Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1996-12-20
2001-10-16
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S485000
Reexamination Certificate
active
06303382
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods for obtaining an integrant(s) of
Bacillus thuringiensis
which produces a larger quantity of a crystal delta-endotoxin with greater pesticidal activity and optionally a larger crystal size as compared to a corresponding parental strain. The crystal delta-endotoxin produced by the integrant of
Bacillus thuringiensis
will have activity directed to the same pest(s) as its parental
Bacillus thuringiensis
crystal delta-endotoxin. The invention further relates to such integrant(s), spores or crystal delta-endotoxins thereof, compositions comprising such integrant(s), as well as methods for controlling a pest(s) using these compositions.
BACKGROUND OF THE INVENTION
Every year, pests detrimental to agriculture, forestry, and public health cause losses in the millions of dollars. Various strategies have been used in attempting to control such pests.
One strategy is the use of chemical pesticides with a broad range or spectrum of activity. However, there are a number of disadvantages to using such chemical pesticides. Specifically, because of their broad spectrum of activity, these pesticides may destroy non-target organisms such as beneficial insects and parasites of destructive pests. Additionally, chemical pesticides are frequently toxic to animals and humans. Furthermore, targeted pests frequently develop resistance when repeatedly exposed to such substances.
Another strategy has involved the use of biopesticides, which make use of naturally occurring pathogens to control insect, fungal and weed infestations of crops. An example of a biopesticide is a bacterium which produces a substance toxic to the infesting pest A biopesticide is generally less harmful to non-target organisms and the environment as a whole than chemical pesticides.
The most widely used biopesticide is
Bacillus thuringiensis. Bacillus thuringiensis
is a motile, rod-shaped, gram-positive bacterium that is widely distributed in nature, especially in soil and insect-rich environments. During sporulation,
Bacillus thuringiensis
produces a parasporal crystal inclusion(s) which is insecticidal upon ingestion to susceptible insect larvae of the orders Lepidoptera, Diptera, and Coleoptera. The inclusion(s) may vary in shape, number, and composition. They are comprised of one or more proteins called delta-endotoxins, which may range in size from 27-140 kDa. The insecticidal delta-endotoxins are generally converted by proteases in the larval gut into smaller (truncated) toxic polypeptides, causing midgut destruction, and ultimately, death of the insect (Höfte and Whiteley, 1989,
Microbiol. Rev.
53:242-255).
There are several
Bacillus thuringiensis
strains that are widely used as biopesticides in the forestry, agricultural, and public health areas.
Bacillus thuringiensis
subsp.
kurstaki
and
Bacillus thuringiensis
subsp.
aizawai
have been found to produce delta-endotoxins specific for Lepidoptera.
Bacillus thuringiensis
subsp.
israelensis
has been found to produce delta-endotoxins specific for Diptera (Goldberg, 1979, U.S. Pat. No. 4,166,112).
Bacillus thuringiensis
subsp.
tenebrionis
(Krieg et al., 1988, U.S. Pat. No. 4,766,203), has been found to produce a delta-endotoxin specific for Coleoptera.
Bacillus thuringiensis
subsp.
tenebrionis
has been deposited with the German Collection of Microorganisms under accession number DSM 2803.
Bacillus thuringiensis
subsp.
tenebrionis
was isolated in 1982 from a dead pupa of the mealworm
Tenebrio molitor
(Tenebrionidae, Coleoptera). The strain produces within each cell one spore and one or more pesticidal parasporal crystals which are of flat platelike form with an edge length of about 0.8 &mgr;m to 1.5 &mgr;m. It belongs to serotype H8a,8b and pathotype C of
Bacillus thuringiensis
(Krieg et al., 1987,
System. Appl. Microbiol.
9, 138-141; Krieg et al., 1988, U.S. Pat. No. 4,766,203). It is only toxic against certain leaf-eating beetle larvae (Chrysomelidae), but ineffective against caterpillars (Lepidoptera), mosquitoes (Diptera) or other insects.
