WO1993003619A1

WO1993003619A1 - Multi-targeted bacillus thuringiensis bioinsecticide

Info

Publication number: WO1993003619A1
Application number: PCT/US1991/005930
Authority: WO
Inventors: Lee A. Bulla, Jr.; Jun Biao Chen; Purnima Ray
Original assignee: Research Corporation Technologies, Inc.
Priority date: 1991-08-19
Filing date: 1991-08-19
Publication date: 1993-03-04

Abstract

Bacillus thuringiensis strains having an enhanced range of insecticidal activity are constructed by genetic engineering and a single colony mating technique. The mating technique allows for expression of chromosomal protoxin genes in the recipient strain. The preferred strain comprises a Bacillus thuringiensis subspecies israelensis that has been genetically modified by the stable incorporation of a protoxin gene from Bacillus thuringiensis subspecies kurstaki.

Description

MULTI-TARGETED BACILLUS THURINGIENSIS BIOINSECTICIDE

Background of the Invention

This invention was made with U.S. government support under Grant 58-319R-7-014 from OICD (Office of International Cooperation and Development) awarded by the U.S. Department of Agriculture. The government has certain rights in the invention.

This invention relates to improved Bacillus thuringiensis strains that have broadened insecticidal activity, biologically pure cultures of such strains, and methods of making and using such strains.

As environmental and health hazards of certain chemical pesticides become increasingly evident, greater attention is being given to the use of alternative agents for pest control. The indiscriminate use of chemical pesticides has led to disruption of many plants' natural defensive agents. Additionally, chemical pesticides may destroy beneficial insects and parasites as well as the pests they are meant to destroy. Further, many of the targeted pests have developed resistance to chemical pesticides after repeated exposure to them. Therefore, not only is the utility of the pesticide reduced, but also there is an increase in resistant strains of the pests, both targeted and minor, due to the reduction of beneficial parasitic organisms. Therefore, there has been an increasing turn towards the use of biopesticides for controlling insects, fungal and weed infestations of agricultural crops.

Certain bacteria have been found useful as biopesticides. Generally, such biopesticidal substances comprise the bacteria, which produce a substance toxic to the pest, i.e.. a toxin. The bacteria may be present with or without a bacterial growth medium or a carrying medium. The substances may be directly applied to the plants, and they are typically less harmful to non-target organisms and to the environment than their chemical counterparts.

One such alternative biopesticidal substance comprises the bacterium Bacillus thuringiensis (B.t. ) and is currently one of the most.widely used biopesticides. B.t. is a rod-shaped, aerobic, spore-forming microorganism which produces proteins known as protoxins. The protoxins are deposited in B.t. as crystalline structures, or they are present in B.t. as part of the spore coat. These protoxin crystals are pathogenic to a variety of sensitive insects. However, the crystals are only active, i.e. , pathogenic in the insect, after ingestion. They are cleaved in the insect's gut to produce the active toxin. Therefore, B.t. is harmless to plants and other nontargeted insects or animals.

Different strains of B.t. produce different crystals, which are toxic to different types of insects. Generally, a single strain of B.t. produces a protoxin for a certain type of pests. It would therefore be advantageous to construct a strain of B.t. which would be toxic to a variety of pests and thereby limit the amount of biopesticides necessary for control of pests. Construction of a bacterial strain for a variety of pests would also allow for the specific design of an insecticide to the varying needs of different geographical areas.

The use of different strains of B.t. as biopesticides for various insect pests is known in the art. Also known is the use of genetic engineering to alter B.t. strains in order to combine activity against more than one insect pest in one strain.

United States Patent 5,024,837 to Donnovan et al., incorporated herein by reference, discloses B.t. strains having insecticidal activity against both lepidopteran and coleopteran insects. The coleopteran active endotoxin is produced by an acquired plasmid containing the cryC gene that encodes for the active endotoxin. Prior to insertion into a recipient B.t. strain, the plasmid may first be transferred to an intermediate Bacillus subtilis (B.s.) strain. After final transformation, the recipient B.t. strain has a combination of insecticidal activity not normally found in non-recombinant B.t. strains.

United States Patent 4,935,353 to Burges et al., incorporated herein by reference, discloses a strain of B.t. having entomicidal activity against lepidopterous insects. The new strain is produced by combining plasmids of two starting strains using plasmid transfer into a new strain. The new strain will express both of the plasmid toxin genes. One of the parent strains of Burges et al. is an asporogenic mutant which does not exhibit entomocidal activity on its own, although the strain does contain the genetic material which would enable it to exhibit entomocidal activity if it were not asporogenic.

European Patent Application 0202470 of Abbott Laboratories, incorporated herein by reference, discloses a toxin crystal protein which is located on the plasmid of Bacillus thuringiensis subspecies kurstaki (B.t.k.) . This endotoxin is active against lepidopteran insects and is expressed in a B.s. strain following channeling into Escherichia coli (E. coli) .

Japanese Abstract JP63017687 discloses expression of a B.t.k. toxin gene in B.s. The B.t.k. protoxin HD-1 gene is cloned, channelled through E___. coli and incorporated in a B.s. strain by plasmid transfer.

