Truncated gene of Bacillus thuringiensis encoding a polypeptide toxin

The invention relates to genetically engineered plant-colonizing microorganisms which prolife-rate 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-colonizing microorganisms of the invention and their progeny are active against a variety of lepidopterous pests. The invention further relates to the use of such plant-colonizing microorganisms in a method of killing or inhibiting lepidopterous pests and to insecticidal compositions containing the plant-colonizing microorganism as the active insecticidal agent.

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 
thurinqiensis 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. Hybridization 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.RTM. and 
Thuricide.RTM.. 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 method 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-colonizing 
microorganisms of the invention and their progeny are active against a 
variety of lepidopterous pests. The invention further relates to the use 
of such plant-colonizing microorganisms in a method of killing or 
inhibiting lepidopterous pests and to insecticidal compositions containing 
the plant-colonizing microorganism as the active insecticidal agent.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is directed to a genetically engineered 
plant-colonizing microorganism containing heterologous DNA which expresses 
a protein having insecticidal activity and having substantially the 
immunological properties of the crystal protein toxin of Bacillus 
thuringiensis. The invention further relates to the use of such 
plant-colonizing microorganisms in a method of inhibiting the growth and 
development of lepidopterous pests and to insecticidal compositions 
containing these plant-colonizing microorganisms as the active 
insecticidal agent. 
As used herein, the term "plant-colonizing microorganism" refers to a 
microorganism which is capable of colonizing the "plant environment" and 
which can express the insecticidal protein in the "plant environment". The 
plant associated microorganism is one which can exist in symbiotic or 
non-detrimental relationship with the plant in the "plant environment". As 
used herein, the term "plant-colonizing microorganism" does not include 
spore forming organisms of the family Bacillaceae as for example, Bacillus 
thuringiensis var. kurstaki, Bacillus thuringiensis var. israeliensis and 
Bacillus subtilis. 
The term "plant environment" refers to the surface of the plant, e.g., 
leaf, stem, stalk, floral parts or root surface and to the "rhizosphere", 
i.e., the soil which surrounds and which is influenced by the roots of the 
plant. 
Exemplary of the plant-colonizing microorganisms which may be engineered as 
taught herein are bacteria from the genera Pseudomonas, Agrobacterium, 
Rhizobium, Erwinia, Azotobacter, Azospirillum, Klebsiella, Flavobacterium 
and Alcaligenes. Rhizosphere colonizing bacteria from the genus 
Pseudomonas are preferred for use herein, especially the flourescent 
pseudomonads, e.g., Pseudomonas fluorescens which is especially 
competitive in the plant rhizosphere and in colonizing the surface of the 
plant roots in large numbers. Another group of particularly suitable 
plant-colonizing microorganisms for use herein are those of the genus 
Agrobacterium; Agrobacterium radiobacter is particularly suitable for use 
herein. 
As used herein, the term "heterologous DNA" refers to any DNA fragment 
isolated from B. thuringiensis which codes for a protein that is 
immunologically cross-reactive to the insecticidally active crystal 
protein toxin produced by B. thuringiensis. Both plasmid and chromosomal 
DNA, or a sub-fragmentation sequence thereof, may be used to genetically 
engineer the plant-colonizing microorganisms described herein. The 
synthetically produced equivalents may likewise be used and such use is 
contemplated herein. Stated another way, DNA from whatever source, which 
expresses an insecticidally active protein which is substantially 
immunologically cross-reactive with the crystal protein toxin of B. 
thuringiensis is contemplated for use in genetically engineering the 
plant-colonizing microorganisms described herein. 
Plasmid DNA from B. thuringiensis var. kurstaki HD-1 was used herein as the 
source of the crystal protein toxin gene. This strain was obtained from 
Dr. T. Yamamoto of the USDA-- Brownsville, Tex. There are a variety of 
publicly available B. thuringiensis strains which may likewise be used; 
e.g., B. thuringiensis var. kurstaki HD-1 (NRRL B-3792) and B. 
thuringiensis var. kurstaki HD-73 (NRRL B-4499). See also U.S. Pat. No. 
4,277,564. 
