Expression of a Bacillus thuringiensis crystal protein gene in Pseudomonas cepacia 526

Abstract

An insecticidal crystal protein gene, cryIA(C), from Bacillus thuringiensis HD-1 was cloned into a broad host range vector, pSUP204, and the resulting plasmid, pSUP89A, was conjugated into a plant-colonizing bacterium, Pseudomonas cepacia 526. Southern blot analysis detected the presence of the cry gene in Pseudomonas cepacia 526 transconjugants. Production of a truncated insecticidal crystal protein in Pseudomonas cepacia was detected by Western blotting and ELISA techniques and was found to have insecticidal activity against the tobacco hornworm, Manduca sexta, in an artificial diet assay. We also followed the establishment of the Pseudomonas cepacia 526/Bt transconjugant on axenically grown tobacco plants and found that approximately 1% of the Pseudomonas cells retained pSUP89A 3 days after application. In spite of this instability, treated tobacco plants were protected from Manduca sexta infestation. Key words: Bacillus thuringiensis, Pseudomonas cepacia, insecticidal crystal protein, Cry protein.

Insect Pathogens as Biological Control Agents: Do They Have a Future?

“…Plant-colonizing bacteria including Pseudomonas fluorescens Migula, P. cepacia (Burkholder) Palleroni and Holmes, Rhizobium leguminosarum Jordan, and Azosporillium spp. have also been used to produce and deliver B. thuringiensis insecticidal proteins (Obukowicz et al, 1986a,b;Skot et al, 1990;Stock et al, 1990;Udayasuryian et al, 1995;Schnepf et al, 1998). Specific delivery systems based on the hosts developing in aquatic habitats have also been proposed to control mosquito larvae (Porter et al, 1993).…”

Expression of a Bacillus thuringiensisdelta-endotoxin cry1Ab gene in Bacillus subtilis and Bacillus licheniformis strains that naturally colonize the phylloplane of tomato plants (Lycopersicon esculentum, Mills)

“…The second approach is the use of recombinant DNA techniques to express cry genes in suitable host bacterial strains. The introduction of cry genes into bacteria such as Pseudomonas (Stock et al 1990), cyanobacteria (Murphy and Stevens 1992) and Rhizobium (Skot et al 1990) has been successfully developed. A less explored approach is the utilization of plant‐associated microorganisms that naturally colonize the plant phylloplane as recombinant microbial delivery systems of toxin genes of B. thuringiensis (Bora et al 1994; Moar et al 1994; Downing et al 2000).…”

Genetic Improvement of Bt Strains and Development of Novel Biopesticides

“…This approach has been used by Mycogen, to produce two commercial products: MVP ® for controlling lepidopterans and M-TRAK ® for controlling coleopterans. In order to control root feeding insects inaccessible to conventional spray technology, the cry1Ac gene has also been transferred into the plant colonizers Pseudomonas cepacia (Stock et al 1990) and Azospirillum spp. (Udayasuriyan et al 1995), two bacteria that colonize the roots and leaves of many plants.…”