United States Patent 4,797,279 to Karamata et al., incorporated herein by reference, discloses bacterial cells that contain a plasmid containing a gene coding for a B.t.k. endotoxin and a plasmid that contains a gene coding for a Bacillus thuringiensis subspecies tenebrionis (B.t.t. ) endotoxin. The resulting hybrids which are obtained in a conventional manner by conjugation of a B.t.k. strain and a B.t.t. strain produce both endotoxins. Conjugation between the two strains is mediated by a conjunctive plasmid and all transfers are plasmid transfers. PCT Publication No. WO 91/01087, incorporated herein by reference, discloses a B.t. hybrid gene which is obtained by in vivo recombination of the hypervariable regions of the genes coding for the B.t. toxin. The preferred pesticidal toxin producing genes are HD-1 Dipel and the HD-73 from slightly different strains of B.t.k. Both of the above-mentioned genes are cloned in a plasmid vector which is then introduced into a strain of E_j_ coli for production of the pesticidal toxins. The application also discloses several different strains of B.t.. including Bacillus thuringiensis subspecies israelensis (B.t.i. ) , which is disclosed as toxic to several dipteran species. It further discloses the isolation and characterization of various genes encoding B.t. toxins. According to the application, this work has shown that the genes reside in the chromosome as well as plasmid DNA and that some strains contain multiple genes. In addition, the application discloses that an endotoxin gene from Bacillus thuringiensis berliner (B.t.b.) is nearly identical to one endotoxin gene from B.t.k. but not to another.

Raymond et al.. Journal Cell Biology. 107:421a (1988) discloses a comparison of the B.t.k. and the B.t.b. protoxin genes. The amino acid sequences of the two genes were 90% homologous.

Crickmore et al., Biochem Journal. 270:133-136 (1990) notes the lack of an efficient transformation system for B.t. as well as the authors' previous success with electroporation. The authors used shuttle vectors and electroporation to introduce cloned B.t. toxin genes into various B.t. strains. In one case, a toxin gene from B.t.t. was introduced into B.t.i. using electroporation. The resulting strain had activity against an insect that neither parent possessed. The authors noted that a major problem with the use of shuttle vectors has been the structural instability of the plasmid within the B.t. recipient strains. They found that some plasmids were stable while others were not.

Klier et al., Mol. Gen. Genet.. 191:257-262 (1983) discloses heterospecific mating between a B.s. strain which contained recombinant plasmids having delta-endotoxin genes from B.t.b. 1750, and different B.t. strains. The berliner strain contains two endotoxins, one of plasmid origin and another of chromosomal origin. When the endotoxin gene of host plasmid origin was introduced into a B.t.i. strain, the gene was expressed. On the other hand, when the endotoxin gene of chromosomal origin was introduced into a different B.t.k. strain, it was apparently incorporated into the chromosomal DNA of the transcipient strain, but it was either not expressed or was expressed in quantities below the sensitivity of the detection method used. Additionally, the transcipient strain was unstable; it had to be kept under antibiotic pressure.

Therefore, there is a need for a B.t. strain that has an expanded insect host range by virtue of containing at least one acquired protoxin gene of chromosomal origin that is stably integrated into the microorganism's genome and that is adequately expressed so as to provide sufficient quantities of protoxin for insecticidal activity. There is also a need to provide a method for constructing a transcipient bacterium, containing genes of both chromosomal and plasmid origin, which is stable and can exist and grow without constant antibiotic pressure. Such strains would result in biopesticides having a broader range of activity.

The present invention overcomes the problems in the prior art by constructing a Bacillus thuringiensis strain having an expanded insect host range using a novel method for genetically transforming gram-positive spore-forming bacilli as well as gram-negative bacteria. The method involves the use of a single colony mating technique. The technique allows for the stable incorporation and expression of chromosomal protoxin genes in B.t. strains.

Summary of the Invention

It is an object of the present invention to provide a Bacillus thuringiensis bacterium having an expanded host range.

It is a further object of the invention to provide a method for the stable incorporation and expression of Bacillus thuringiensis chromosomal protoxin genes into other Bacillus thuringiensis hosts.

Another object of the invention is to provide an insecticide which contains the Bacillus thuringiensis bacterium having an expanded host range. A still further object of the invention is to provide a method of controlling insects using the Bacillus thuringiensis bacterium having an expanded host range.

Yet another object of the invention is to provide a transformed B.s. bacterium which stably incorporates and expresses a recombinant chromosomal protoxin gene of a B.t. bacterium.

-Another object of the invention is to provide a method for transforming a B.s. bacterium resulting in the stable incorporation and expression of a recombinant chromosomal Bacillus thuringiensis protoxin gene.

A still further object of the invention is to provide a method of transferring DNA from a donor bacterium to a recipient bacterium which combines the selection and conjugation steps into a single step.

A still further object of the invention is to provide expression vectors containing Bacillus thuringiensis protoxin genes and host cells transformed by such genes and vectors.

Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the invention. The objects and advantages of the invention will be obtained by means of the instrumentalities and combinations particularly pointed out in the appended claims. To achieve the objects and in accordance with the purpose of the invention, as embodied and broadly described herein, the present invention provides a Bacillus thuringiensis strain having an expanded insect host range. The strain is a genetically modified Bacillus thuringiensis bacterium that produces a protoxin by expression of a foreign chromosomal protoxin gene. Preferably, the bacterium also produces a second protoxin by expression of a gene that differs from the foreign chromosomal protoxin gene. Preferably, such second gene occurs naturally in the strain that is the recipient for the foreign chromosomal protoxin gene. Thus, the preferred bacterium will have the combined insecticidal activity of both the recipient strain and the donor strain. Most preferably, such activity is directed against lepidopteran insects and dipteran insects.

In an alternative preferred embodiment, the genetically modified Bacillus thuringiensis bacterium further produces a third protoxin by expression of a gene that differs from the first and second protoxins. This third gene may be a foreign chromosomal protoxin gene or a protoxin gene of plasmid origin. This permits the modified bacterium to have even further expanded activity, such as against coleopteran insects.

The genetically modified bacteria of the invention are used as biopesticides. The insecticide comprises a mixture of an insecticidally effective amount of one or more of the genetically modified B.t. strains in a pesticidally acceptable carrier. Thus, the invention further provides a method of controlling or mitigating insect pests by applying an effective amount of the insecticide of the invention to the pests or their habitats.

