U.S. patent application number 10/195542 was filed with the patent office on 2003-05-15 for novel strains of bacillus thuringiensis and pesticidal composition comprising them.
This patent application is currently assigned to INSTITUT PASTEUR. Invention is credited to Agaisse, Herve, Bravo, Alejandra, Lereclus, Didier, Salamitou, Sylvie, Sanchis, Vincent.
Application Number | 20030091550 10/195542 |
Document ID | / |
Family ID | 9484017 |
Filed Date | 2003-05-15 |
United States Patent
Application |
20030091550 |
Kind Code |
A1 |
Bravo, Alejandra ; et
al. |
May 15, 2003 |
Novel strains of bacillus thuringiensis and pesticidal composition
comprising them
Abstract
The present invention relates particularly to a strain of
Bacillus thuringiensis characterized in that it expresses the gene
sigma E (.sigma.E) and does not sporulate at all or little
sporulates or does not produce any viable spores. The invention
also relates to a pesticide composition containing said strain of
Bacillus thuringiensis.
Inventors: |
Bravo, Alejandra; (Morelos,
MX) ; Lereclus, Didier; (Paris, FR) ; Agaisse,
Herve; (Paris, FR) ; Salamitou, Sylvie;
(Maule, FR) ; Sanchis, Vincent; (Paris,
FR) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND MAIER & NEUSTADT PC
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
INSTITUT PASTEUR
Paris Cedex
FR
|
Family ID: |
9484017 |
Appl. No.: |
10/195542 |
Filed: |
July 16, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10195542 |
Jul 16, 2002 |
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09533693 |
Mar 23, 2000 |
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09533693 |
Mar 23, 2000 |
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09051914 |
Jul 2, 1998 |
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6096306 |
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09051914 |
Jul 2, 1998 |
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PCT/FR96/01684 |
Oct 28, 1996 |
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Current U.S.
Class: |
424/93.461 ;
435/252.31 |
Current CPC
Class: |
C12N 1/205 20210501;
C07K 14/325 20130101; C12R 2001/075 20210501; C07K 2319/00
20130101 |
Class at
Publication: |
424/93.461 ;
435/252.31 |
International
Class: |
A01N 063/00; C12N
001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 27, 1995 |
FR |
95,12741 |
Claims
1) Bacillus thuringiensis characterized in that it expresses the
sigma E (.theta.E) gene and does not sporulate or sporulates little
or does not produce viable spores.
2) Bacillus thuringiensis according to claim 1, characterized in
that the strain does not sporulate.
3) Bacillus thuringiensis according to one of claims 1 and 2,
characterized in that it is a strain not expressing the sigma K
gene.
4) Bacillus thuringiensis according to claim 3, characterized in
that the SigK gene has been interrupted by introduction of a DNA
sequence or has been at least partially deleted.
5) Bacillus thuringiensis according to claim 4, characterized in
that the sigK gene has been interrupted by introduction of a DNA
sequence conferring a positive selection character on the
strain.
6) Bacillus thuringiensis according to claim 5, characterized in
that the positive selection character is resistance to an
antibiotic.
7) Bacillus thuringiensis according to one of claims 1 to 6,
characterized in that the Bt strain expresses one or more Cry
genes.
8) Bacillus thuringiensis, according to claim 7, characterized in
that the Cry gene(s) is/are carried by a vector, for example a
plasmid.
9) Bacillus thuringiensis according to claim 7, characterized in
that the Cry genes are integrated into the chromosome.
10) Bacillus thuringiensis according to one of claims 1 to 9,
characterized in that it expresses a protein of interest carried by
a self-replicating plasmid or by a DNA sequence integrated into the
chromosome.
11) Bacillus thuringiensis according to claim 10, characterized in
that it contains a DNA sequence in the SigK gene expressing a
protein of interest.
12) Bacillus thuringiensis according to claim 10, characterized in
that it contains in replacement of all or part of the SigK gene a
DNA sequence expressing a protein of interest.
13) Bacillus thuringiensis 407 SigK.sup.- (pHT410) deposited at the
National Collection of Microorganism Cultures of the Institut
Pasteur on Oct. 26, 1995 under the number I-1634.
14) Bacillus thuringiensis Kto SigK.sup.-
(pHTF3-1C/A(b)-IRS-T-.DELTA.) deposited at the National Collection
of Microorganism Cultures of the Institut Pasteur on Oct. 22, 1996
under the number I-1776.
15) Pesticidal composition, characterized in that it contains a Bt
strain according to one of claims 1 to 14.
16) Composition according to claim 15, characterized in that the Bt
strain has been inactivated.
17) Composition according to one of claims 15 and 16, characterized
in that it has been inactivated by physical means.
18) Composition according to one of claims 15 and 16, characterized
in that it has been inactivated by irradiation.
19) Composition according to one of claims 15 and 16, characterized
in that it has been inactivated by chemical means.
20) Composition according to one of claims 15 to 19, characterized
in that the Bt strain has been treated to improve the digestibility
of the strain or alternatively to improve the accessibility of the
protein.
21) Composition according to claim 20, characterized in that the Bt
strain has been treated by sonication.
22) Nucleotide sequence containing the SigE gene, not containing
any active SigK gene and containing a sequence coding for a gene of
interest.
Description
[0001] The present invention relates to novel strains of Bacillus
thuringiensis, pesticidal compositions employing them as well as
the use of these strains for the expression of proteins of
interest.
[0002] Bacillus thuringiensis (Bt) is a Gram-positive bacterium
which produces proteins having insecticidal properties, especially
against larvae of a large number of insects. These bacteria,
possibly after inactivation, are used in pesticidal compositions
intended to combat insects harmful to crops or vectors of disease,
especially mosquitoes.
[0003] At present, Bt serotype 3a 3b especially is used against
crop pests, and the serotype H14 is used to destroy mosquito
larvae.