The plasmid DNA fragment isolated from the B. thuringiensis donor strain 
was a 16 Kb BamHI fragment which expresses protein that is immunologically 
cross-reactive with antibody made to the 134,000 dalton crystal protein 
toxin of B. thuringiensis. The 16 Kb BamHI fragment was subcloned to 
produce an 8.1 Kb BamHI-PstI fragment. This fragment was further subcloned 
to produce a 4.6 Kb HpaI-PstI fragment. All of these DNA fragments coded 
for an insecticidally active protein toxin of about 134,000 daltons in 
size and which was immunologically cross-reactive with antibody made to 
the crystal protein toxin of B. thuringiensis. Deletions of the 4.6 Kb 
fragment are contemplated for use herein to the extent that the deletions 
do not result in the loss of the insecticidal properties of the protein 
capable of being coded by the deletion fragments. DNA fragments have been 
made by deletion ranging from 4.1-2.4 Kb in size and coding for an 
insecticidally active protein of about 110,000 to about 80,000 daltons. 
As would be recognized by skilled artisans, there are inherent advantages 
in using the smallest possible DNA fragment which will still express 
insecticidally active protein. For example, a higher yield of B.t. DNA is 
obtained in the cloning steps and introduction of superfluous DNA not 
coding for the insecticidally toxin into the genome of the 
plant-colonizing microorganism is reduced. 
Cloning vectors used herein are known in the art and are generally 
available. Choice of a particular vector is within the skill of the art 
and is largely a matter of individual preference. Plasmid cloning vectors 
which may be mentioned as being suitable for use herein are identified in 
Table I. 
TABLE I 
______________________________________ 
Plasmid Brief 
Vector Description Reference 
______________________________________ 
pBR328 -- Bolivar, F., 
(1978) Gene 
4:121 
pUC7 -- Vieira, J. and 
Messing, J. 
(1982) 
Gene 19:259 
pUC8 Multi-site pBR322 
Vieira, J. and 
(ATCC 37017) Messing, J. 
like Vector (1982) Gene 
19:259 
pMON5008 Derivative of USSN 592,158 
pKT230 filed 3/21/84 
______________________________________ 
The plant-colonizing microorganisms of the invention are useful in a method 
of combatting lepidopteran pests wherein an insecticidally effective 
amount of the plant-colonizing microorganism is applied to the plant 
environment or to the plant seed. The plant-colonizing microorganisms of 
the invention will have the same spectrum of insecticidal activity as the 
crystal protein toxin of Bacillus thuringiensis Berliner var. kurstaki. 
That is, the microorganisms of the invention are insecticidally active 
against such lepidopteran larvae as, for example, Spruce budworm, wax 
moth, cabbage looper, imported cabbage worm, gypsy moth and tobacco 
hornworm. 
The insecticidal plant-colonizing microorganisms of the invention may be 
applied directly to the plant environment, e.g., to the surface of the 
leaves, roots or floral parts or to the plant seed. When used as a seed 
coating, the plant-colonizing microorganisms of the invention are applied 
to the plant seed prior to planting. Generally, small amounts of the 
insecticidally active microorganism will be required to treat such seeds. 
The determination of an insecticidally effective amount of plant-colonizing 
microorganisms useful in the method of the invention required for a 
particular plant is within the skill of the art and will depend on such 
factors as the plant species, method of planting, and the soil type, 
(e.g., pH, organic matter content, moisture content). 
Compositions containing the insecticidally active plant associated 
microorganism of the invention are prepared by formulating the 
biologically active microorganism with adjuvants, diluents, carriers, etc. 
to provide compositions in the form of finely-divided particulate solids, 
granules, pellets, wettable powders, dusts, aqueous suspensions, gels, 
dispersions, and emulsions. Illustrative of suitable carrier vehicles are: 
solvents e.g., water or organic solvents and finely divided solids, e.g., 
kaolin, chalk, calcium carbonate, talc, silicates and gypsum. 
It is contemplated herein to use the insecticidal microorganisms in the 
methods and compositions of the invention in encapsulated form; e.g., the 
plant-colonizing microorganism can be encapsulated within shell walls of 
polymer, gelatin, lipid and the like or other formulation aids as for 
example emulsifiers, dispersants, surfactants, wetting agents, anti-foam 
agents and anti-freeze agents, may be incorporated into the insecticidal 
compositions, especially if such compositions will be stored for any 
period of time prior to use. 
In addition to the insecticidally active plant-colonizing microorganism the 
compositions of the invention may additionally contain other known 
biologically active agents, for example, a herbicide, fungicide, or other 
insecticide. Also, two or more insecticidally active plant-colonizing 
microorganisms may be combined. 
The application of insecticidal compositions containing the genetically 
engineered plant-colonizing microorganisms of the invention as the active 
agent can be carried out by conventional techniques utilizing, for 
example, spreaders, power dusters, boom and hand sprayers, spray dusters 
and granular applicators. 