The invention further comprises a method of making the genetically modified bacteria. In its broadest terms, the method comprises using genetic engineering (recombinant DNA) techniques to transform an intermediate microorganism, preferably Bacillus subtilis, with the foreign chromosomal protoxin gene. The chromosomal protoxin gene is then transferred from the transformed Bacillus subtilis to the recipient Bacillus thuringiensis bacterium by our novel single colony mating technique.

More specifically, the Bacillus subtilis bacterium is transformed by the steps of: (1) ligating a Bacillus thuringiensis chromosomal protoxin gene into a shuttle vector to produce an expression vector; (2) transferring the expression vector into a Bacillus subtilis bacterium by protoplast transformation; and (3) recovering the transformed Bacillus subtilis bacterium. Preferably, the expression vector is first replicated in a compatible host, such as Escherichia coli. before being transferred into the Bacillus subtilis bacterium.

The single colony mating technique comprises several steps. First, the recipient bacterium, in this case the Bacillus thuringiensis that is being genetically modified, is placed onto a solid growth medium that contains at least two different antibiotics. The recipient bacterium contains a gene encoding resistance to one of the antibiotics. Second, single colonies of the donor bacterium are separately inoculated onto the medium. The donor bacterium contains a gene encoding resistance to the other of the two antibiotics, and the resistance gene is part of the DNA being transferred. Third, the agar plate is incubated for a sufficient period of time and under appropriate, standard conditions to effect transfer of the DNA from the donor bacterium to the recipient bacterium. Fourth, a transcipient colony is selected after the incubation by selecting a colony that grows on the medium. Only those transcipient bacteria; i.e. those that contain the DNA from the donor, are able to grow because only they will be resistant to both antibiotics. Preferably, transcipient colonies are further evaluated for insecticidal activity, particularly activity showing the expression of the protoxin chromosomal gene.

The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate one embodiment of the invention and, together with the description, serve to explain the principles of the invention.

Description of the Figures

Figure 1 is an agarose gel electrophoretogra of pUW39 isolated from transformed J3__ coli. Lane l contains pUW39 DNA digested with the restriction enzyme, EcoRI. Lane 2 contains pHV33 DNA digested with EcoRI. Figure 2 is an electrophoretic and Southern hybridization analysis of pUW39 isolated from transformed B.s. Panel A is an agarose gel electrophoretogram of EcoRI digest of Charon 4A DNA (lane 1) , pHV33 DNA (lane 2) , and pUW39 DNA (lanes 4-6) . Lane 3 is a partial digest of pUW39. Panel B is an autoradiogram of the ³²P-labeled 4.6 kbp DNA fragment hybridized to the EcoRI digest contained in A.

Figure 3 is an Ouchterlony double diffusion immunological analysis of transformed B.s. containing the 130 kDa protoxin gene of B.t.k. Well 1 contains alkaline solubilized cell extract of well-type B.t.k. Well 2 contains alkaline solubilized cell extract of B.s. transformed with pUW39. Well 3 contains alkaline solubilized cell extract of untransformed wild-type B.s. Well 4 contains B.t.k. toxic protein antibody.

Figure 4 is an Ouchterlony double diffusion immunological analysis of B.t.i. transformed with pUW39 containing the 130 kDa protoxin gene of B.t.k. Wells A and F contain alkaline solubilized B.t.i. toxic protein from wild-type B.t.i. Well B contains B.t.i. toxic protein antibody. Wells C and D contain alkaline solubilized B.t.k. toxic protein from wild-type B.t.k. Well E contains B.t.k. toxic protein antibody. Well G contains alkaline solubilized toxic protein from B.t.i. transformed with pUW39. Figure 5 is an immunochemical detection of 130 kDa protoxins in toxic proteins from wild-type B.t.i.. B.t.k. and B.t.i. transformed with pUW39.

Detailed Description of the Invention

Reference now will be made in detail to the presently preferred embodiments of the invention, which, together with the following examples, serve to explain the principles of the invention.

The invention relates to a genetically modified Bacillus thuringiensis bacterium that produces a protoxin by expression of a foreign chromosomal protoxin gene. As used herein, the phrase foreign chromosomal protoxin gene means a gene of chromosomal origin (i.e., a gene in the chromosome) in a Bacillus thuringiensis strain, which gene is not found in the Bacillus thuringiensis recipient strain and which codes for a protein protoxin (also known as a delta-endotoxin) in the donor strain.

The foreign gene is stably incorporated into the genome of the recipient (host) bacterium. That is, it remains incorporated in the progeny of the modified bacterium without the need for antibiotic pressure. The gene may be incorporated into the chromosome of the recipient bacterium or into a plasmid in the recipient bacterium.

Moreover, the gene expresses at detectable levels, and such expression is stable. That is, the expression of the gene in the recipient strain provides sufficient protoxin for insecticidal activity in standard insect bioassays used to determine such activity, and any variation in the level of expression is within the range normally found in the expression of protoxins by naturally occurring Bacillus thuringiensis strains. Preferably, the LC-50 values for the transcipient strain are virtually the same as those of the parental strains.

Preferably, prior to modification, the recipient strain contains a second protoxin gene that differs from the foreign gene. The second gene most preferably occurs naturally in the recipient strain (i.e., it is inherent), but it could also be an acquired gene incorporated into the genome of the recipient strain by various genetic techniques. In the latter case, the gene could be of chromosomal origin in the donor B.t.. or it could be of plasmid origin in the donor B.t. Therefore, the genetically modified bacterium preferably produces two different protoxins, thereby providing activity against types of insects that neither the recipient bacterium nor the donor bacterium individually had activity against. For example, the recipient bacterium preferably produces a protoxin that is active against dipteran insects, such as mosquitos, black flies, and tsetse flies, and the donor bacterium provides a chromosomal gene that codes for a protoxin against lepidopteran insects, such as cabbage looper, tobacco hornworn, spruce budworm, and corn rootworm. The recipient strain may be any Bacillus thuringiensis strain. Preferably, the recipient is a strain of Bacillus thuringiensis subspecies israelensis (B.t.i.) . Such strains are well known and widely available to those skilled in the art. They naturally produce a protoxin that has activity against dipteran insects.