[0004] The proteins with pesticidal activity produced by Bacillus
thuringiensis are called .delta.-endotoxins and are produced
abundantly during sporulation. They accumulate in the form of
parasporal crystalline inclusions, and can represent up to 25% of
the dry weight of the sporulated cells.
[0005] Numerous genes of .delta.-endotoxins have been cloned,
sequenced and classified in five groups and in various subgroups on
the basis of sequence homologies and of the toxicity spectrum. The
corresponding genes are called cry genes.
[0006] Formulations based on Bacillus thuringiensis have been used
as biopesticides for close to 30 years under different trade names.
The use of Bacillus thuringiensis as a biological control agent has
numerous advantages with respect to chemical pesticides; in fact,
it has a narrow and very specific host spectrum and it is without
effect on the insects which are not targets and it is without
unfavorable effect on vertebrates or on the environment.
[0007] However, the slight persistence of the .delta.-endotoxins in
the environment and the presence of spores in the formulations
represent two disadvantages for the marketing of products based on
Bacillus thuringiensis. In order to resolve these two problems, it
has been proposed in the patent application EP-192 319 to
encapsulate the toxins in cell membranes, in particular using
Pseudomonas fluorescens-type cells expressing the Cry1Ac toxin, or
alternatively in the patent application PCT WO94/25612, by
expressing the Cry1IIA toxin in an affected nonsporulating mutant
in the spoOA gene. This latter strategy is possible because the
mode of expression of the cryIIIA gene is different from the mode
of expression of other cry genes, in fact this cryIIIA gene is
expressed from a promoter whose activation is independent of all
the genes involved in the initiation of sporulation or of the
factors involved in sporulation.
[0008] In Bacillus thuringiensis, the sporulation is dependent on
the expression of two sigma factors respectively called sigma
[.theta.]35 and sigma [.theta.]28; account having been taken of
their great homology with the sigma (.theta.)E and sigma (.theta.)K
factors in Bacillus subtilis, it is this latter terminology which
will be used below, in the same way as the corresponding genes will
be called sigE and sigK.
[0009] The present invention relates to a strain of Bacillus
thuringiensis which expresses .theta.E but does not sporulate or
sporulates little or does not produce viable spores.
[0010] The present invention is based on the demonstration of the
fact that a mutant of Bacillus thuringiensis which expresses sigE
and which does not express sigK produces a quantity of toxins
virtually identical to the corresponding wild strain, but, on the
other hand, does not sporulate or does not produce viable
spores.
[0011] This is particularly the case when the strain is a
sigK.sup.- strain.
[0012] The construction of such strains has two advantages: 1)
avoiding the dissemination of spores into the environment during
treatment with biopesticides; 2) increasing the persistence of the
toxins in the environment because of their encapsulation.
[0013] Tests have shown that a Bt strain not expressing the sigma K
gene sigK.sup.-) was capable of accumulating a quantity of toxins
equivalent to the source strain, while not producing spores. It was
capable of producing the virtual totality of the toxins encoded by
the cry genes or the related genes whose expression is dependent on
the production of the .theta.E protein.
[0014] In order to obtain sigK.sup.- mutants of Bt, it is
particularly advantageous to use an interruption technique, by
insertion or deletion or change of phase of the sigK gene by
introducing any DNA sequence, it being possible, in addition, to
choose this DNA sequence so as to confer a selection character on
the mutant; it could be, for example, resistance to an antibiotic,
especially to kanamycin, which would allow strains which have been
subjected to interruption to be selected.
[0015] The sigK.sup.- mutants can likewise be obtained by the
deletion of all or part of the nucleotide sequence corresponding to
that of the sigK gene with or without regulatory regions.
[0016] The techniques of interruption of genes are known, they
consist essentially in introducing, at the level of a DNA sequence
carrying the sigK gene, any DNA sequence, the whole being
introduced into the strain produces a homologous recombination, the
sigK gene being replaced by the interrupted sigK gene. The
selection character allows mutants of interest to be selected at
this time.
[0017] Of course, it is particularly advantageous to choose, as
strains intended to be transformed, strains of Bacillus
thuringiensis having a very varied or very significant toxin
production. In fact, as has been indicated above, the fact of
interrupting the sigK gene only blocks sporulation but does not
block the production of the toxins.
[0018] Bt is understood as meaning any strain of Bacillus
thuringiensis.
[0019] Thus, among the strains intended to be subjected to
interruption, the industrial strains could be used. For example, Bt
subsp. kurstaki HD-1 described by Dulmage H. T. (1970), or Bt
israelensis, or a wild strain such as Bt aizawai 7-29 (this strain
is accessible to IEBC under the No. T07029).
[0020] The present invention relates more particularly to the
strain Bacillus thuringiensis 407 SigK.sup.- (pHT410) as well as
the recombinant strain Bacillus thuringiensis Kto SigK.sup.-
(pHTF3-1C/A (b)-IRS-T-.DELTA.) deposited in the National Collection
of Microorganism Cultures of the Institut Pasteur, on Oct. 26, 1995
under the No. I-1634 and on Oct. 22, 1996 under the No. I-1776
respectively.
[0021] It is likewise possible, in order to increase the production
of toxins, to introduce into the sigK.sup.- strains according to
the invention self-replicating plasmid systems ensuring the
expression of the said toxins according to constructs which are
likewise known to the expert.
1 INFORMATION RELATIVE TO A DEPOSITED MICROORGANISM (rule 13a of
the PCT) A. The information refers to the microorganism found in
the description page 3, line 32 B. IDENTIFICATION OF THE Other
deposits are the DEPOSIT object of a supplementary sheet X Name of
the depositing institution INSTITUT PASTEUR Address of the
depositing institution (including the Zip code and the country) 28
Rue du Docteur Roux 75015 PARIS FRANCE Date of deposition Order No.