The compositions of the invention are applied at an insecticidally 
effective amount which will vary depending on such factors as, for 
example, the specific lepidopteran larvae to be controlled, the specific 
plant to be treated and method of applying the insecticidally active 
compositions. 
The following examples further illustrate various specific embodiments of 
the invention described herein. As would be apparent to skilled artisans, 
various changes and modifications from these examples are possible and are 
contemplated within the scope of the invention described here. 
The insertion of heterologous DNA derived from B. thuringiensis coding for 
a high molecular weight protein having insecticidal activity into a 
plant-colonizing microorganism was carried out as follows: 
Starting Microorganism 
Bacillus thuringiensis var. kurstaki HD-1 utilized herein as the source of 
plasmid DNA for the recombinant plasmids was obtained from Dr. Takashi 
Yamamoto of the United States Department of Agriculture (USDA). B. 
thuringiensis strains were maintained as sporulated stock cultures 
according to standard procedures. Cultures were routinely monitored for 
crystal production by phase contrast microscopy. 
Preparation of Synthetic Oligonucleotide Probes 
The amino acid sequence of the crystal protein toxin gene isolated from 
Bacillus thuringiensis var. kurstaki HD-1 was partially determined 
according to the method of Hunkapiller et al (5). These sequences were 
verified using the DNA sequence of the NH.sub.2 -terminal portion of the 
crystal protein gene disclosed by Wong et al (4). Synthetic 
oligonucleotide sequences based on an amino acid sequence determined from 
the crystal protein polypeptide were prepared according to the procedure 
of Beaucage et al (6). The oligonucleotide probes prepared are as shown in 
Table II. 
TABLE II 
______________________________________ 
SYNTHETIC OLIGONUCLEOTIDE PROBES 
Size Probe Sequence Area of B.t. Protein 
______________________________________ 
14-mer TGG GGA CCG GAT TC 
1200 bp region 
14-mer GAA AGA ATA GAA AC 
* 27-31 amino acid region 
21-mer CCT GAA GTA GAA- * 19-25 amino acid region 
GTA TTA GGT 
______________________________________ 
* numbered from NH.sub.2 -- terminal end 
Preparation and Isolation of Plasmid DNA From B. Thuringiensis 
Plasmid DNA from B. thuringiensis var. kurstaki HD-1 was purified from 1 to 
2 liters of culture according to the procedure of Kronstad et al (7). All 
plasmid preparations were banded at least once in CsCl/ethidium bromide 
gradients. Plasmids 30 megadaltons and larger in size were preferentially 
isolated. 
Digestion with restriction enzymes EcoRI, PstI, HindIII, BamHl and SmaI, 
was carried out according to conditions recommended by the supplier 
(Boehringer Mannheim). Escherichia coli strain JM 101 (8) and strain 
SR-200 (9) were used as the recipients for the transformation step. 
Competent cells were prepared according to standard procedures (10). 
Colonies transformed with plasmid pUC8, were plated on L-agar with 100 
.mu.g/ml of ampicillin and 40 .mu.l of 4% 
5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside (x-gal). 
Preparation of Nitrocellulose Filters and Hybridization 
Plasmid DNA was transferred to nitrocellulose according to the procedure of 
Southern (11). Prehy-bridization was done by incubating the nitrocellulose 
paper with the bound transferred DNA in pre-hybridization fluid, 10 X 
Denhardt's (0.2% BSA, 0.2% Ficoll, 0.2% polyvinylpyrrolidone) and 6 X SSC 
(0.9M NaCl, 0.09M sodium citrate) for 2-4 hours at 37.degree. C. 
Hybridization was done by incubating the nitrocellulose paper for 8-10 
hours with 10-11 ml of the pre-hybridization fluid and the labelled probe. 
After several washes with 6X SSC at increasing temperatures (30-45.degree. 
C.) the paper was exposed to X-ray film. 
Cloning of the B.t. toxin gene in E. coli 
BamHI-restricted pBR328 (100 ng), treated with alkaline phosphatase 
(Boehringer Mannheim) was mixed and ligated with 500 ng of B. 
thuringiensis plasmid DNA restricted with BamHI. CaCl.sub.2 prepared 
competent E. coli SR200 were transformed and selected by ampicillin 
resistance and screened for tetracycline sensitivity. Analysis by 
mini-plasmid prep procedures (12) identified two clones which had the 
correct 16 Kb insert. Southern hybridization analysis with radio-labelled 
probes from Table II demonstrated that the DNA fragment which contained 
the sequence hybridizing to the synthetic probe had been sub-cloned. The 
two plasmids, designated pMAP1 and pMAP2, differed only in the orientation 
of the DNA fragment within the vector. These plasmid constructs produced 
material cross-reactive to B.t. crystal protein toxin antibody when 
analyzed according to Western blot procedures (13). A restriction map of 
the inserted B.t. fragment was prepared and four EcoRI (E) sites and three 
Hind III (H) sites were located between the BamHI (B) sites. This is 
schematically illustrated as: 
##STR1## 
E. coli SR200 containing pMAP2 has been deposited in compliance with MPEP 
608.01(p) with the American Type Culture Collection, 12301 Parklawn Drive, 
Rockville, Md. 20852 USA and has been designated ATCC #39800. 