The donor strain is any Bacillus thuringiensis strain that naturally contains one or more chromosomal protoxin genes. Preferably, the donor strain is a strain of Bacillus thuringiensis subspecies kurstaki (B.t.k.) . This subspecies contains a chromosomal protoxin gene that encodes a protoxin with activity against lepidopteran insects. Preferably, the gene is from a strain that produces the known 130 kilodalton (kDa) lepidopteran protoxin. Alternatively, the following strains may be used as the source of chromosomal protoxin genes: Bacillus thuringiensis subspecies berliner. tolworthi , aizawaii. morrosonii. and galleriae.

In a particularly preferred embodiment, the genetically modified Bacillus thuringiensis bacterium of the invention comprises a bacterium that has the insecticidal activity of the bacterium deposited with the American Type Culture Collection (ATCC) under Accession No. 68665 and derivatives and mutants thereof that retain the insecticidal activity. This strain was deposited with the ATCC, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under the Budapest Treaty on August 13, 1991. This preferred strain is a B.t.i. strain that naturally contains a protoxin against dipteran insects. The strain has been modified by stable insertion of a chromosomal gene from B.t.k. that codes for and expresses a 130 kDa protoxin that has activity against lepidopteran insects. Thus, this strain is active against both dipteran and lepidopteran insects, which is not the case with nonengineered B.t.i. or B.t.k. strains. This strain also contains the preferred expression vector of the invention, which is the plasmid designated pUW39.

Given the teachings contained herein and this particular strain, persons skilled in the art can use known techniques to obtain mutants and derivatives that still have the insecticidal activity of the parent strain. For example, such mutants or derivatives may have enhanced expression of one or both of the strain's protoxins or may have different nutritional requirements. However, as long as they are derived from the strain and have lepidopteran and dipteran activity, they are within the scope of this invention.

In an alternative preferred embodiment, the preferred Bacillus thuringiensis bacterium that produces two different protoxins has been further modified to produce a third protoxin by expression of a gene that differs from the first and second protoxin genes. The third protoxin gene may be from any Bacillus thuringiensis bacterium that contains the gene of interest. Preferably, this third protoxin is active against coleopteran insects, such as Colorado potato beetle and pine bark beetle. The gene for such protoxin may be obtained from any Bacillus thuringiensis strain having anti-coleopteran activity, but it is preferably obtained from a Bacillus thuringiensis subspecies tenebrionsis (B.t.t) strain. The third protoxin gene may be of chromosomal or plasmid origin, and it may be stably integrated into the chromosome or a plasmid of the previously modified Bacillus thuringiensis bacterium. Accordingly, the alternative preferred modified Bacillus thuringiensis bacterium produces protoxins against lepidopteran, dipteran, and coleopteran insects.

The preferred bacteria of the invention are described as a bacterium having anti-dipteran and anti- lepidopteran activity and another bacterium having anti- dipteran, anti-lepidopteran, and anti-coleopteran activity. However, other combinations are also possible, given the teachings contained herein. These include modified bacteria having anti-dipteran and anti-coleopteran activity or anti-lepidopteran and anti-coleopteran activity.

The Bacillus thuringiensis bacteria of the invention are made by a combination of standard genetic engineering techniques and a novel technique that we call single colony mating. The genetic engineering (recombinant DNA) techniques are among those disclosed in Maniatis et al.. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory (1982) , incorporated herein by reference. The genetic engineering techniques are used to transform another bacterium, preferably Bacillus subtilis. with the protoxin encoding gene that is ultimately intended for the recipient Bacillus thuringiensis strain. The gene is then transferred from the transformed Bacillus subtilis to the recipient Bacillus thuringiensis by our single colony mating technique.

Accordingly, the invention also comprises a transformed Bacillus subtilis bacterium that contains a chromosomal protoxin gene of a Bacillus thuringiensis bacterium, wherein the protoxin gene is expressed in the transformed Bacillus subtilis. Preferably, the protoxin gene is the previously discussed gene from B.t.k. Alternatively, the transformed Bacillus subtilis bacterium may also contain a second gene that codes for a different protoxin. This second gene may also be of chromosomal origin or plasmid origin. It may be on an acquired plasmid, i.e., on a plasmid that has been transformed into the Bacillus subtilis. Preferably, the second gene encodes a protoxin having anti-coleopteran activity.

In the genetic engineering aspect of the methodology, the chromosomal protoxin gene of interest is obtained from the appropriate Bacillus thuringiensis strain by known techniques, primarily utilizing restriction enzymes. The gene is preferably cloned into a known cloning vector, such as Charon 4A Lambda Phage. It is then ligated into a shuttle vector. The cloned protoxin gene is preferably purified prior to ligation into the vector. The preferred shuttle vector is the commercially available plasmid pHV33, which was made by fusing pBR322 and pC194. The further ligation of the gene into the shuttle vector in proper orientation and correct reading frame for expression produces an expression vector for expressing the gene in the appropriate host cell. The preferred expression vector is the plasmid pUW39, which is the plasmid formed when the gene for the 130 kDa lepidopteran protoxin from B.t.k. is ligated into the shuttle vector pHV33.

The expression vector is then replicated. This is accomplished by transformation of the expression vector into a compatible host microorganism. With pUW39, the preferred host is E. coli. Other host cells may be chosen by persons skilled in the art, once given the teachings contained herein and the desired expression vector. The vector is put into the E. coli by standard transformation techniques. The E. coli is cultured under standard conditions for a sufficient period of time known or determinable by those skilled in the art to provide for the desired replication of the expression vector.