Oct. 26, 1995 I-1634 C. SUPPLEMENTARY INFORMA- A supplementary
sheet is TION (if necessary) attached for the continu- ation of
this information D. INTENDED STATES FOR WHICH THE INFORMATION IS
GIVEN (if the information is not given for all the desig- nated
States) E. INFORMATION SUPPLIED SEPARATELY (if necessary) The
numbered information below will be subsequently supplied to the
International Office (specify the general nature of the
information, e.g. "order no. of the deposit") Reserved for the
Reserved for the receiving office International Office X This sheet
has been This sheet arrived at received at the same the
International time as the inter- Office on: national application
Authorized official Authorized official J. LE PICAUD A. The
information refers to the microorganism found in the description
page 3, line 33 B. IDENTIFICATION OF THE Other deposits are the
DEPOSIT object of a supplementary sheet Name of the depositing
institution INSTITUT PASTEUR Address of the depositing institution
(including the Zip code and the country) 28 Rue du Docteur Roux
75015 PARIS FRANCE Date of deposition Order No. Oct. 22, 1996
I-1776 C. SUPPLEMENTARY INFORMA- A supplementary sheet is TION (if
necessary) attached for the continu- ation of this information D.
INTENDED STATES FOR WHICH THE INFORMATION IS GIVEN (if the
information is not given for all the desig- nated States) E.
INFORMATION SUPPLIED SEPARATELY (if necessary) The numbered
information below will be subsequently supplied to the
International Office (specify the general nature of the
information, e.g. "order no. of the deposit") Reserved for the
Reserved for the receiving office International Office X This sheet
has been This sheet arrived at received at the same the
International time as the inter- Office on: national application
Authorized official Authorized official J. LE PICAUD
[0022] Form PCT/RO/134 (July 1992)
[0023] The proteins expressed by the strain could depend on the
type of pesticidal activity sought, thus Cryl is toxic for
Lepidoptera, Cry1I against Lepidoptera and Diptera and Cry1V
against Diptera.
[0024] Generally speaking, it is possible to produce sigK.sup.- of
the wild strains which naturally express a certain number of toxins
such as cryIC, cryIA, cryIVA,B,D. It is likewise possible to use
mutant strains such as described according to the invention which
are sigK.sup.- and which express, after chromosomal or plasmid
integration, genes coding for homologous or heterologous proteins
with respect to the Bt genome.
[0025] An illustration of the technique utilizable is described in
Biotechnology, 1992, vol. 10, p. 418 (Lereclus et al.).
[0026] It is possible to introduce a gene, for example the cryIC
gene, into the Bt sigK.sup.- strain by homologous recombination.
The recombinant acquires the cryIC gene and does not retain any
foreign DNA.
[0027] Several toxin genes can thus be added, either in the
bacterial chromosome by homologous recombination, or integrated on
resident plasmids.
[0028] By way of example, a strain of Bt kurstaki at the same time
expressing the cryIAc gene under the control of its own promoter
and the cryIC gene under the control of the promoter of the cryIIIA
gene.
[0029] Among the cry genes utilizable in these constructs, it is
possible to cite: cryI, cry II, cry IV and cyt.
[0030] Another method of introduction of a gene to be expressed
consists in using Gram-positive bacterial plasmids having a
functional replication origin in Bt which is described, for
example, in the patent application PCT WO93/02199 concerning the
pHT304 and pHT315 plasmids.
[0031] The sigK.sup.- strains obtained according to the process
according to the invention are utilizable, optionally after
inactivation, in pesticidal compositions, in particular in
insecticidal compositions intended to be used in order to destroy
larvae, in particular insect larvae. The pesticidal compositions
will be prepared according to techniques known per se, that is to
say, if this is necessary, as a mixture with an inert or noninert
support ensuring an optimum activity of the Bacillus toxins
concerned.
[0032] The inactivation of the strains, which is essential for the
utilization of the sporulating strains in certain countries, is
optional in the case of the sigK.sup.- mutants constructed in the
context of the present invention. This inactivation can be carried
out by any physical or chemical method, especially by irradiation,
which ensures the non-viability of the strains. The strains
according to the invention do not have any viable spores, their
inactivation is easier than in the sporulated strains case.
[0033] As has been indicated above, the toxins being kept in the
interior of the bacteria allows the length of life of the toxins in
the environment (the toxin only being liberated during the
digestion of the bacterium by the larva) to be increased. However,
it has been possible to demonstrate the fact that certain mutants
according to the invention have a resistance which is quite
exceptional, in this case it is necessary to provide for either the
use of selected specific strains for their good digestibility in
the insects to be treated, or alternatively to provide for chemical
treatments, surface-active agents for example, physical treatments,
treatments with ultrasound, or biological treatments, introduction
of particular elements into the walls of the microorganism (by
genetic recombination technique or others) in order to ensure a
better digestibility or an easier accessibility of the toxin when
the microorganism has been ingested.
[0034] This novel strain of Bt is capable of supplying one of the
elements to a pesticidal composition, but is likewise useful as a
vector for expressing homologous or heterologous genes with respect
to the genome of Bt, the said genes being cloned in the sigK.sup.-
mutant of Bt, either with the aid of a self-replicating plasmid, or
by homologous recombination with respect to the genome of the
bacterium.
[0035] The construction of the vector system which can express, for
example, proteases, lipases or any other type of protein, may be
similar to that described in the patent application PCT
WO94/25612.
[0036] The invention likewise relates to a nucleotide sequence
containing the SigE gene, not containing any active SigK gene and
containing a sequence coding for a gene of interest.
[0037] Other characteristics and advantages of the present
invention will appear on reading the examples below and in
referring to the figures in which:
[0038] FIG. 1 represents the interruption of the sigE and sigK
chromosomal genes of Bacillus thuringiensis; the pAB1 and pAB2
plasmids are integrated into the Bt chromosome by homologous
recombination, the second event of homologous recombination leads
to the loss of all the pRN5101 sequence; the arrows indicate the
transcription direction of the Ap.sup.R, EM.sup.R and Km.sup.R
genes which correspond to the genes conferring, respectively,
resistance to ampicillin, erythromycin and kanamycin, the triangles
represent, respectively, the replication origin of pBR322 (oriEc)
and the replication origin of pE194ts (orits);
[0039] FIG. 2 represents the construction of the plasmids for the
analysis of transcription in Bacillus thuringiensis; pHT304-18Z is
[sic], as has been previously described (Agaisse and Lereclus
1994b); the arrows indicate the transcription direction of ermC,
bla and lacZ and the functional replication direction in E. coli
(oriEc); ori1030 is the replication origin of the Bt pHT1030
plasmid (Lereclus and Arantes 1992); the broken arrows indicate the
transcription direction initiated starting from the promoter psigE,
psigK, Bt I and Bt II, as has been indicated above (Rong et al.
