Insecticidal protein toxins from Xenorhabdus

Details Insecticidal protein toxins from Xenorhabdus Insecticidal protein toxins from Xenorhabdus

2000-02-28

2002-04-30

Bugaisky, Gabrielle (Department: 1653)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S822000, C424S093400

Reexamination Certificate

active

06379946

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to toxins isolated from bacteria and the use of said toxins as insecticides.
BACKGROUND OF THE INVENTION
In the past there has been interest in using biological agents as an option for pest management. One such method explored was the potential of insect control using certain genera of nematodes. Nematodes, like those of the Steinernema and Heterorhabditis genera, can be used as biological agents due in part to their transmissible insecticidal bacterial symbionts of the genera Xenorhabdus and Photorhabdus, respectively. Upon entry into the insect, the nematodes release their bacterial symbionts into the insect hemolymph where the bacteria reproduce and eventually cause insect death. The nematode then develops and reproduces within the cadaver. Bacteria-containing nematode progeny exit the insect cadaver as infective juveniles which can then invade additional larvae thus repeating the cycle leading to nematode propagation. While this cycle is easily performed on a micro scale in a laboratory setting, adaptation to the macro level, as needed to be effective as a general use insecticide, is difficult, expensive, and inefficient to produce, maintain, distribute and apply.
In addition to biological approaches to pest management such as nematodes, there are now pesticide control agents commercially available that are naturally derived. These naturally derived approaches can be as effective as synthetic chemical approaches. One such naturally occurring agent is the crystal protein toxin produced by the bacteria
Bacillus thuringiensis
(Bt). These protein toxins have been formulated as sprayable insect control agents. A more recent application of Bt technology has been to isolate and transform into plants the genes that produce the toxins. Transgenic plants subsequently produce the Bt toxins thereby providing insect control, (see U.S. Pat. Nos. 5,380,831; 5,567,600; and 5,567,862 to Mycogen in San Diego, Calif.).
Transgenic Bt plants are quite efficacious and usage is predicted to be high in some crops and areas. This has caused a concern that resistance management issues may arise more quickly than with traditional sprayable applications. Thus, it would be quite desirable to discover other bacterial sources distinct from Bt which produce toxins that could be used in transgenic plant strategies, or could be combined with Bts to produce insect controlling transgenic plants.
It has been known in the art that bacteria of the genus Xenorhabdus are symbiotically associated with the Steinernema nematode. Unfortunately, as reported in a number of articles, the bacteria only had pesticidal activity when injected into insect larvae and did not exhibit biological activity when delivered orally (see Jarosz J. et al. “Involvement of Larvicidal Toxins in Pathogenesis of Insect Parasitism with the Rhabditoid Nematodes,
Steinernema Feltiae
and
Heterorhabditis Bacteriophora” Entomophaga
36 (3) 1991 361-368; Balcerzak, Malgorzata “Comparative studies on parasitism caused by entomogenous nematodes,
Steinernema feltiae
and
Heterorhabditis bacteriophors
I. The roles of the nematode-bacterial complex, and of the associated bacteria alone, in pathogenesis”
Acta Parasitologica Polonica,
1991, 36(4); 175-181).
For the reasons stated above it has been difficult to effectively exploit the insecticidal properties of the nematode orits bacterial symbiont. Thus, it would be quite desirable to discover proteinaceous agents derived from Xenorhabdus bacteria that have oral activity so that the products produced therefrom could either be formulated as a sprayable insecticide or the bacterial genes encoding said proteinaceous agents could be isolated and used in the production of transgenic plants. Until applicants' invention herein there was no known Xenorhabdus species or strains that produced protein toxin(s) having oral activity.
SUMMARY OF THE INVENTION
The native toxins are protein complexes that are produced and secreted by growing bacterial cells of the genus Xenorhabdus. The protein complexes, with a native molecular size ranging from about 800 to 3000 kDa, can be separated by SDS-PAGE gel analysis into numerous component proteins. The toxins exhibit significant toxicity upon exposure to a number of insects. Furthermore, toxin activity can be modified by altering media conditions. In addition, the toxins have characteristics of being proteinaceous in that the activity thereof is heat labile and sensitive to proteolysis.
The present invention provides an easily administered functional protein.
The present invention also provides a method for delivering insecticidal toxins that are functionally active and effective against many orders of insects.
Objects, advantages, and features of the present invention will become apparent from the following specification.


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David Joseph Bowen, “Characterization of a High Molecular Weight Insecticidal Protein Complex Produced by the Entomopathogenic BacteriumPhotorhabdus luminescens”Ph.D. Thesis May 1995 U. Wisc.
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Akhurst et al, “A numerical taxonomic study of the genus Xenorhabdus (Enterobacteriaceae) and proposed elevatio of the subspecies ofX. nematophilusto species.” Journal of General Microbiology, vol. 134, No.7, Jul. 1988, pp. 1835-1845.
J. Jarosz et al. “Involvement of larvicidal toxins in pathogenesis of insect parasitism with the rhabditoid nematodes,Steinernema feltiaeandHeterorhabditis bacteriophora.”Entomophaga, vol. 36, No. 3, 1991. pp. 361-368.
M. Balcerzak, “Comparative studies on parasitism caused by entomogenous nematodes,Stenernema feltiaeandHeterorhabditis bacteriophora.”Acta Parasitologica Polonica, 1991, vol. 36, No. 4, pp. 175-181.
S. Frost et al. “Molecular Biology of the Symbiotic-Pathogenic Bacteria Xenorhabdus spp. and Photrhabdus spp.” Microbiological Reviews, Mar. 1996, vol. 60, pp. 21-43.
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Creighton, T.E. Proteins: Structures and Molecular Properties, W.H. Freeman and Company, New York, pp. 23-27 (1993).

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