The expression vector is then recovered from the E. coli by known techniques and transferred into Bacillus subtilis by protoplast transformation, using standard techniques, whereby the transferred genetic material is incorporated into the B. subtilis genome and expressed by the transformed bacterium. Preferably, subclones of the transformed B. subtilis are then identified and confirmed by standard immunological analyses and insect bioassays. Transformation of the B.s. is not limited to protoplast transformation as other known methods, such as electroporation, may be used.

The expression vector is then transferred from the intermediate bacterial strain to the recipient Bacillus thuringiensis strain by use of the single colony mating technique. This technique combines the previously separate steps of conjugation and selection into a single step.

In this technique, the recipient bacterium is placed onto a solid growth medium, preferably an agar plate, containing two different antibiotics. The recipient strain is chosen or genetically engineered so that it contains a gene encoding resistance to one of the antibiotics. Single colonies of the donor bacterium, in this case the transformed Bacillus subtilis, are separately inoculated onto the medium so that they come into contact with the recipient strain. The donor strain contains a gene encoding resistance to the second of the two antibiotics in the medium, i.e., the antibiotic to which the recipient bacterium is not resistant. This second antibiotic resistance gene is on the expression plasmid. Preferably, significant quantities of the donor and recipient bacteria are first grown up in liquid medium that contains no antibiotics.

The solid medium is then incubated for a sufficient period of time and under appropriate conditions known to those skilled in the art or determinable by those skilled in the art, once given the teachings contained herein, to effect a transfer of the expression plasmid from the donor bacterium to the recipient bacterium. The expression vector may integrate with the chromosome of the recipient bacterium or it may remain as a separate plasmid in the recipient bacterium. Colonies of transcipient bacteria are identified and selected after incubation by simply selecting a colony that grows on the medium and isolating the colony. The transcipient colonies will comprise bacteria resistant to both antibiotics. Preferably, the identified transcipient colonies are further evaluated by testing them for insecticidal activity, using standard bioassays.

The sequence of placing the bacteria colonies in the single colony mating technique is not limited to the sequence of the donor colony placed on the recipient colony. Transformation would also occur if the recipient colony is placed on the donor.

The single colony mating technique of the invention is not limited to Bacillus subtilis and Bacillus thuringiensis. It can be applied to transfer DNA from any donor bacterium to any recipient bacterium, including gram- negative bacteria as well as gram-positive bacteria, provided that the recipient bacteria contain a genotypic or phenotypic marker, such as a resistance gene to one of the antibiotics in the medium, and the DNA being transferred from the donor bacterium contains a different genotypic or phenotypic marker, such as a gene for antibiotic resistance to the second antibiotic in the medium. Additional genotypic or phenotypic markers may also be used so long as all such characterizing markers are not contained in either the donor bacteria or the recipient bacteria. In the case of a marker different from antibiotic resistance, such as an ability or inability to metabolize a particular nutrient, at least two markers are used. The donor strain will have one, and the recipient strain will have the other. The medium will have or lack the appropriate nutrient so that only the transcipient can grow on it.

The methods discussed above can be used to add additional toxic activity as well as other phenotypic attributes to the recipient strain, thereby creating a further genetically modified strain that has additionally enhanced insecticidal or other types of activities. One or more additional chromosomal or plasmid protoxin genes can be added. The gene may be identical to one previously added, thereby enhancing the newly created insecticidal activity, but not increasing the range of the transcipient, or a protoxin gene that differs from those already in the transcipient can be added. Preferably, the preferred transcipient of the invention is further genetically modified by the addition of a protoxin gene that encodes a protoxin having activity against coleopteran insects.

The additional gene or genes need not be added by the single colony mating of transformed Bacillus subtilis with the first transcipient. Instead, the gene may be added by known techniques, such as conventional DNA transformation, protoplast fusion, and electroporation.

Once the transcipient strain is isolated and grown, it can then be used as a biopesticide. Preferably, the biopesticide comprises an effective amount of one or more of the modified strains in a pesticidally acceptable carrier. As used herein, the term pesticidally acceptable carrier means a carrier substance for the bacteria whose effects, if any, on the activity of the bacteria, are acceptable to persons skilled in the art and whose effects, if any, on humans, plants, and other living organisms are also acceptable to persons skilled in the art. The formulation for use of the biopesticide is not limited to any specific type of formulation or amount. For example, a biopesticide formulation may be administered as a dust or wettable powder, using methods known in the art. The amount of bacteria in the mixture will vary depending upon the type of pests for which the biopesticide is to be used. What is necessary, however, is that an effective amount of the biopesticide be used in the mixture. Usually the effective amount is between 0.1 to 99% with the remaining composition made up of the carriers or other inactive ingredients. The effective amount can be determined by persons skilled in the art, given the teachings contained herein. Preferably, the biopesticide is in the form of a suspension concentrate.

The bacteria may be added to a suitable carrier as a dust or in a suspension, for instance a suspension in oil or water. Suitable carriers may be either solid or liquid and may correspond to substances normally used in the agricultural field. Such carriers may include but are not limited to natural or regenerated mineral substances, solvents, dispersents, wetting agents, tackifiers, binders or fertilizers. The formulations are prepared in a manner known in the art, such as by mixing or grinding the active ingredients with extenders, i.e., solvents, solid carriers or surfactants. The formulations are then applied to the target crops or to the habitats of the targeted insects in an effective amount. The effective dosage, based upon the LC-50 value, can be determined by persons skilled in the art, based on the teachings contained herein.

The process of the invention produces new strains having a modified activity. It also provides the ability to tailor a specific strain for specific geographical areas, or ecological niches.

The invention will be further explained by the following examples.