1986; Sandman et al. 1988; Wong et al. 1983); the HindIII-BamHI
fragments carrying the promoter regions of the spoIID, cotA and
cryIAa genes have been cloned in pHT304-18Z;
[0040] FIG. 3 represents the expression of .beta.-galactosidase in
Bt under the control of the promoters of the spoIID and cotA genes;
the cells are grown on SP medium at 30.degree. C.; the time zero
indicates the end of the exponential phase, t.sub.n is the number
of hours before or after time zero; A is the .beta.-galactosidase
activity of the Bt strain carrying pHTspoIID; B is the
.beta.-galactosidase activity of the Bt strain carrying pHTcotA;
the specific activity of .beta.-galactosidase is determined at the
times indicated in Spo.sup.+ 407 (.box-solid.), 407 SigE.sup.-
(.circle-solid.) and 407 sigK.sup.- (.largecircle.);
[0041] FIG. 4 represents the expression of .beta.-galactosidase
directed under the control of cryIAa in the strains of Bt carrying
pHTcryIA2, grown on SP medium at 30.degree. C. and the
.beta.-galactosidase activity being determined at the times
indicated in Spo.sup.+ 407 (.box-solid.), 407 SigE.sup.-
(.circle-solid.) and 407 SigK.sup.- (.largecircle.);
[0042] FIG. 5 represents the pHTF3-1C/A(b)-IRS-T plasmid; this
plasmid derives from pBluescript II KS.sup.- (the DNA of
pBluescript II KS.sup.- being represented by the "bla+oriEc" box),
it carries two sequences containing the internal resolution site
(IRS of the Tn4430 transposon (Lereclus et al., 1986) located
directly on both sides of the pBluescript II KS.sup.- and of a tet
gene conferring resistance to tetracycline coming from Bacillus
cereus, it contains, in addition, the coding part of the cryIC/A
(b) chimeric gene under the control of the p3 promoter of cryIIIA
(Agaisse and Lereclus, 1994) and the replication origin of the
pHT1030 plasmid of B. thuringiensis (Lereclus and Arantes,
1992);
[0043] FIG. 6 represents the recombination reaction between the two
IRS sites, catalyzed by the TnpI integrase of the Tn4430 transposon
present in the Kto SigK.sup.- strain, the plasmid originating from
the site-specific recombination is designated
pHTF3-IC/A(b)-IRS-T-.DELTA..
EXAMPLE 1
[0044] Material and Methods
[0045] Bacterial Strains and Media
[0046] The strain Bt 407 (H1 serotype) and its acrystal-liferous
derivative (Cry.sup.- ) have been isolated by .largecircle..
Arantes as has been described above (Lereclus et al. 1989). E. coli
K-12 strain TG1 (.DELTA.(lac-proAB) supE thi hsd D5 (F'traD36
pro.sup.+ proB.sup.+ laclq lacZ .DELTA.M15)) is used for the
cloning experiments (Gibson 1984). The Bt strains are cultured at
30.degree. C. in a Luria medium (LB) and in HCT medium (Lecadet et
al. 1980) or in a sporulation nutrient medium (SP medium) (Lereclus
et al. 1995). The E. coli strains are cultured at 37.degree. C. in
an LB medium. The antibiotic concentration for the bacterial
selection is as follows: ampicillin, 100 .mu.g/ml (for E. coli);
erythromycin, 5 .mu.g/ml (for Bt); kanamycin, 10 .mu.g/ml for E.
coli and 200 .mu.g/ml for Bt.
[0047] Plasmids and DNA Fragments
[0048] The pRN5101 plasmid which was supplied by S. Gruss is a
heat-sensitive replication origin plasmid in Gram-positive
organisms, it was constructed by insertion of pE194ts (Villafane et
al. 1987) into the ClaI site of pBR322. The Bluescript plasmid (pBS
KS.sup.-) comes from Stratagene and the pHT304-18Z and pHT410
plasmid constructs have already been described (Agaisse and
Lereclus 1994b; Lereclus et al. 1989). The oligonucleotides
(Cry1A-1 and Cry1A-2) used for the PCR amplification of the 362 bp
fragment containing the promoter region of the Cry1Aa gene (Wong et
al. 1983) are described in Table 1. The Cry1A-1 primer has a 7 bp
extension at the 5' end containing the HindIII restriction site and
the Cry1A-2 primer contains an 8 bp extension with the BamHI
restriction site. The two restriction sites are introduced to
facilitate cloning in pHT304-18Z. To interrupt the Bt sigE and sigK
genes, the 5' and 3' regions of the corresponding genes are
amplified by PCR using oligonucleotides with appropriate
restriction sites at the 5' end (Table 1) and sub-cloned separately
in pBS KS.sup.-. The 5' regions of the sigE and sigK genes are 857
and 611 bp restriction fragments of BamHI-XbaI respectively. The 3'
regions are 807 and 606 bp EcoRI-BamHI restriction fragments
respectively. The DNA fragments containing the 5' and 3' regions of
each of the genes are purified and bound to a 1.5 kb XbaI-EcoRI
fragment carrying the aphA3 gene of Enterococcus faecalis (Km.sup.R
cassette) (Trieu-Cuot and Courvalin 1983) in the BamHI restriction
site of the pRN5101 plasmid. The resultant heat-sensitive plasmids
pAB1 and pAB2 carry a copy interrupted by a kanamycin resistance
gene in the sigE and sigK genes respectively.
