Bacillus thuringiensis mutants which produce higher yields...

Details Bacillus thuringiensis mutants which produce higher yields... Bacillus thuringiensis mutants which produce higher yields...

1997-07-29

2001-04-03

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

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

Bacteria or actinomycetales; media therefor

C435S252310, C435S173300, C424S093461, C424S093460

Reexamination Certificate

active

06210952

ABSTRACT:

1. FIELD OF THE INVENTION
The invention relates to a mutant of
Bacillus thuringiensis
which produces a larger amount of crystal delta-endotoxin with a greater pesticidal activity as compared to the corresponding parental strain. The mutant may also have a larger crystal size as compared to the corresponding parental strain. The crystal delta-endotoxin produced by the mutant
Bacillus thuringiensis
will have an activity directed towards the same pest(s) as its parental
Bacillus thuringiensis
crystal delta-endotoxin. The invention further relates to a method for producing such a mutant, compositions comprising such a mutant as well as methods for controlling a pest(s) using these compositions.
2. 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 order Lepidoptera, Diptera, or Coleoptera. The inclusion(s) may vary in shape, number, and composition. They are comprised of one or more proteins called crystal delta-endotoxins, which may range in size from 27-140 kDa. The insecticidal crystal 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 crystal delta-endotoxins specific for Lepidoptera.
Bacillus thuringiensis
subsp.
israelensis
has been found to produce crystal 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 crystal delta-endotoxin specific for Coleoptera. Several
Bacillus thuringiensis
crystal delta-endotoxin proteins are also reportedly pesticidal to nematodes, Acari, Hymenoptera, Phthiraptera, Platyhelminthes, Homoptera, Blattodea, and Protozoa.
The isolation of another coleopteran toxic
Bacillus thuringiensis
strain was reported in 1986 (Herrnstadt et al., 1986,
Bio/Technology
4:305-308; Herrnstadt 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 (Herrnstadt 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 delta-endotoxins 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,
Microbial. 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).
The utility of
Bacillus thuringiensis
strains for the control of pests is dependent upon efficient and economical production of the active toxins. This in turn is dependent upon the amount of crystal delta-endotoxins which can be produced by fermentation of the active
Bacillus thuringiensis
strains.
Consequently a recognized need for products of improved strength exists.
One way to fulfill this need would be to concentrate the preparations. However, this would add considerably to the production cost in comparison to the savings obtained in storage and transportation.
A much more elegant solution would be to create mutants of existing B.t. strains which produce substantially larger amounts of delta-endotoxin and have a substantially higher amount of pesticidal activity compared to its parental strain. Such mutants would give a more efficient and economical production of active delta-endotoxins and a possibility for manufacture of B.t. products with increased potency at equal or lower cost. This in turn would be an advantage for the user as reduced volumes of pesticide formulation have to be stored and handled for a given acreage. In addition, the users will have less container material to dispose of, thereby reducing the impact on the environment.
3. SUMMARY OF THE INVENTION
The invention is directed to a mutant that produces larger amounts of crystal delta-endotoxin than the corresponding parental strain, preferably more than about 1.25 times and most preferably more than about 1.5 times the amount with greater pesticidal activity and has activity directed towards the same pest as a corresponding parental strain.
As defined herein, a “parental strain” is the original
Bacillus thuringiensis
strain before mutagenesis. In a specific embodiment, the parental strain is a wild-type strain.
“Greater Pesticidal activity” as defined herein means at least 1.25 times and preferably more than about 1.5 more activity against a pest, times through killing or stunting of the growth of the pest, than the corresponding parental strain. In a preferred embodiment, the pesticidal activity of the mutant is between about 1.5 to about 5 times greater than the pesticidal activity of the corresponding parental
Bacillus thuringiensis
strain.
In a specific embodiment, the mutant may also have a larger crystal volume, preferably more than 1.25 times and most preferably more than twice the volume than the corresponding parental strain. The volume may be determined by photographing spore/crystal preparations using a microscope with a camera attachment. Measurements of the crystals in millimeters may be made, and then normalized to the average length of the spores in each photo to account for any differences in photo enlargement. The volume for the bipyramidal crystals is calculated using the following formula: V=(width
2
×length)/3. Additionally, the mutant

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