Example 1

B.t.i. Having Activity Against Lepidopteran and Dipteran Insects

The chromosomal 130 kDa protoxin gene of B.t.k. HD-l from the Northern Regional Research Laboratory, which encodes a protoxin against lepidopteran insects, was cloned in a Charon 4A lambda phage. The cloned material was then purified from the recombinant phage and digested with restriction enzyme EcoRI. A fragment of the 4.6 kbp DNA from the Charon 4A was then ligated into vector pHV33, according to the method of Palla, M.B., et al., Gene 12:147-154 (1980) which incorporated herein by reference, with T4DNA ligase, resulting in the formation of an expression vector designated pUW39. The plasmid pHV33 is a hybrid plasmid which has been previously constructed by the fusion of pBR322, USB catalog number 14386, and pC194, Ehrilich, S.D., et al., PNAS, 74:1680-1682 (1988), incorporated herein by reference. The plasmid pUW39 was then introduced into Escherichia coli HB101 for replication by conventional straightforward transformation.

Figure 1 shows that the 4.6 kbp fragment, which is equivalent to the Charon 4A fragment, was present in the pUW39 isolated from |__ coli. Two bands of DNA corresponding to 7.4 kbp (equivalent to pHV33) and 4.6 kbp (equivalent to the Charon 4A fragment) were visible in lane 1, whereas only one band, corresponding to 7.4 kbp (equivalent to pHV33) , was visible in lane 2.

The recombinant plasmids were then isolated and transferred into Bacillus subtilis BD224 by protoplast fusion. Subclones in the Bacillus subtilis were identified and confirmed by known immunological analyses and insect bioassays.

Figures 2 and 3 show the presence of the B.t.k. protoxin gene, which is contained in the constructed plasmid pUW39. Figure 2 shows that hybridization occurred with the 4.6 kbp DNA that contains the 130 kDa protoxin gene of B.t.k. (lane 1, arrow) , the dimer consisting of the 4.6 kbp fragment and pHV33 (lane 3, arrow) and to the 4.6 kpb fragment of pUW39 (lanes 4-6, arrow) . No hybridization occurred with pHV33 alone (lane 2) . Figure 3 shows that the B.t.k. toxic protein antigen (well 1) reacted strongly with B.t.k. toxic protein antibody (well 4) creating an intense precipitin band between wells 1 and 4. Likewise, alkaline solubilized cell extract of B.s. transformed with pUW39 that contains the B.t.k. protoxin gene (well 2) reacted with B.t.k. toxic protein antibody (well 4) forming a precipitin band between wells 2 and 4.

The cloned pUW39 containing the B.t.k. protoxin gene was introduced into B.t.i 1884, provided by H. deBarjec, Pasteur Institute, by single colony mating. Prior to and after genetic transfer, the B.t.i. recipient strain contained a gene encoding a protoxin having insecticidal activity against dipteran insects.

The single colony mating method was carried out on an LB solid medium. First, a LB liquid medium was constructed according to the following recipe prior to the mating step. The following ingredients were combined:

10 grams of tryptone 5 grams yeast extract 5 grams sodium chloride 3 ml of 1.0M NaOH

and then diluted with water to a 1 liter liquid medium. Solid LB medium was formed by adding 15 grams of agar to one liter of liquid LB medium.

After construction of the mediums, a 1% innoculum of the recipient B.t.i. strain was grown in liquid LB medium for 24 hours and was then placed into fresh liquid LB medium and incubated for 6 hours. One/tenth ml of the 6 hour culture was then spread evenly onto an LB solid medium which also contained streptomycin, chloramphenicol and casein hydrolysate. Prior to genetic transfer, the recipient B.t.i. strain contained a gene encoding resistance to streptomycin.

Concurrently, Bacillus subtilis containing pUW39 was spread onto a second LB agar plate which contained no antibiotics and was incubated for 24 hours. Single colonies of the Bacillus subtilis were isolated and inoculated onto the surface of the first LB agar plate containing the antibiotics and the B.t.i. The first agar plate had been previously divided into 50 sections and a single colony of B.s. was placed in each section. The agar plate containing both strains was then incubated for 48 hours at 30°C to effect genetic transfer. The B.s. transferred the genetic material originally from B.t.k.. which included the gene encoding resistance to chloramphenicol. Thus, following genetic transfer, only the transcipient strain could grow in the presence of the antibiotics whereas the donor strain, i.e.. B.s. and the recipient strain, i.e.. B.t.i. could not grow due to the absence of both antibiotic resistances.

Transcipient colonies were selected and were then grown several times in the presence of both antibiotics and were then bioassayed for their immunological and insecticidal activities. The immunoassays used were Ouchterlony double diffusion and Western blot analyses and the insect bioassays involved testing activity against tobacco hornworm larvae, (lepidopteran) and mosquito larvae, i.e.. anopheles and aedes spp. (dipteran) insects. The LC-50 values for tobacco hornworm larvae and aedes larvae were 1.0 micrograms/cm² for 5 ml medium and 0.25 micrograms/20 ml, respectively. These values were virtually identical to those values of the parental B.t.k. and B.t.i. strains when tested separately against the same insects under identical conditions.

Figures 4 and 5 depict the results of the immunoassays. Figure 4, an Ouchterlony and im unoassay, shows that the B.t.i. and B.t.k. toxic protein antigens (wells A and D) reacted strongly with the respective B.t.i. and B.t.k. toxic protein antibodies (wells B and E) creating intense precipitin bands between wells A and B and wells D and E. Likewise, alkaline solubilized toxic protein crystals of B.t.i. transformed with pUW39 that contains both the B.t.i. and B.t.k. protoxin genes (well A) reacted with B.t.i. and B.t.k. toxic protein antibodies (wells B and E) forming precipitin bands between wells B and G and wells E and G. No cross-reactivity occurred between B.t.k. crystal protein antigen (well C) and B.t.i. crystal protein antibody (well B) nor between B.t.i. crystal protein antigen (well F) and B.t.k. crystal protein antibody (well E) . Precipitin bands were not observed between wells B and C and wells E and F.