Sub-Cloning of B.t. Toxin 
An 8.1 Kb BamHI-PstI fragment was isolated after BamHI-PstI digestion of 
pMAP2 by electroelution from a preparative agarose gel onto DEAE paper 
used according to the directions of the manufacturer Schleicher & Schuell 
(14). Plasmid pUC8 was used to sub-clone the BamHI-PstI fragment of pMAP2 
carrying the B.t. gene. Ligation of PUC8 digested with BamHI and PstI with 
the purified 8.1 Kb BamHI-PstI fragment was followed by transformation of 
competent E. coli JM101. Transformants were selected on the basis of 
ampicillin resistance and a lack of .beta.-galactosidase activity. A clone 
was isolated and was confirmed to contain the desired plasmid. This 
construct was designated pMAP3. E. coli JM101 containing pMAP3 has been 
deposited in compliance with MPEP 608.01 (p) with ATCC and has been 
designated ATCC #39801. 
Reduction of the B. thuringiensis DNA insert of pMAP3 from 8.1 Kb to 4.6 Kb 
was done by deleting a SmaI-HpaI fragment. Plasmid pMAP3 DNA, purified by 
CsCl gradient centrifugation was digested with SmaI and HpaI restriction 
enzymes and religated. The resulting DNA fragment was utilized to 
transform competent E. coli JM101 cells. Ampicillin resistant 
transformants were screened by agarose electrophoresis of mini-plasmid 
preparations. A clone was identified which contained a plasmid with the 
expected DNA restriction enzyme digestion pattern. This construct was 
labelled pMAP4. The above-described sub-cloning of the 16 Kb insert of 
pMAP2 containing the B. thuringiensis toxin gene to an 8.1 Kb insert 
(pMAP3) and a 4.6 Kb insert (pMAP4) is illustrated in FIG. 1. 
Insertion of DNA Isolated From B.t. Into Cloning Vector pMON5008 
Plasmid pMON5008 constructed by B. C. Hemming and D. J. Drahos of Monsanto 
Company was used as a cloning vector to transform competent cells of E. 
coli with a 4.6 Kb fragment of plasmid DNA isolated from pMAP3. Plasmid 
pMON5008 is a derivative of plasmid pKT230; construction of pMON5008 is 
described in U.S. Ser. No. 592,158 filed Mar. 21, 1984, which is commonly 
assigned to Monsanto Company and the disclosure of which is herein 
incorporated by reference. 
In order to get proper insertion of the 4.6 Kb fragment isolated from pMAP3 
into pMON008 adjustments to the ends of the 4.6 Kb fragment were required. 
A PstI linker (CCTGCAGG) was added to the 4.6 Kb HpaI-PstI fragment as 
described below. 
Plasmid pMAP3 (10 .mu.g) was digested with HpaI; complete digestion was 
confirmed by agarose gel analysis. The digest was extracted with mixture 
of phenol/chloroform (1:1), followed by chloroform extraction and finally 
by ethanol extraction. The resulting precipitate was washed with TE 
buffer, (0.01 M TRIS/0.001M EDTA, pH 8.0 and thereafter resuspended in 
same. Two .mu.g of PstI linker (CCTGCAGG) obtained from New England 
Biolabs was combined with 2 units of T4 DNA kinase in a total volume of 10 
.mu.l of kinase/ligase buffer (11). The mixture was incubated at 
37.degree. C. for 1 hour. Thereafter, 2 .mu.g of the kinase/linker mixture 
was added to 2 .mu.g of HpaI digested pMAP3 and 2 .mu.l of T4 DNA ligase 
(2 units) and the resulting mixture was incubated for 18 hours at 
22.degree. C. after which 1 .mu.l of 0.5 M EDTA (pH 8.0) was added and the 
mixture was extracted as described above. The resulting precipitate was 
washed with TE buffer and resuspended in 90 .mu.l of fresh TE buffer. The 
precipitate was digested with PstI and the digest was mixed with 6.0 .mu.l 
of 5 M NaCl and run through a Sepharose CL-4B column. The fractions were 
collected and were screened by agarose gel electrophoresis. Fractions 
containing high molecular weight DNA were combined, precipitated, and the 
resulting precipitate was washed with TE buffer and thereafter resuspended 
in fresh TE buffer. 