The isolation of another coleopteran toxic
Bacillus thuringiensis
strain was reported in 1986 (Herrnstadt et al., 1986,
Bio/Technology
4:305-308; Hermstadt and Soares, 1988, U.S. Pat. No. 4,764,372). This strain, designated “
Bacillus thuringiensis
subsp.
san diego”,
M-7, has been deposited at the Northern Regional Research Laboratory, USA under accession number NRRL B-15939. However, the assignee of the '372 patent, Mycogen, Corp. has publicly acknowledged that
Bacillus thuringiensis
subsp.
san diego
is
Bacillus thuringiensis
subsp.
tenebrionis.
Furthermore, the '372 patent has been assigned to Novo Nordisk A/S. A spo-cry
+
(asporogenous crystal forming) mutant of M-7 has purportedly been obtained by culturing M-7 in the presence of ethidium bromide (Hermstadt and Gaertner, 1987, EP Application No. 228,228). However, there was no indication of increased production of delta-endotoxin, increased parasporal crystal size, and/or increased pesticidal activity relative to the parental, M-7 strain.
The crystal proteins are encoded by cry (crystal protein) genes. The cry genes have been divided into six classes and several subclasses based on relative amino acid homology and pesticidal specificity. The six major classes are Lepidoptera-specific (cryI), Lepidoptera- and Diptera-specific (cryII), Coleoptera-specific (cryIII), Diptera-specific (cryIV) (Höfte and Whiteley, 1989, Microbiol. Rev. 53:242-255), Coleoptera- and Lepidoptera-specific (referred to as cryV genes by Tailor et al., 1992,
Mol. Microbiol.
6:1211-1217); and Nematode-specific (referred to as cryV and cryVI genes by Feitelson et al., 1992, Bio/Technology 10:271-275).
Delta-endotoxins have been produced by recombinant DNA methods. The delta-endotoxins produced by recombinant DNA methods may or may not be in crystal form. Various cry genes have been cloned, sequenced, and expressed in various hosts, e.g.,
E. coli
(Schnepf et al., 1987,
J. Bacteriol.
169:4110-4118),
Bacillus subtilis
(Shivakumar et al., 1986,
J. Bacteriol.
166:194-204), and maize plants (Koziel et al., 1993, Bio/Technology 11:194-200).
Amplification of cry genes has been achieved in
Bacillus subtilis.
The delta-endotoxin gene of
Bacillus thuringiensis
subsp.
kurstaki
HD73 has been cloned into
Bacillus subtilis
using an integrative plasmid and amplified (Calogero et al., 1989,
Appl. Environ. Microbiol.
55:446-453). However, no increase in crystal size was observed as compared to
Bacillus thuringiensis
subsp.
kurstaki
HD73. Furthermore, no difference in pesticidal activity was reported.
The level of expression of delta-endotoxin genes appears to be dependent on the host cell used (Skivakumar et al., 1989, Gene 79:21-31). For example, Skivakumar et al. found significant differences in the expression of the cryIA and cryIIA delta-endotoxin genes of
Bacillus thuringiensis
subsp.
kurstaki
in
Bacillus subtilis
and
Bacillus megaterium.
The cryIA gene was expressed when present on a multicopy vector in
Bacillus megaterium,
but not in
Bacillus subtilis.
The cryIIA gene was expressed in both hosts, but at a higher level in
Bacillus megaterium.
Sections of
Bacillus megaterium
cells expressing these delta-endotoxin genes were examined by electron microscopy; the presence of large bipyramidal crystals in these cells was detected. However, there is no indication that these crystals are any larger than crystals found in
Bacillus thuringiensis
subsp.
kurstaki
which normally contain these genes. Results from bioassays of the
Bacillus megaterium
cells expressing these delta-endotoxin genes indicate that there was no increase in pesticidal activity as compared to
Bacillus thuringiensis
subsp.
kurstaki.
Indeed, five times the concentration of
Bacillus megaterium
than
Bacillus thuringiensis
subsp.
kurstaki
was required to obtain the same insect killing effect.
Recombinant
Bacillus thuringiensis
strains have also been disclosed. Shuttle vectors with various c
Adams Lee Fremont
Sloma Alan P.
Thomas Michael David
Widner William R.
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