Integration of the delta-endotoxin gene of Bacillus thuringiensis into the chromosome of root-colonizing strains of pseudomonads using Tn5

Obukowicz, Mark G.; Perlak, Frederick J.; Kusano-Kretzmer, Kuniko; Mayer, Ernest; Watrud, Lidia S.

doi:10.1016/0378-1119(86)90031-4

Cited by 86 publications

(32 citation statements)

References 14 publications

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“…Construction of the genes (25,26) and purification of the Escherichia coli-expressed gene products (27,28) have been described. The Pseudomonas fluorescens strain expressing the cryIA(b) gene, Psll2-12a, was constructed (29) and fermented (8), as described. CryIA(c) protein was purified from B. thuringiensis ssp.…”

Section: Methodsmentioning

confidence: 99%

Binding of Bacillus thuringiensis proteins to a laboratory-selected line of Heliothis virescens.

MacIntosh

Stone

Jokerst

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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A laboratory-selected colony of Heliothis virescens displaying a 20-to 70-fold level of resistance to Bacilus thuringiensis proteins was evaluated to identify mechanism(s) of resistance. Brush-border membrane vesicles were isolated from larval midgut epithelium from the susceptible and resistant strains ofH. virescens. Two B. thuringiensis proteins, CryIA(b) and CryIA(c), were iodinated and shown to specifically bind to brush-border membrane vesicles of both insect strains. Multiple changes in the receptor-binding parameters were seen in the resistant strain as compared with the susceptible strain. A 2-to 4-fold reduction in binding affinity was accompanied by a 4-to 6-fold increase in binding-site concentration for both proteins. Although these two B. thuringiensis proteins competed for the same high-affinity binding site, competition experiments revealed different receptor specificity toward these proteins in the resistant H. virescens line. The H. virescens strains were not sensitive to a coleopteran-active protein, CryMA, nor did these proteins compete with the CryLA proteins for binding. Complexity of the mechanism of resistance is consistent with the complex mode of action of B. thuringiensis proteins.Insect-control proteins from Bacillus thuringiensis ssp. kurstaki are active against a wide range of agronomically important lepidopteran larvae (1, 2). Use of these proteins in optimized microbial strains and genetically improved plants will become increasingly important for insect control (3), particularly as the popularity of many commercial chemical insecticides is declining due to the onset of resistance by target pests (4). Although commercial preparations of B. thuringiensis strains have been used for >25 yr, only recently have insects with reduced susceptibility been identified (5) and obtained in laboratory-selection experiments (6-8). Management strategies are being developed to prevent or delay the onset of insect resistance to assure the long-term efficacy of B. thuringiensis proteins. Biochemical characterization of B. thuringiensis proteins, their mode of action, and mechanisms of increased resistance are critical for the development of appropriate management strategies.The mode of action of B. thuringiensis protein insecticides is complex, as evidenced in a number of reports over the last few years (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Upon ingestion by the insect, the proteins are proteolytically processed (9-14), cross the peritrophic membrane, and bind to high-affinity receptors on the midgut epithelium (15)(16)(17)(18)(19)(20). The membrane-bound B. thuringiensis protein disrupts the membrane integrity by forming a pore, causing an electrolyte imbalance that ultimately kills the insect (21,22). Receptor binding has been analyzed by usingmidgut brush-border vesicles from the gut epithelium from various lepidopteran larvae and for several B. thuringiensis proteins (16,(18)(19)(20)23). High-affinity binding sites were initially identified that directly correlated with the ob...

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Section: Methodsmentioning

confidence: 99%

Binding of Bacillus thuringiensis proteins to a laboratory-selected line of Heliothis virescens.

MacIntosh

Stone

Jokerst

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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“…are the insect control proteins deposited as crystals within the cell. Insect control protein genes are expressed well in Escherichia coli (7) or Pseudomonas (8). Poor expression in plants is a well-reported characteristic of the B.t.k.…”

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confidence: 99%

Modification of the coding sequence enhances plant expression of insect control protein genes.

Perlak

Fuchs

Dean

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

505

263

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Increased expression of the insect control protein genes ofBacilus thunrngiensis in plants has been critical to the development of genetically improved plants with agronomically acceptable levels of insect resistance. The expression of the cryIA(b) gene was compared to partially modIfied (3% nucleotide difference) and to fully modified (21% nucleotide difference) crylA(b) and crylA(c) genes in tobacco and tomato. The modified genes increased the frequency of plants that produced the proteins at quantities sufficient to control insects and dramatically increased the levels of these proteins. Among the most highly expressing transformed plants for each gene, the plants with the partially modified crylA(b) gene had a 10-fold higher level ofinsect control protein and plants with the fully modified crylA(b) had a 100-fold higher level of CryIA Insect control proteins from a prokaryotic source, Bacillus thuringiensis var. kurstaki (B.t.k.; ref. 1) are specific for lepidopteran insects and exhibit no activity against humans, other vertebrates, and beneficial insects (2). These properties have made the genes of these insect-specific proteins attractive candidates for genetic modification of crops for protection against lepidopteran pests. Genes encoding lepidopteran-specific insect control proteins have been cloned and sequenced. Truncated genes, which produce insecticidally active protein, have been expressed in tomato (3), tobacco (4), and cotton (5). Field tests of these plants revealed that higher levels of insect control protein in the plant tissue would be required to obtain commercially useful plants (6).The insect control proteins are highly expressed in their natural host, B. thuringiensis. Up to 50% of the total protein in sporulated cultures ofB.t.k. are the insect control proteins deposited as crystals within the cell. Insect control protein genes are expressed well in Escherichia coli (7) or Pseudomonas (8). Poor expression in plants is a well-reported characteristic of the B.t.k. insect control proteins. Truncating the gene, keeping essentially the N-terminal half of the protein intact, results in improved expression of the gene in plants to barely detectable levels (3, 4). The use ofdifferent promoters, fusion proteins, and leader sequences has not significantly increased insect control protein gene expression (4, 9).We hypothesized that a gene with a sequence adapted for a Gram-positive prokaryote may not be the appropriate coding sequence for efficient plant expression. Examination of the insect control protein gene coding sequence indicated that it differs significantly from plant genes in G+C content. Multiple sequences motifs that are not common in the coding region of plant genes were found to be common in the wild-type (WT) crylA(b) sequence. These included localized regions of A+T richness resembling plant introns (10), potential plant polyadenylylation signal sequences (11), ATTTA sequences, which have been shown to destabilize mRNA in other systems (12), and codons rarely used in plants...