[0049] The plasmids pDG675 and pDG676 respectively carrying the
promoter region of the spoIID and cotA genes of B. subtilis were
supplied by Dr. P. Stragier (Institut de Biologie Physico-Chimique,
Paris, France). pHTspoIID was constructed by sub-cloning the 300 bp
HindIII-BamHI restriction fragment of pDG675 between the HindIII
and BamHI restriction sites of pHT304-18Z. pHTcotA was constructed
as follows: the 400 bp of the EcoRI-BamHI fragment of pDG676 are
initially sub-cloned in pBS KS.sup.-, giving pKScotA. The
HindIII-BamHI restriction fragment of pKScotA is then sub-cloned
between the HindIII and BamHI restriction sites of pHT304-18Z, the
resultant plasmid being designated by the name pHTcotA.
[0050] Construction and Transformation
[0051] The plasmid DNA is extracted from E. coli by the standard
alkaline lysis process. The chromosomal DNA is extracted from Bt as
has been described previously (Msadek et al. 1990). The restriction
enzymes and the T4 ligase come from New England Biolabs, Beverly,
Mass. The DNA fragments are purified on agarose gel using the
Prep-A-Gene kit (BioRad Laboratories, Richmond, Calif.). The
oligonucleotide primers are synthesized by Genset (Paris, France)
and the PCR amplification is carried out using the GeneAmp PCR 2400
system (Perkin-Elmer, Foster City, Calif.). The DNA matrix used in
the PCR amplficiation is either the cryIAa gene already cloned from
the Bt 407 strain (Lereclus et al. 1989) or the chromosomal DNA
extracted from the 407 Cry.sup.- strain. The reaction conditions
are as follows: a 5 min. incubation at 95.degree. C., followed by
30 one min. cycles at 57.degree. C. for the hybridization, one min.
at 72.degree. C. for the extension and one min. at 92.degree. C.
for the denaturation; finally, a new incubation at 72.degree. C.
for 10 min. is carried out. The Taq polymerase comes from USB
Laboratories (Cleveland, Ohio). The standard procedure is used for
the transformation of E. coli and the Bt strains are transformed by
electroporation, as has already been described (Lereclus et al.
1989).
[0052] The protein analysis is carried out after culture of the Bt
strains and sonication, the analysis being carried out on 0.1% SDS
-12% PAGE.
[0053] Bioassays of Insecticidal Activity
[0054] The toxicity of the preparations is estimated using the
larva of Plutella xylostella in the second stage and the free
ingestion technique as has been described previously (Sanchis et
al. 1988).
EXAMPLE 2
[0055] Construction of the SigE.sup.- and sigK.sup.- mutants of
Bt
[0056] The pAB1 and pAB2 heat-sensitive plasmids containing the
copy of the gene interrupted by Km.sup.R of sigE and sigK
respectively are introduced into the Bt 407 Cry.sup.- strain by
electroporation. The replacement of the sigE and sigK genes by the
sigE::Km and sigK::Km interrupted copy is obtained by successive
cultures of transformants in the presence of kanamycin at a
non-permissive temperature (40.degree. C.) (see FIG. 1). As emerges
from FIG. 1, the Bt strains transformed by the pAB1 or pAB2
plasmids are all resistant both to erythromycin and to kanamycin at
37.degree. C.
[0057] The transformants in which the sigE or sigK gene has been
exchanged for its interrupted copy are cultured at a non-permissive
temperature, that is to say at which the replication of the
plasmids has been blocked. They can be selected by their resistance
to kanamycin. The Spo.sup.- mutants (407-SigE.sup.- and
407-SigK.sup.- below) are resistant to kanamycin but sensitive to
erythromycin. The replacement of the Bt sigE and sigK genes by
their interrupted copy is checked by PCR analysis, the chromosomal
DNA of the selected mutants is used as a matrix for the PCR and the
complementary external sequences of each gene are used as a primer
in combination with sigE-4 and sigK-4 oligonucleotides
respectively. The size of the PCR products corresponds to genes
interrupted by Km.sup.R.
[0058] The Bt SigE.sup.- and SigK.sup.- mutant strains are
incapable of sporulating. No heat-resistant spore is produced after
72 hours of growth at 30.degree. C. in HCT or SP medium. Using
similar growth conditions, at least 90% of the cells of the wild
strain sporulate after 24 or 48 hours. The examination of the cells
by phase-contrast microscopy indicates that the sigE.sup.- mutant
strain is blocked at an early sporulation stage (stage II), after
the formation of the asymmetric septum dividing the mother cell and
the spore compartment. The sigK.sup.- mutant strain is blocked in a
later sporulation stage (stage IV). A gray prespore situated at one
of the poles of the cell can be observed in the interior of the
cells.
[0059] The pHTspoIID and pHTcotA plasmids (see FIG. 2) carrying the
promoter regions of the spoIID and cotA genes of Bacillus subtilis
fused with the lacZ gene are constructed to follow the appearance
and the disappearance of the .theta.E and .theta.K factors during
the sporulation of Bt. spoIID is transcribed by an RNA polymerase
containing the .theta.E factor (Lopez-Diaz et al. 1986; Rong et al.
1986). This gene is involved in the morphological development of
the spores at stage II (Young and Mandelstam 1979). The cotA gene
codes for a spore envelope protein (Donavan et al. 1987) and its
transcription depends on .theta.K (Sandman et al. 1988). The
pHTspoIID and pHTcotA plasmids are introduced into Bt 407 Cry.sup.-
Spo.sup.+, 407-SigE.sup.- and 407-SigK.sup.- by electroporation and
the synthesis of .beta.-galactosidase is followed during growth in
SP medium (FIGS. 3A and 3B). In the Spo.sup.+ strain, the synthesis
of .beta.-galactosidase under the control of the spoIID promoter
starts at t2 to attain a maximum of approximately 10,000 U/mg of
proteins at t5 and then decreases. In the strain in which the
synthesis of .beta.-galactosidase is under the control of the cotA
promoter, this is detected only at t6 and attains a maximum of 4000
U/mg of proteins at t11. There is no detectable expression of
.beta.-galactosidase (less than 10 U/mg of proteins) in the
407-SigE.sup.- mutant for the spoIID' or cotA'-'lacZ
transcriptional fusions. As the transcription of sigK depends on
.theta.E (Sandman et al. 1988) there is no production of the factor
K in this mutant strain. There is no detectable expression of lacZ
starting from the cotA promoter in the 407-SigK.sup.- mutant and
the expression starting from the spoIID promoter has a maximum at
t6 as in the wild strain.