Figure 5, panel 1 shows that the 130 kDa B.t.i. protoxin, as well as several other immunoreactive polypeptides, was present in both the transformed and wild- type B.t.i. strains (lanes A and B, respectively) whereas it was absent in wild-type B.t.k. (lane C) . Panel 2 shows that transformed B.t.i. (lane A) , wild-type B.t.i. (lane B) and wild-type B.t.k. (lane C) contained the 130 kDa protoxin as well as other immunoreactive polypeptides. Both B.t.i. strains (lanes A and B) also contained a 28 kDa polypeptide whereas B.t.k. (lane C) did not. Panel 3 shows that the 130 kDa B.t.k. protoxin was present in the transformed B.t.i. (lane A) and wild-type B.t.k. (lane C) strains but not in wild-type B.t.i. (lane B) .

Table 1 depicts the insecticidal activity of the transformed B.s. against the silkworm larva Bombvx mori. All cells were grown until fully sporulated and then were lysed mechanically. Equal dry weights of cell extracts were treated with 2N NaOH (pH 12) for 5 hours, the insoluble cell debris was removed by centrifugation and the resulting supernatants were dialyzed to pH 8.0. Equal volumes of the supernatants were spread evenly onto equal surface areas of mulberry leaves and 2nd instar larvae of B. mori were released singly onto the mulberry leaves. Mortality counts were taken at 72 hours.

Table 1

Strain % dead larvae at 72 hrs. transformed B.s. 90 wild B.t.k. 100 nontransformed B.s. 0

B.t.i. 1884 0

As can be seen from Table 1, transformed B.s. containing the B.t.k. 130 kDa protoxin gene was quite effective at killing silkworm larvae compared to wild-type B.t.k. Neither wild-type B.s. or B.t.i. 1884 had any toxic effect and the silkworm larvae developed to full maturation.

Table 2 depicts the insecticidal activity of the transcipient B.t.i. against the silkworm Bombyx mori and the mosquito Anopholes spp. All cells tested were grown and handled as described in Table 1. Bioassays with B. mori also were done as in Table 1.

Mosquito bioassays were performed with 2nd instar Anopholes spp. larvae. Single larvae were placed in individual cups holding water plus equal amounts of supernatant to a volume of 20 ml. Twenty larvae were used to determine toxicity of each respective supernatant. Mortality counts were taken at 72 hours.

Table 2

As seen in Table 2, the transcipient B.t.i. containing the 130 kDa B.t.k. protoxin gene killed silkworm larvae as effectively and efficiently as did wild-type B.t.k.. while retaining its capacity to kill mosquito larvae as well. Table 3 depicts the obtained LC-50 values which show that the new transcipient strain contained protoxins resulting in insecticidal activity against both lepidopteran (i.e., tobacco hornworm, manduca sexta) and dipteran (i.e., aedes aegypti) insects at levels comparable to the wild parental strains.

Table 3

Strain Pest LC-50 Value wild B.t.k. tobacco hornworm 0.30 ug/cm² for 5 ml medium transcipient tobacco hornworm 1.00 ug/cm² for B.t.i. 5 ml medium wild B.t.i. mosquito larvae 0.25 ug/20 ml transcipient mosquito larvae 0.25 ug/20 ml B.t.i.

As used in the LC-50 values in Table 3, u stands for micro.

Example 2

B.t.i. Having Activity Against Lepidopteran. Dipteran and Coleopteran Insects

A second example of the invention involves forming a bacterium which contains insecticidal activity against dipteran, lepidopteran, and coleopteran insects. The strain of bacterium described above is first formed and into such strain is introduced a coleopteran toxic gene from Bacillus thuringiensis subsp. tenebrionis. The gene cloned in E. coli encoding for the coleopteran protoxin is first isolated from E_j. coli that contains the toxin gene in a plasmid. The fragment is then ligated into the vector pHV33. Once the gene fragment is ligated into vector pHV33, that recombinant plasmid is then incorporated into the above-obtained B.t.i. strain of Example 1 by electroporation.

An alternative method is to first transfer the recombinant plasmid containing the B.t.t. gene into B.s. by protoplast transformation as described in Example 1 and then transfer the gene from B.s. into B.t.i. by the single colony mating described above. If the single colony mating method is used, an additional antibiotic must be selected and placed on the agar plate. The antibiotic must be selected such that a gene encoding for resistance would be transferred from Bacillus subtilis to the recipient strain along with the gene coding for the coleopteran protoxin.

While the invention has been described with reference to specific embodiments, it will be apparent to those skilled in the art that many alternatives, modifications and variations may be made. Accordingly, it is intended to embrace all such alternatives, modifications and variations that may fall within the spirit and scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A genetically modified Bacillus thuringiensis bacterium that produces a protoxin by expression of a foreign chromosomal protoxin gene.

2. The Bacillus thuringiensis bacterium of claim

1, wherein said bacterium produces a second protoxin by expression of a gene that differs from said foreign chromosomal protoxin gene.

3. The Bacillus thuringiensis bacterium of claim

2, wherein at least one of said protoxins has insecticidal activity against lepidopteran insects.

4. The Bacillus thuringiensis bacterium of claim 2, wherein one of said protoxins has insecticidal activity against dipteran insects.

5. The Bacillus thuringiensis bacterium of claim 1, wherein said bacterium has the insecticidal activity of the bacterium deposited with the ATCC under Accession No. 68665 and derivatives and mutants thereof that retain said insecticidal activity.

6. The Bacillus thuringiensis bacterium of claim 1, wherein said bacterium is a Bacillus thuringiensis subspecies israelensis bacterium that has been genetically modified by the stable incorporation of said foreign chromosomal protoxin gene into its genome.