The DNA which was collected from the column was mixed with PstI digested 
plasmid vector pUC7 which had been treated with alkaline phosphatase and 
column-purified. The PstI-PstI fragment was ligated into the PstI site of 
pUC7 and used to transform competent E. coli JM101. Inserting the B.t. 
gene into the unique PstI site of pUC7 positioned the gene between two 
BamHI sites. 
Ampicillin resistant .beta.-gal negative transformants were selected and 
were analyzed for the correct plasmid construct by mini-plasmid 
preparations and restriction endonuclease digestion. A plasmid with a 4.6 
Kb fragment flanked by both PstI and BamHI sites was isolated and 
designated pMAP8. 
Plasmid pMON5008 DNA was isolated, digested with BamHI or BglII, treated 
with alkaline phosphatase and purified on a Sepharose CL-4B column. A 
mixture of 1 ug of this vector DNA and 2 ug of pMAP8 digested with BanHI 
was ligated and used to transform competent E. coli cells. Transformants 
were selected by their kanamycin resistance and screened by restriction 
endonuclease digestion of the plasmid DNA isolated by mini-plasmid 
preparation. Constructs with B.t. DNA inserted at both the BamHI and BglII 
sites of pMON5008 in both orientations were obtained and identified as 
pMAP12, pMAP13, pMAP14 and pMAP15. 
Selection of Plant-Colonizing Microorganism 
Pseudomonas fluorescens 3732 (Ps.3732) was isolated from St. Charles, Mo. 
farm soil. A rifampicin resistant strain designated Pseudomonas 
fluorescens 3732-3 was identified by plating 1.times.10.sup.9 colony 
forming units (CFU) on an L-agar plate with 100 .mu.g/ml rifampicin. A 
nalidixic acid resistant mutant, designated Ps. 3732-3-7,was obtained by 
exposing to UV light 1.times.10.sup.10 CFU of Ps.3732-3 in 5 ml of L-broth 
in an open petri plate on a gently rotating shaker. Exposure times ranged 
from 1 to 8 minutes and exposed colonies were plated on L-agar with 100 
.mu.g/ml of nalidixic acid. Colonies were streaked to isolation several 
times, grown under non-selective conditions at 30.degree. C. in L-broth, 
and plated on media with and without nalidixic acid. 
Engineering Of Plant-Colonizing Microorganisms 
Plasmids pMAP12, 13, 14 and 15 were transferred into Ps.3732-3-7 by a 
tri-parental mating system (16). The system consists of two donor strains 
and a recipient strain. The donors are two E. coli strains; one with the 
pMAP plasmid (a pMON5008 derivative with kanamycin resistance) to be 
transferred into Ps. 3732-3-7 and the other an E. coli strain with 
pRK2013. The transfer (tra) genes of pRK2 are located on pRK2013 and will 
mediate the transfer of plasmids into Ps. 3732-3-7 but will not replicate 
in Pseudomonads. The recipient strain is resistant to rifampicin and 
naladixic acid but sensitive to kanamycin. 
The three strains involved in the 3-part mating system (E. coli with 
pRK2013, E. coli with the pMON5008-derivative, and Ps.3732-3-7) were grown 
separately overnight in L-broth. One-tenth ml of culture was transferred 
to fresh L-broth and grown for three hours at 37.degree. C. (30.degree. C. 
for Ps.3732-3-7). One ml of each was pelleted by centrifugation and washed 
with L-broth supplemented with 0.1% glucose. All three cultures were 
resuspended in a total of 200 ul of L-broth and plated into the center of 
a freshly poured L-agar plate. The plates were incubated for 16 hours at 
30.degree. C. Cells were resuspended from the plates with 1 ml of 10 mM 
MgSO.sub.4 and plated on Pseudomonas F (PF) agar (Difco catalogue 
#0448-01) with 100 ug/ml of rifampicin and 50 ug/ml of kanamycin. 
Trans-conjugants were selected on PF agar with 50 .mu.g/ml of kanamycin and 
100 .mu.g/ml of rifampicin. Desirable P. fluorescens 3732-3-7 colonies on 
PF agar were fluorescent under long wave UV light resistant to rifampicin 
and kanamycin resistant due to the presence of pMAP 12, 13, 14 or 15. 
