Production of Bacillus thuringiensis integrants

Details Production of Bacillus thuringiensis integrants Production of Bacillus thuringiensis integrants

1995-01-25

2001-08-28

Degen, Nancy (Department: 1636)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Bacteria or actinomycetales

C435S252310, C435S320100, C536S023100, C536S023710

Reexamination Certificate

active

06280721

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to methods for obtaining an integrant(s) of
Bacillus thuringiensis
. The invention further relates to such integrant(s), or spores thereof, compositions comprising such integrant(s), as well as methods for controlling a pest(s) using these compositions.
BACKGROUND OF THE INVENTION
Pests may be controlled using either chemical pesticides or biopesticides. However, because of their broad spectrum of activity, chemical pesticides may destroy non-target organisms such as beneficial insects and parasites and predators 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.
Biopesticides 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 pest-rich environments. During sporulation,
Bacillus thuringiensis
produces a parasporal crystal inclusion(s) which is toxic upon ingestion to susceptible larvaea. 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 delta-endotoxins are generally converted by proteases in the larval gut into smaller (truncated) toxic polypeptides, causing midgut destruction, and ultimately, death of the pest (Höfte and Whiteley, 1989
, Microbiol. Rev
. 53:242-255).
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
, 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). Several
Bacillus thuringiensis
crystal delta-endotoxins are also reportedly pesticidal to Acari, Hymenoptera, Phthiraptera, Platyhelminthes, Homoptera, Blattodea, and Protozoa.
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) and
Bacillus subtilis
(Shivakumar et al., 1986
, J. Bacteriol
. 166:194-204).
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 megateriun 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.
In the prior art methods, a host cell is transformed with a recombinant DNA vector carrying a DNA sequence encoding a delta-endotoxin and DNA replication sequences. The expression of the delta-endotoxin is dependent on the replication of the recombinant DNA vector in the host. When, for the purpose of producing a desired polypeptide by recombinant DNA procedures, bacterial cells are transformed with a recombinant plasmid vector which carries inserted genetic information coding for the delta-endotoxin, it has often been observed that such plasmids become unstable even though they may, in themselves, be stably inherited in the cell. This instability may either take the form of unstable maintenance of the plasmid in the cells so that the plasmid will eventually be lost from a cell population, or so that the DNA coding for the protein in question may be deleted from the plasmid. A traditional way of solving the former problem has been to grow the transformed cells under selection pressure, that is, typically in the presence of an antibiotic to which the cells in question have been made resistant due to the presence of a gene coding for a product mediating resistance to that antibiotic on the plasmid transformed to the cells. This approach, however, is neither economically feasible in large-scale production due to the high cost of the antibiotics in question, nor is it desirable for environmental reasons. The use of antibiotics in culture media also makes it more difficult to obtain product approval from health authorities and the like.
It has previously been suggested that plasmids could be stabilized by inserting into them a DNA sequence encoding a partitioning function which ensures the even distribution of plasmids to progeny cells on cell division. An alternative method of achieving the stable inheritance of cloned DNA sequences is to provide for the integration of such DNA sequences in the genome of the host bacterium. Integration of DNA sequences present on plasmid vectors may take place by the so-called “crossing-over” procedure, e.g. as described by A. Campbell,
Advances Genet
. 11, 1962, pp. 101-145. According to this procedure, the plasmid vector is provided with a DNA sequence which is homologous to a region on the bacterial genome, or alternatively with two homologous sequences placed on either side of the heterologous DNA sequence to be integrated. In a subsequent recombination event, the homologous sequence and adjacent sequences on the vector are integrated into the host genome at the region of homology.
In some cases, however, it has been found that the integrated DNA sequences are deleted from the cells in the absence of selection pressure, for instance by a similar type of homologous recombination event as that responsible for the integration of the DNA. In particular, it has previously been observed that recombination between homologous DNA sequences is stimulated in the proximity of replicative DNA present on or near the DNA integrated in the host cell genome, cf. Ph. Noirot et al.,
J. Mol. Biol
. 196, 1987, pp. 39-48; and M. Young and S. D. Ehrlich,
J. Bacteriol
. 171(5), May 1989, pp. 2653-2656.
An object of the present invention is therefore to provide stable integration of DNA

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