7. The Bacillus thuringiensis bacterium of claim

6, wherein said Bacillus thuringiensis subspecies israelensis bacterium produces a protoxin against dipteran insects.

8. The Bacillus thuringiensis bacterium of claim

7, wherein said chromosomal protoxin gene is acquired from Bacillus thuringiensis subspecies kurstaki.

9. The Bacillus thuringiensis bacterium of claim

8, wherein said chromosomal protoxin gene encodes a 130 kDa lepidopteran protoxin.

10. The Bacillus thuringiensis bacterium of claim 2, wherein said gene that produces said second protoxin occurs naturally in said bacterium.

11. The Bacillus thuringiensis bacterium of claim 2, wherein said second protoxin is produced by an acquired gene of plasmid origin.

12. An insecticide comprising a mixture of an insecticidally effective amount of the Bacillus thuringiensis bacterium of claim 1 and a pesticidally acceptable carrier.

13. The Bacillus thuringiensis bacterium of claim 2, wherein said bacterium produces a third protoxin by expression of a gene that differs from said first and second protoxin genes.

14. The Bacillus thuringiensis bacterium of claim

13, wherein one of said protoxins has activity against coleopteran insects.

15. The Bacillus thuringiensis bacterium of claim

14, wherein said coleopteran protoxin is produced by expression of a gene acquired from a Bacillus thuringiensis subspecies tenebrionis bacterium.

16. The Bacillus thuringiensis bacterium of claim

15, wherein said coleopteran protoxin is produced by expression of a foreign chromosomal protoxin gene.

17. The Bacillus thuringiensis bacterium of claim

16, wherein said coleopteran protoxin is produced by expression of an acquired plasmid protoxin gene.

18. The Bacillus thuringiensis bacterium of claim 13, wherein said bacterium produces protoxins against lepidopteran, dipteran and coleopteran insects.

19. An insecticide for controlling lepidopteran, dipteran and coleopteran insects comprising a mixture of an insecticidally effective amount of the Bacillus thuringiensis bacterium of claim 18 and a pesticidally acceptable carrier.

20. A transformed Bacillus subtilis bacterium, wherein said bacterium contains a chromosomal protoxin gene of a Bacillus thuringiensis bacterium, and wherein said protoxin gene expresses in said transformed bacterium.

21. The transformed Bacillus subtilis bacterium of claim 20, wherein said chromosomal protoxin gene is acquired from Bacillus thuringiensis subspecies tenebrionis and encodes a coleopteran endotoxin.

22. The transformed Bacillus subtilis bacterium of claim 20, wherein said chromosomal protoxin gene is acquired from Bacillus thuringiensis subspecies kurstaki and encodes a lepidopteran protoxin.

23. A method for transforming a Bacillus subtilis bacterium comprising the steps of:

a. ligating a Bacillus thuringiensis chromosomal protoxin gene into a shuttle vector to produce an expression vector; and

b. transferring said expression vector into a Bacillus subtilis bacterium by protoplast transformation.

24. The method of claim 23 further comprising the step of replicating said expression vector prior to said step of transferring said expression vector into a Bacillus subtilis bacterium.

25. The method of claim 24 further comprising the steps of purifying said cloned protoxin gene prior to ligation into a vector.

26. The method of claim 25, wherein said protoxin gene is first cloned in a Charon 4A lambda phage.

27. The method of claim 25, wherein said cloned protoxin gene is ligated into the vector pHV33.

28. The method of claim 27, wherein said protoxin gene encodes a 130 kDa lepidopteran protoxin of a Bacillus thuringiensis subspecies kurstaki bacterium.

29. The method of claim 23, wherein said protoxin gene is a coleopteran protoxin gene of a Bacillus thuringiensis subspecies tenebrionis bacterium.

30. A method of transferring DNA from a donor bacteria to a recipient bacteria comprising the steps of:

a. placing said recipient bacteria onto a solid growth medium containing two different antibiotics, wherein said recipient bacteria contains a gene encoding resistance to one of said antibiotics;

b. separately inoculating single colonies of said donor bacteria onto said solid growth medium, wherein said donor bacteria contains a gene encoding resistance to the other of said antibiotics, said resistance gene being part of said transferred DNA;

c. incubating said solid growth medium for a sufficient period of time and under appropriate conditions to effect transfer of said DNA from said donor bacteria to said recipient bacteria; and

d. selecting a transcipient colony after said incubation by selecting a colony that grows on said solid growth medium, wherein said transcipient colony comprises bacteria resistant to both antibiotics.

31. A method for constructing the Bacillus thuringiensis bacterium of claim 1 comprising the step of transferring the Bacillus thuringiensis chromosomal protoxin gene from the Bacillus subtilis bacterium of claim 20 to a recipient Bacillus thuringiensis bacterium.

32. The method of claim 31, wherein said Bacillus subtilis bacterium is constructed according to the method of claim 23.

33. The method of claim 32, wherein said transfer of said Bacillus subtilis chromosomal protoxin gene occurs according to the method of claim 30.

34. The method of claim 33, wherein said recipient Bacillus thuringiensis bacterium is a Bacillus thuringiensis subspecies israelensis.

35. A Bacillus thuringiensis bacterium made according to the method of claim 31.

36. A method of controlling or mitigating insecticidal pests comprising the step of applying an effective amount of the insecticide of claim 12 to the pests or their habitats.

37. A method of controlling or mitigating insecticidal pests comprising the step of applying an effective amount of the insecticide of claim 19 to the pests or their habitats.

38. An expression vector for expressing DNA that codes for a protoxin of Bacillus thuringiensis in a compatible host comprising a vector capable of transforming a procaryotic cell and chromosomal DNA encoding for a protoxin of Bacillus thuringiensis inserted into said vector in proper orientation and correct reading frame for expression.

39. The expression vector of claim 37 wherein said chromosomal DNA is inserted into the plasmid pHV33.