EXAMPLE 3
[0060] Expression of cry1Aa'-'lacZ in SigE.sup.- and SigK.sup.-
Mutants of Bt
[0061] To determine the temporal regulation of the promoters of the
cry1Aa gene in the wild strain of Bt and in the Spo.sup.- mutants,
a plasmid containing the cry1Aa'-lacZ [sic] transcriptional fusion
was constructed. A region containing the promoter region of the
cry1Aa gene is amplified by PCR, as has been described, then cloned
in pHT304-18Z upstream of the lacZ reporter gene. The resultant
plasmid, designated by the name pHTcry1A2 (FIG. 2), is introduced
into the Bt 407 Cry.sup.- Spo.sup.+, 407 SigE.sup.- and 407
SigK.sup.- strains by electroporation. The production of
.beta.-galactosidase in the Spo.sup.+ 407 Cry.sup.- strain starts
at t2 and has two peaks, the first at t7 and the second at t11
(FIG. 4). As has been indicated for the spoIID'-`lacZ and
cotA`-'lacZ fusions, t7 and t11 correspond to the maximal periods
of expression of .theta.E and .theta.K. The expression of the
synthesis of .beta.-galactosidase directed by the promoter region
of cry1Aa is severely reduced in the 407-SigE.sup.- mutants (FIG.
4). However, a slight .beta.-galactosidase activity is detected at
t2 with a maximum of 200 U/mg of proteins at t10. The
.gamma.-galactosidase synthesis directed by the promoter region of
cry1Aa starts at t2 and shows a maximum of 9000 U/mg of proteins at
t7 in the mutant 407-SigK.sup.- (FIG. 4). The second expression
peak at a later sporulation time in the Spo.sup.+ strain is not
apparent in the SigK.sup.- mutant, which indicates a participation
of the .theta.K factor in the transcription of the cry1Aa gene
during the late sporulation phase.
EXAMPLE 4
[0062] Production of the Cry1Aa Toxin in the SigE and SigK Mutants
of Bt
[0063] The pHT410 plasmid carrying the cry1Aa gene of the wild
strain of Bt 407 (Lereclus et al. 1989) is introduced into the Bt
407 Cry.sup.- Spo.sup.+ 407-SigE.sup.- and 407-SigK.sup.- strains
by electroporation.
[0064] The transformants are cultured on HCT and SP medium at
30.degree. C. and the production of crystalline inclusions is
examined by phase-contrast microscopy and electron microscopy.
After 48 hours' growth in the HCT medium, the wide bipyramidal
crystals are observed in the 407-Spo.sup.+ and 407-SigK.sup.-
transformants. However, the crystals in the Spo.sup.+ strain are
liberated while those of the SigK.sup.- mutants remain encapsulated
in the cell wall. Even after 72 hours' growth in the HCT medium,
there is no liberation of the crystalline inclusions from the
SigK.sup.- mutant. No crystal is observed in the 407-SigE.sup.-
strain carrying the pHT410 plasmid.
[0065] An SDS-PAGE analysis of the proteins contained in the
crystal-cell and spore-crystal preparations from cells grown on HCT
medium show that the 407-SigE.sup.- strain carrying pHT410 does not
produce the Cry1Aa polypeptide of 130 kDa, unlike the
407-SigK.sup.- strain carrying pHT410 which produces a toxin
similar to that obtained from the 407 Cry.sup.- Spo.sup.+ strain
containing the same plasmid.
[0066] The insecticidal activity of the spore-crystal and
cell-crystal preparations is analyzed using larvae of Plutella
xylostella Lepidoptera at the second stage (Table 2). Having taken
account of the presence of proteins other than Cry1Aa in the
407-SigK.sup.- mutant, it is not possible to determine the precise
concentration of toxin in the crystalline preparation of this
strain. This is why the LD50 is defined in terms of culture volume
to estimate the insecticidal activity of these products. The
bioassays indicate that the Cry1Aa toxin produced in 407-SigK.sup.-
is very toxic for the larvae of P. xylostella. However, the
insecticidal activity of these products is significantly increased
by sonication.
[0067] The Bacillus thuringiensis strain, which does not express,
or only very weakly expresses, the sigma K protein under the
experimental conditions above and which is deposited at the CNCM
under the No. I-1634 is constructed under the following
conditions:
[0068] Bacterial strain including the sigK gene is interrupted by
the aphA3 gene conferring resistance to kanamycin. The strain thus
constructed in transformed by the pHT410 plasmid carrying the
Cry1Aa gene and the ermC gene, conferring resistance to
erythromycin. This nonsporulating strain produces significant
quantities of Cry1Aa toxin during stationary phase la.
EXAMPLE 5
[0069] Construction of a Recombinant Strain of B. thuringiensis
Designated Kto SiqK.sup.- (pHTF3-IC/A(b)-IRS-T-.DELTA.) Expressing
a Gene Coding for a CrY1C/Cry1A(b) Chimeric .delta.-endotoxin Under
the Control of the Promoter of the cryIIIA Gene
[0070] The Kto strain is a natural sporulating strain of B.
thuringiensis; this strain synthesizes a .delta.-endotoxin of
Cry1A(c) type. This .delta.-endotoxin has an insecticidal activity
against the larvae of Ostrinia nubilalis (European corn borer), a
major pest of maize crops in the United States and in Europe. This
.delta.-endotoxin (and thus the strain Kto) is, on the other hand,
not very active against other important pests belonging to the
Noctuidae family such as Spodoptera littoralis, Spodoptera exigua
or Mamestra brassicae (see Table 3). Conversely, the Cry1C
.delta.-endotoxin or the Cry1C/Cry1A(b) chimeric .delta.-endotoxin,
designated Cry1C/A(b) below, whose construction (PHT81 plasmid) is
additionally described by Sanchis et al. (1989), are active against
S. littoralis but not very active against O. nubilalis (Table
3).
