Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-06-30
2001-03-06
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S350000
Reexamination Certificate
active
06197747
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention is directed to a plant-colonizing microorganism, which has been engineered to contain heterologous DNA coding for a high molecular weight protein having insecticidal activity against lepidopterous larvae. The invention is also directed to insecticidal compositions containing such microorganisms as the active agent and to the use of such plant-colonizing microorganisms in a method of combatting lepidopterous pests.
Bacillus thuringiensis
is a spore forming soil bacterium which is known for its ability to produce a parasporal crystal which is lethal to a wide variety of lepidopteran larvae. The crystals, which account for 20-30% of the dry weight of sporulated cultures, are composed primarily of a single, high molecular weight protein (134,000 daltons) which is synthesized only during sporulation.
Whiteley et al (1) reported the isolation of plasmid DNA from
Bacillus thuringiensis var. kurstaki
HD-1, insertion of said DNA into the cloning vector pBR322 and transformation into
Escherichia coli
strain HB101. Colonies presumed to contain recombinant plasmids were screened for production of an antigen that would react with an antibody made against
B. thuringiensis
crystal protein toxin. One recombinant strain, identified as ES12, was isolated which synthesized a polypeptide of 130,000 daltons which reacted with antibody directed to the crystal protein. Protein extracts of ES12 were toxic to larvae of the tobacco hornworm,
Manduca sexta
. The amounts of polypeptide produced were very low compared to those that can be produced by
B. thuringiensis
. This appeared to be due to the different methods of regulation of protein production in
B. thuringiensis
and
E. coli.
Klier et al (2) reported that the crystal protein gene of
Bacillus thuringiensis
strain berliner 1715 occurred on both a large host plasmid and on the chromosomal DNA. A DNA sequence corresponding to the chromosomal sequence was inserted into plasmid pBT 15-88. The inserted sequence of pBT 15-88 was not expressed in
E. coli
. A 14 Kb BamHI DNA fragment from the 42 megadalton host plasmid was cloned into the BamHI site of pHV33 and this vector was inserted into
E. coli
. Extracts of
E. coli
containing the recombinant plasmid were immunologically cross-reactive against antibodies directed against purified crystal protein. The polypeptide synthesized by
E. coli
containing the recombinant plasmid had approximately 10% the activity of that synthesized by sporulating cells of
B. thuringiensis
. Five-fold concentrated extract of
E. coli
harboring the recombinant plasmid when spread on cabbage leaves and fed ad libitum were toxic to the larvae of
Pierris brassica
. Klier also inserted pHV33 containing the 14 Kb insert into
B. subtilis
. The crystal protein gene was not expressed in vegetative cells of
B. subtilis
although it was expressed in sporulating cells, the amount of crystal protein produced by the sporulating cells was about 10% of that produced by sporulating
B. thuringiensis.
Held et al (3) obtained DNA fragments of
B. thuringiensis var. kurstaki
by EcoRI digestion and cloned these fragments into the vector Charon 4A.
E. coli
were infected with a recombinant bacteriophage, C4R6C, consisting of cloning vector Charon 4A and DNA from
B. thuringiensis
. These infected cells produced protoxin antigen which was the same size as the
B. thuringiensis
protoxin and protein extracts were toxic to neonate larvae of
Manduca sexta
. Hybridiza-tion of C4K6C DNA to
B. thuringiensis
plasmids indicated that the original Charon 4A clone contained the genes of chromosomal, not plasmid origin.
Wong et al (4) reported the nucleotide sequence of the promoter region and part of the coding region of the crystal protein gene from
B. thuringiensis var. kurstaki
HD-1-Dipel. A potential ribosome binding site of 11 nucleotides was located three nucleotides upstream from the initiator ATG codon. The deduced sequence for the first 333 amino acids of the crystal protein was reported.
U.S. Pat. No. 4,448,885 describes plasmids capable of replicating in an
E. coli
bacterial host species which contains expressible heterologous DNA coding for a polypeptide of 130,000 daltons which has the immunological properties of the crystal protein of
B. thuringiensis
. Also disclosed is an
E. coli
bacterial strain transformed to express a polypeptide of 130,000 daltons which reportedly has immunological properties of the crystal protein of
B. thuringiensis
. A method of using said bacterial strains to produce an insecticidal effect is also disclosed.
Commercial insecticidal preparations containing spores and crystalline protein produced by
Bacillus thuringiensis
are available as wettable powders and aqueous suspensions under such names as Dipel® and Thuricide®. These materials are used for the control of lepidopteran larvae such as Spruce budworm, cabbage looper, imported cabbage worm, gypsy moth, etc., which prey upon tobacco, cotton, soybeans, etc.
Significant limitations to the use of commercial preparations of crystalline endotoxin of
Bacillus thuringiensis
include the need for repeated applications of the insecticidal preparations and limitation of the insect target range. Another disadvantage is that the crystal protein is only produced during the sporulation stage of the
B. thuringiensis
life cycle. Such a growth phase limitation, particularly in an industrial process, can result in inconvenience and excessive time requirements during manufacture. At the completion of sporulation, the self-lysing cells release both spores and crystals into the culture medium. Because of environmental concerns it is desirable that commercial insecticidal preparations be substantially free of spores. However, because of the similarity in size and density of the spores and crystal protein toxin, separation of the crystals from the spores is complicated and laborious and thus, costly. Further, pressures resulting from growth phase limitations or other factors may result in strains of
B. thuringiensis
losing their ability to produce the crystals; such acrystalliferous strains do not have insecticidal activity.
Although the isolation of DNA from
B. thuringiensis
coding for the crystal protein toxin and the insertion of this DNA into expression vectors for the transformation of
E. coli
or
B. subtilis
is known, the prior art does not teach that such DNA can be inserted into plant-colonizing microorganisms, that such DNA will be expressed and that the plant-colonizing microorganism will have insecticidal activity against lepidopteran pests. Nor does the art teach that such plant-colonizing microorganisms can live and grow in the “plant environment” and give contact or systemic season long insect control avoiding the need for repeated applications of the insecticidal crystal protein. The delivery of insecticidal protein via a genetically engineered plant-colonizing microorganism which colonizes the “plant environment” and which expresses the insecticidal protein in the plant environment, i.e., on the leaf, stem, stalk, floral parts or root surface is unexpected in view of the prior art which is directed to the production of insecticidal crystal protein in culture.
The insecticidally active genetically engineered plant-colonizing microorganisms of the present invention thus provide a superior methods of combatting certain lepidopterous insects which avoids the problems associated with the use of conventional chemical insecticides and which avoids the problems and expense related to the production of the insecticidally active protein in culture and separation and purification of the insecticidal protein from the culture medium.
SUMMARY OF THE INVENTION
The invention relates to genetically engineered plant-colonizing microorganisms which proliferate in symbiotic or non-detrimental relationships with the plant in the plant environment. Such microorganisms contain DNA derived from
Bacillus thuringiensis
which codes for the insecticidal crystal protein toxin. The engineered plant-col
Perlak Frederick J.
Watrud Lidia S.
Ball, Esq. Timothy K.
Howrey Simon Arnold & White , LLP
Low Christopher S. F.
Monsanto Company
Srivastava Devesh
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