insertion cassette MIC231 from Bacillus cereus was identified. The transcriptional regulation mechanism of the cry1 gene upstream sequence was reported. Some small plasmids from B. thuringiensis were compared from different strains, and these might also someday serve as vectors for cry genes.
4. Mechanism of action and insect resistance The mechanism of binding of the B. thuringiensis Cry1Ac toxin to Lymantria dispar APN was reported. It was found that the binding of Cry1Ac to gypsy moth APN is a sequential two-step binding mechanism. The first step is a fast-on, fast-off binding of domain III to the GalNAc moiety on the APN receptor. This step determines the on-rate of the overall reaction and must precede the second step, which is a slow-on, slow-off binding of domain III to amino acid residues of the APN receptor. Several basic and hydrophobic residues are known to be important in binding to the APN receptor, and hydrophobic residues play a critical role in the off-rate. Substitution of domain III of the Cry1Ac protein confers toxicity against the lepidopteran Trichoplusia ni to the coleopteran-specific Cry3A protein. The function of Cry4A, and the role of interdomain salt bridges in the pore-forming ability of this protein were analyzed. The pore formation activity of Cry toxins in membranes isolated from differ-
Meeting Report/Biotechnology Advances 18 (2000) 143–145
145
ent insect orders was compared. A comparison of the binding properties of Cry1A and Cry9Ca proteins to the midgut brush border membranes of Culex pipiens and Bombyx mori was reported. Finally, insect resistance of Spodoptera exigua and Plutella xylostella to B. thuringiensis was reported. 5. Transgenic plants The properties and the application of transgenic cotton, canola and rapeseed, poplar, coffee, rice, and tropical maize, all expressing B. thuringiensis toxins, were reported. This work used a synthetic cry1C gene, a partially modified cry1Ac gene, an arrowhead proteinase inhibitor gene, a synthetic cry1Ac gene, and a fully modified cry1B gene. For transgenic cotton in Thailand, the efficacy ranged from 0 to 60%. The potential for using transgenic rice in China to protect rice from insect attacks was shown. The development of insect resistance to transgenic plants expressing B. thuringiensis toxins is a concern and is currently being monitored and analyzed in Australia. 6. Production and application Production strategies for B. thuringiensis toxins have been developed in China using various types of media including organic wastewater. Fermentation strategies for the production of recombinant B. thuringiensis were also studied. The application and efficacy of B. thuringiensis products in China, Canada, Germany, Egypt, Brazil, and Malaysia were reported. Finally, human health surveillance monitoring in Canada was reported. Next meeting The 4th Pacific Rim Conference on the Biotechnology of Bacillus thuringiensis will be held in Sydney, Australia, in 2001.
Ming Sun and Ziniu Yu Department of Microbial Science and Technology Huazhong Agricultural University Wuhan, Hubei 430070 P.R. China