Colonies were streaked on plates containing 64 ug/l of X-gal indicator for 
.beta.-galactosidase to confirm the presence of this marker. P. 
fluorescens 3732-3-7 containing pMAP15 has been deposited in compliance 
with MPEP 608.01(p) with ATCC and is designated ATCC #39802. 
Utilizing the procedure described above, Agrobacterium radiobacter was 
engineered to contain various of the novel plasmids described herein. A. 
radiobacter 212-4 containing pMAP15 has been deposited in compliance with 
MPEP 608.01(p) with ATCC and is designated ATCC #39803. 
Preparation of Deletion Derivatives of the B.t. Toxin Gene 
Deletion derivatives of the B.t. crystal protein toxin gene were prepared 
by deleting DNA fragments of pMAP8 within the coding region of the 134,000 
dalton toxin. Plasmid pMAP8 (1-1.5 ug in 20 uL of TE buffer was cut with 
the appropriate enzyme(s), extracted with a phenol/chloroform mixture 
(1:1), diluted to 40 uL with TE buffer, religated and used to transform 
CaCl.sub.2 --competent JM101 cells. Plasmids with deletions were 
identified by screening mini-prep plasmid preparations on agarose gels 
after electrophoresis. Two deletion derivatives, designated pMAP10 and 
pMAP11, were constructed by deleting a 1.4 Kb KpnI fragment (pMAP10) and a 
0.5 Kb NruI-ScaI fragment (pMAP11) from pMAP8. E. coli with either of 
these constructs produced material toxic to Manduca sexta. The restriction 
map of the deletion fragments is shown in FIG. 2. The 2.4 Kb BamHI-KpnI 
fragment of pMAP10 was sub-cloned in pUC18 (18). pMAP10 and pUC18 were 
digested with BamHI and KpnI, mixed, ligated and used to transform E. coli 
JM101. A clone was isolated which contained a plasmid with a single 2.4 Kb 
BamHI-KpnI fragment. This plasmid was designated pMAP18. E. coli 
containing this plasmid were toxic to Manduca sexta. 
Insertion of The Deletion Derivatives Into Ps. 3732-3-7 
The procedure described above for the introduction of the B.t. gene into 
Pseudomonas fluores cens 3732-3-7 was repeated for the deleted B.t. DNA 
fragment. Plasmid DNA (pMAP10) was digested with BamHI and cloned into 
pMON5008 at both the BamHI and BglII sites in both orientations. These 
constructs were designated pMAP20, 21, 22 and 23. Imumunological analysis 
(13) confirmed the production of CRM with anti-B.t. toxin antibody by P. 
fluorescens 3732-3-7. 
The plant-colonizing microorganisms of the invention were tested for 
insecticidal activity according to the following examples. In the examples 
which follow, protein extracts of the plant-colonizing microorganism or 
unlysed whole cells were used. Protein extracts were prepared as shown in 
Example 1. 
EXAMPLE 1 
Preparation of Protein Extract 
Fifty milliliters of L-broth containing 100 .mu.g/ml ampicillin was 
inoculated with the microorganism (control or engineered plant-colonizing 
microorganism) and the inoculum was maintained overnight at 37.degree. C. 
(30.degree. C. for the pseudomonads) on a shaker. The inoculum was 
centrifuged for ten minutes in SS-34 10 K. The pellet was resuspended in 5 
ml of Ellis buffer) 0.05 M citric acid, 0.05 M NaH.sub.2 PO.sub.4.H.sub.2 
O, 0.05 M Na.sub.2 CO.sub.3, 0.05 M 2-amino-3-methyl-1,3-propanediol, pH 
10.5) .01 M (dithiothreitol). The suspension was quick frozen on dry ice 
and thereafter thawed in a water bath maintained at 30.degree. C. 
Thereafter, 1 ml of glass beads (Thomas Scientific #5663 R50) was added to 
the suspension and the mixture vortexed for about 15 seconds. This 
procedure was repeated 8 times. The glass beads were removed by 
centrifuging through glass wool. The lysed cell sample was collected and 
added to an equal volume of Ellis buffer (pH 6.5). The extract was then 
used in the insect assay(s). 
The amount of insecticidal protein expressed in several of the 
plant-colonizing microorganism of the invention were estimated based on 
ELISA (17) immunological analysis of soluble protein and Western Blot (13) 
analysis of total protein. Estimates for several plasmid constructs are 
shown in Table III. 
TABLE III 
______________________________________ 
Picograms of B.t. 