[0071] In order to increase the spectrum of activity of the Kto
strain, it was of interest to introduce the cry1C gene or the
chimeric cry1C/A(b) gene into the Kto strain. However, it has been
shown that the introduction of a cry1-type gene (dependent on
sporulation) into a strain of B. thuringiensis already containing
one or more other .delta.-endotoxin genes, whose expression
likewise depends on sigma E and sigma K sporulation factors, is not
interpreted by an increase in the total production of
.delta.-endotoxins. Consequently, a recombinant strain containing
different genes of cry1 type will have a wider spectrum of activity
but will produce less of each of the .delta.-endotoxins; it will
thus have a lower effectiveness with respect to each of the target
insects than strains producing a sole .delta.-endotoxin specific
for each of the targeted insects. This phenomenon can be explained
by a titration effect of the sigma factors of sporulation by the
promoters of different cry1 genes present in the strain. In order
to resolve this problem, it has recently been shown (Sanchis et
al., 1996) that it is possible to place the gene coding for the
Cry1C protein under the control of the promoter of the cryIIIA
gene, whose expression is independent of the sigma sporulation
factors (Agaisse and Lereclus, 1994).
[0072] The cry1c gene, under the control of the cryIIIA promoter,
was introduced into the Kto strain to give the recombinant
Kto(pHTF3-1C-IRS-.DELTA.) strain (Sanchis et al., 1996). This
recombinant strain at the same time produces the Cry1A(c) and Cry1C
toxins and the quantity of .delta.-endotoxins produced is increased
by a factor of 1.5 to 2 with respect to the parent strain. The
increase in the total production of the two .delta.-endotoxins
Cry1A(c) and Cry1C obtained in the strain Kto(pHTF3-1C-IRS-.DELTA.)
probably results from the fact that the expression of the cry1C
gene in this strain does not depend on specific sigma factors of
sporulation; it thus does not interfere with that of the cry1A(c)
gene which is dependent on sporulation. In order to construct the
Kto SigK.sup.- (pHTF3-1C/A(b)IRS-T-.DELTA.) strain described here,
the gene coding for the chimeric .delta.-endotoxin Cry1C/A(b) whose
activity with respect to S. littoralis is slightly superior to that
of Cry1C has been placed under the control of the promoter of the
cryIIIA gene, as described previously for the cry1C gene (Sanchis
et al., 1996).
[0073] Likewise, a sigK.sup.- mutant (whose sigK gene is
interrupted by the aphA3 gene) of the Kto strain was constructed as
described in Example 2 with the aid of the pAB2 plasmid (see FIG.
1). When the Kto SigK.sup.- strain, which is an Spo.sup.- mutant of
Bt, is cultured at 30.degree. C. in HCT medium for 48 hours, it
produces significant quantities of the Cry1A(c) .delta.-endotoxin
which accumulates in the form of a crystalline inclusion which
remains encapsulated in the cell, which does not lyze. The activity
of the Kto SigK.sup.- strain with respect to O. nubilalis is
equivalent to that of the Kto parent strain, whether the Kto
SigK.sup.- strain has previously been sonicated or not.
[0074] The Kto SigK.sup.- strain was then transformed with the
pHTF3-1C/A(b)-IRS-T plasmid (see FIG. 5). This plasmid, derived
from pBluescript II KS.sup.-, carries two sequences containing the
internal resolution site (IRS) of the Tn4430.sub.-- transposon
(Lereclus et al., 1986). These two IRSs are located directly on
both sides of the pBluescript II KS and of a tet gene conferring
resistance to tetracycline and coming from Bacillus cereus. In
addition, the pHTF3-1C/A(b)-IRS-T contains the coding part of the
chimeric cry1C/A(b) gene under the control of the p3 promoter of
cryIIIA and the replication origin of the pHT1030 plasmid of B.
thuringiensis (Lereclus and Arantes, 1992).
[0075] After transformation, the TnpI integrase of the Tn4430
transposon present in the Kto SigK.sup.- strain catalyzes a
recombination reaction between the two IRS sites and the DNA
contained between these two sites is excised. Of the two cyclic
molecules resulting from the reconbination, only that which carries
the replication origin of the pHT1030 plasmid and the chimeric
cry1C/A(b) gene can be replicated and the plasmid thus obtained,
designated pHTF3-1C/A(b)-IRS-T-.DELTA., has lost the DNA
corresponding to the pBluescript II KS.sup.- and to the tet gene
(see FIG. 6). The Kto SigK.sup.- (pHTF3-1C/A(b)-IRS-T-.DELTA.)
recombinant strain produces both the Cry1A(c) and the cry1C/A(b)
.delta.-endotoxins in significant quantity and thus has the
advantage of having a wider spectrum of activity than the parent
Kto or Kto SigK.sup.- strain (Table 4).
[0076] In addition, such a strain has two other advantages:
[0077] 1.) The Cry1A(c) and Cry1C/A(b) .delta.-endotoxins remain
encapsulated in the cell. This could be interpreted by an increase
in the persistence of the toxins in the treated crop zone, because
of the physical protection which this could confer on them against
degradation and UV radiation after spreading.
[0078] 2.) The sigK.sup.- mutant is an Spo.sup.- mutant blocked at
stage IV of the sporulation process and thus does not produce a
viable spore; the use of such a mutant allows the dissemination of
spores into the environment during insecticidal treatment to be
avoided.