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“…thuringiensis cry genes have been introduced into bacteria other than B. thuringiensis, such as the root colonizers P. fluorescens and Agrobacterium radiobacter and Ancylobacter aquaticus, a bacterium isolated from aquatic habitats. These strains were toxic against the larvae of the tobacco hornworm (Manduca sexta), the malaria mosquito Anopheles stephensi, and the leatherjacket (Tipula oleracea) (16,23,24,35). The introduction of the cryIA(c) gene from B. thuringiensis subsp.…”

Section: Discussionmentioning

confidence: 99%

“…It also carries a disabled IS1 element which enables transposition of the Omegon-Km cassette although it cannot transpose itself, resulting in stably integrated genes. Although the stability of the ptac-cry1Ac7 cassette in P. fluorescens 14 was not investigated, Herrera (12) showed that the Omegon-Km-cry cassette in P. fluorescens 14 was stably integrated for at least 100 generations and stable integration of cry genes into rootcolonizing P. fluorescens strains using a transposon Tn5-mediated system or suicide vectors for integration by homologous recombination have been described in the literature (23,24,35).…”

Section: Discussionmentioning

confidence: 99%

Biocontrol of the Sugarcane Borer Eldana saccharina by Expression of the Bacillus thuringiensis cry1Ac7 and Serratia marcescens chiA Genes in Sugarcane-Associated Bacteria

Downing

Leslie

Thomson

2000

Appl Environ Microbiol

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The cry1Ac7 gene of Bacillus thuringiensis strain 234, showing activity against the sugarcane borer Eldana saccharina, was cloned under the control of the tac promoter. The fusion was introduced into the broad-hostrange plasmid pKT240 and the integration vector pJFF350 and without the tac promoter into the broad-hostrange plasmids pML122 and pKmM0. These plasmids were introduced into a Pseudomonas fluorescens strain isolated from the phylloplane of sugarcane and the endophytic bacterium Herbaspirillum seropedicae found in sugarcane. The ptac-cry1Ac7 construct was introduced into the chromosome of P. fluorescens using the integration vector pJFF350 carrying the artificial interposon Omegon-Km. Western blot analysis showed that the expression levels of the integrated cry1Ac7 gene were much higher under the control of the tac promoter than under the control of its endogenous promoter. It was also determined that multicopy expression in P. fluorescens and H. seropedicae of ptac-cry1Ac7 carried on pKT240 caused plasmid instability with no detectable protein expression. In H. seropedicae, more Cry1Ac7 toxin was produced when the gene was cloned under the control of the Nm r promoter on pML122 than in the opposite orientation and bioassays showed that the former resulted in higher mortality of E. saccharina larvae than the latter. P. fluorescens 14::ptac-tox resulted in higher mortality of larvae than did P. fluorescens 14::tox. An increased toxic effect was observed when P. fluorescens 14::ptac-tox was combined with P. fluorescens carrying the Serratia marcescens chitinase gene chiA, under the control of the tac promoter, integrated into the chromosome.The gram-positive, aerobic, spore-forming bacterium Bacillus thuringiensis has been used as a safe alternative and supplement to chemical pesticides for over 2 decades. It is a pathogen of insect larvae which produces highly specific crystal inclusions during sporulation. These parasporal crystals consist predominantly of protoxin molecules known as ␦-endotoxins, Cry toxins, or Cry proteins. The crystal inclusions dissolve in the larval midgut, where one or more protoxins are released and proteolytically converted into smaller toxic polypeptides. The activated toxins are highly specific to the insect and very specific in their activity (14). Despite the success of conventional B. thuringiensis-based products, they have several disadvantages as bioinsecticides. In the case of the sugarcane borer Eldana saccharina Walker (Lepidoptera: Pyralidae), a widespread sugarcane pest which causes considerable crop loss in the cane-growing areas of South Africa and Swaziland, these include instability in the environment and on the surface of sugarcane, as well as difficulty in reaching the internal regions where the larvae feed. The use of recombinant DNA technology has provided solutions to the problems through the development of two approaches, namely, genetically modified microorganisms and transgenic plants (18,21,22,25,26).As part of an integrated pest management approach to th...

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Integration of the delta-endotoxin gene of Bacillus thuringiensis into the chromosome of root-colonizing strains of pseudomonads using Tn5

Cited by 86 publications

References 14 publications

Binding of Bacillus thuringiensis proteins to a laboratory-selected line of Heliothis virescens.

Binding of Bacillus thuringiensis proteins to a laboratory-selected line of Heliothis virescens.

Modification of the coding sequence enhances plant expression of insect control protein genes.

Biocontrol of the Sugarcane Borer Eldana saccharina by Expression of the Bacillus thuringiensis cry1Ac7 and Serratia marcescens chiA Genes in Sugarcane-Associated Bacteria

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