Protein per 
Plant-Colonizing 
Microgram of 
Plasmid Microorganism Total Protein 
% 
______________________________________ 
pMAP12 Ps 3732-3-7 816 .08 
pMAP13 Ps 3732-3-7 252 .02 
pMAP14 Ps 3732-3-7 1460 .14 
pMAP15 Ps 3732-3-7 1860 .18 
pMAP15 P. fluorescens 
11584 1.1 
112-12 
pMAP8 E. coli JM101 120000 12 
______________________________________ 
EXAMPLE 2--DIET ASSAY 
A standard artificial diet medium was dispensed into 3.5.times.1.0 cm flat 
bottom wells (50 wells/tray--Flow Laboratories Inc.) to a volume of ca. 5 
mls. The agar based diet hardened within a short period of time and was 
thereafter treated with the test (or control) material. 100 ul of test (or 
control) material was applied with an automatic pipettor to the surface of 
each of 10 wells of diet. An alcohol flamed glass spreader was used to 
spread the material to insure an even coating. The treated trays were 
allowed to dry under a vertical flow hood before placing one neonate 
larvae on the diet surface of each of 10 wells (10 larvae/treatment). The 
trays were sealed and then incubated at 28.degree. C. for 4 days prior to 
evaluating the percent mortality induced by the treatment. Control 
treatments were included in each assay to check the effects of the diet 
and the un-engineered microorganism. In all cases no toxicity (i.e., 
mortality) was observed from the diet alone or from diet treated with 
non-engineered microorganisms. Table IV summarizes the results observed 
when microorganisms containing novel plasmids of this invention were 
tested for toxicity against larvae of tobacco hornworm (Manduca sexta), 
corn earworm (Heliothi zea) and cabbage looper (Trichoplasia ni). 
TABLE IV 
______________________________________ 
Material % 
Insect Plasmid Microorganism 
Applied Mortality 
______________________________________ 
Corn Earworm 
pMAP1 E. coli SR200 
Protein Extract 
44.4 
pMAP2 " " 55.5 
Tobacco pMAP1 E. coli SR200 
Unlysed Cells 
100 
Hornworm 
pMAP2 " " 100 
pMAP3 E. coli JM101 
" 100 
pMAP4 " " 100 
pMAP6 " " 100 
pMAP7 " " 100 
pMAP8 " " 100 
pMAP12 " " 100 
pMAP13 " " 100 
Tobacco pMAP8 E. coli JM101 
Unlysed Cells 
100 
Hornworm pMAP12 " " 100 
pMAP13 " " 100 
pMAP14 " " 100 
pMAP15 " " 100 
pMAP12 Ps. 3732-3-7 
" 100 
pMAP13 " " 100 
pMAP14 " " 100 
pMAP15 " " 100 
pMAP15 A. radiobacter 
" 100 
212-4 
Cabbage PMAP1 E. coli SR200 
" 100 
Looper pMAP1 " " 100 
______________________________________ 
% 
Plasmid 
Microorganism Preparation 
Mortality 
______________________________________ 
pMAP10 E. coli Unlysed cells 
100 
pMAP10 " " 100 
pMAP11 " " 100 
pMAP20 " " 100 
pMAP21 " " 100 
pMAP20 Ps. 3732-3-7 " 100 
pMAP21 " " 100 
pMAP22 " " 100 
pMAP23 " " 100 
______________________________________ 
EXAMPLE 3 
The procedure of Example 2 was repeated except that larvae of the black 
cutworm (Agrotis ipsilon) were used. In one test no larval mortality was 
observed; however, application of the engineered microorganism resulted in 
significant weight loss of the larvae. In another test, mortality was 
observed. In all cases 100 .mu.l of protein extract or unlysed cell 
preparation was applied. The results are summarized in Table V. 
TABLE V 
______________________________________ 
Average 
% Weight 
Plasmid Microorganism 
Preparation 
Mortality 
(mg) 
______________________________________ 
pMAP12 Ps. 3732-3-7 
Protein Extract 
0 389.6 
pMON5008 
" " 0 656.2 
-- untreated control 
" 0 776.4 
pMAP8 E. coli JM101 
Lysed Cells 
29.7 -- 
pMAP18 " " 55.0 317.0 
pMAP18 " " 10.0 255.0 
-- P. fluorescens 
" 12.5 293.3 
112-12-15 
-- untreated control 
" 0 290.8 
______________________________________ 
EXAMPLE 4--DROPLET ASSAY 
A 2:1 (microbial preparation: FD&C blue dye) mix containing 10% sucrose was 
vortexed, then applied to the surface of a styrofoam plate in about 10 ul 
droplets. Neonate tobacco hornworm larvae were placed in the vicinity of 
the droplets and allowed to feed at will. Satiated larvae, as evidenced by 
their blue abdomens, were removed from the plate and placed on artificial 
diet. Percent mortality was evaluated after four days. The results are 
summarized in Table VI. 