2TABLE 1 Oligonucleotide sequences used as PCR primers Restriction
Position site at the Primer Sequence in bp.sup.a 5' end cryIA-1
5'CCCAAGCTTGCAGGTAAATGGTTCTAAC3' 136-177* HindIII cryIA-2
5'CGCGGATCCATCTCTTTTATTAAGATACC3' 493-518* BamHI sigE-1
5'CGGGATCCCGTTGAAAGCGTAGAGGTCAGAA3' 16-38* BamI sigE-2
5'GCTCTAGAGCCAACGCGATGCATATGTTGCTA3' 834-853* XbaI sigE-3
5'GGAATTCCATTGTCTGACGTGTTAGGTACA3' 961-982* EcoRI sigE-4
5'CGGGATCCCGATACGCAATATCTCGCAATGA3' 1730-1751* BamHI sigK-1
5'CGGGATCCCGTCCAGTTATAATTTGAGCTCCAA3' 31-53* BamHI sigK-2
5'GCTCTAGAGCCCCGATTGTACCAATTGAAAT3' 603-623* XbaI sigK-3
5'GGAATTCCATTAAAGCGATCGAGAGCTATT3' 627-648* EcoRI sigK-4
5'CGGGATCCCGGCACCTTCTAATATTACAGATAGAA3' 1194-1217** BamHI sigE-Ch
5'TTTTCTAAAAAGCGTATTGAA3' 1-22** any sigK-Ch
5'GGAGAAACCATAGTTATGAA3' 1-20** any .sup.aThe position of the
oligonucleotides is determined from: *Wong et al. 1983 and **Adams
et al. 1991
[0079]
3TABLE 2 Insecticidal activity of Bt strains LD50.sup.a .mu.l/ml of
powdered Strain food.sup.b 407 Spo.sup.+ (pHT410) 14.7 (7.9-21.8)
407-SigK.sup.- (pHT410) 124.6 (68.6-1358.7) 407-SigK.sup.- (pHT410)
sonicated 35.6 (15.2-71.2) for 1 min..sup.c 407-SigK.sup.- (pHT410)
sonicated 9.5 (6.2-12.6) for 5 min..sup.d .sup.aThe LD50 is the
volume of preparation necessary to kill 50% of the insect larvae
.sup.b.mu.l of spore-crystal or cell-crystal solution used per ml
of solution spread on the leaves .sup.cThe cells are partially
disrupted by sonication for 1 min., the majority of the crystalline
inclusions remain in the interior of the cells .sup.dThe cells are
totally disrupted by sonication for 5 min., 95% of the crystals are
liberated
[0080]
4TABLE 3 Activity of Cry1A (c), Cry1C and Cry1C/A (b)
.delta.-endotoxins compared with respect to S. littoralis and O.
nubilalis LC50.sup.(1) with respect to larvae of the second stage
in ng of protein/cm.sup.2 .delta.-Endotoxins S. littoralis O.
nubilalis Cry1A (c) 1000 2 Cry1C 70 >250 Cry1C/A (b) 20 87
.sup.(1)The LC50, or lethal concentration 50, is the concentration
of .delta.-endotoxins which is necessary to kill 50% of the treated
population after 5 days; the biological tests were carried out as
described by Sanchis et al. 1996
[0081]
5TABLE 4 Insecticidal activity of strains of Bacillus thuringiensis
LC50.sup.(1) with respect to larvae of the second stage
Characteristics of the in ng of protein/cm.sup.2 Strain strain S.
littoralis O. nubilalis Kto Natural Spo.sup.+ strain 981 1.7
producing Cry1A (c) (758-1270) (0.9-3) Kto (pHTF3-1C- Kto Spo.sup.+
strain 25 <4.2 IRS-.DELTA.) producing Cry1A (c) (13-50) and
Cry1C Kto SigK.sup.- (pHTF3- Kto.sup.- Spo.sup.- strain 11 2.6 1C/A
(b)-IRS-T-.DELTA.) producing Cry1A (c) (3-36) (0.06-110) and
Cry1C/A (b) in encapsulated form .sup.(1)The LC50 is the
concentration which is necessary to kill 50% of the treated
population in 5 days; the values in brackets represent the 95%
confidence intervals; the biological tests were carried out as
described by Sanchis et al. (1996)
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Sequence CWU 1
1
12 1 28 DNA Artificial Sequence Description of Artificial
SequenceSynthetic DNA 1 cccaagcttg caggtaaatg gttctaac 28 2 29 DNA
Artificial Sequence Description of Artificial SequenceSynthetic DNA
2 cgcggatcca tctcttttat taagatacc 29 3 31 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 3 cgggatcccg
ttgaaagcgt agaggtcaga a 31 4 32 DNA Artificial Sequence Description
of Artificial SequenceSynthetic DNA 4 gctctagagc caacgcgatg
catatgttgc ta 32 5 30 DNA Artificial Sequence Description of
Artificial SequenceSynthetic DNA 5 ggaattccat tgtctgacgt gttaggtaca
30 6 31 DNA Artificial Sequence Description of Artificial
SequenceSynthetic DNA 6 cgggatcccg atacgcaata tctcgcaatg a 31 7 33
DNA Artificial Sequence Description of Artificial SequenceSynthetic
DNA 7 cgggatcccg tccagttata atttgagctc caa 33 8 31 DNA Artificial
Sequence Description of Artificial SequenceSynthetic DNA 8
gctctagagc cccgattgta ccaattgaaa t 31 9 30 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 9 ggaattccat
taaagcgatc gagagctatt 30 10 35 DNA Artificial Sequence Description
of Artificial SequenceSynthetic DNA 10 cgggatcccg gcaccttcta
atattacaga tagaa 35 11 21 DNA Artificial Sequence Description of
Artificial SequenceSynthetic DNA 11 ttttctaaaa agcgtattga a 21 12
20 DNA Artificial Sequence Description of Artificial
SequenceSynthetic DNA 12 ggagaaacca tagttatgaa 20
* * * * *