TABLE VI 
______________________________________ 
Plasmid Microorganism CFU/.mu.l* 
% Mortality 
______________________________________ 
pMAP1 E. coli SR200 1.0 .times. 10.sup.5 
50 
pMAP2 " 7.5 .times. 10.sup.4 
100 
pMAP3 E. coli JM101 7.9 .times. 10.sup.4 
100 
pMAP4 " 3.4 .times. 10.sup.1 
100 
pMAP1 E. coli SR200 8.6 .times. 18.sup.3 
0 
pMAP2 " 2.2 .times. 10.sup.4 
75 
pMAP4 E. coli JM101 2.2 .times. 10.sup.4 
80 
______________________________________ 
*Approximate number of Colony Forming Units ingested. 
EXAMPLE 5--LEAF DISC ASSAY 
Two (2) cm discs of tomato leaf tissue was immersed in a solution of live 
cells. The discs were blotted on filter paper and then the discs were 
added individually to wells containing distilled water moistened filter 
paper. The wells were identical to those used in the Diet Assay. One 
neonate tobacco hornworm larvae was added to each of ten wells/treatment. 
Mortality was recorded after 72 hours. The results are summarized in Table 
VIII. 
TABLE VIII 
______________________________________ 
Plasmid Microorganism CFU/ml % Mortality 
______________________________________ 
pMAP12 Ps. 3732-3-7 1 .times. 10.sup.9 
100 
pMAP15 " 1 .times. 10.sup.9 
100 
pMAP15 " 1 .times. 10.sup.9 
100 
pMAP20 " 1.6 .times. 10.sup.7 
0* 
pMAP21 " 3.1 .times. 10.sup.8 
100 
pMAP22 " 4.0 .times. 10.sup.6 
0* 
pMAP23 " 1.9 .times. 10.sup.7 
100 
______________________________________ 
*Insects were alive; however, growth was stunted. 
DNA Sequence of the B.t. Toxin Gene 
The DNA sequence of 3734 nucleotides from pMAP4 including the entire toxin 
protein coding sequence was determined by the chain termination method of 
Sanger et al. (19). The sequence includes 75 nucleotides upstream of the 
translational initiation codon and extends through a KpnI site 188 
nucleotides after the translational termination codon. The DNA sequence 
and the derived amino acid sequence for the toxin protein are shown in 
FIG. 3. The first nucleotide of the protein coding sequence is labeled 
position +1. DNA sequences from nucleotide -75 to nucleotide 220 and from 
nucleotide 3245 to 3650 were also determined by the chemical method of 
Maxam and Gilbert (20). The DNA sequence from -171 to -160 is from the 
known sequence of the plasmid vector pUC7 (Vieira, supra.) DNA sequence 
from -159 to -153 is from a chemically synthesized PstI linker (New 
England Biolabs); the three nucleotides from -152 to -150 are derived from 
the known cleavage site for restriction enzyme EpaI. The sequence from 
nucleotide -149 to -76 (74 nucleotides) has been inferred from known 5' 
-flanking sequences of other B.t. toxin genes (21, 22, 23, 24). 
The DNA sequence of the 2.4 Kb fragment of pMAP10 begins at the BamHI site 
at -171 and terminates at the KpnI site at 2175. The protein toxin 
expressed by this truncated gene represents about 63% of the protein toxin 
expressed by the entire gene. E. coli transformants with pMAP18, which 
contains only the 2.4 Kb gene fragment, were toxic to Manduca sexta and 
black cutworm (Agrotis ipsilon) which data demonstrate that the shortened 
protein is efficacious for insect control. 
Those skilled in the art recognize that certain variations of the DNA 
fragments and genes disclosed and claimed herein can be made by one or 
more nucleotide deletions, substitutions, inversions and/or additions 
using known techniques. It should therefore be understood that such 
variations which do not result in a substantial change in the activity of 
the protein encoded therein are considered within the scope of the present 
invention. 
Although this invention has been described with respect to specific 
embodiments, the details thereof are not to be construed as limitations, 
for it will be apparent that various equivalents, changes and 
modifications may be resorted to without departing from the spirit and 
scope thereof and it is understood that such equivalent embodiments are to 
be included herein. 
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