Patent Publication Number: US-9404121-B2

Title: Bacillus thuringiensis gene with lepidopteran activity

Description:
CROSS REFERENCE 
     This utility application claims the benefit U.S. Provisional Application No. 61/260,430, filed Nov. 12, 2009, and is a continuation of U.S. application Ser. No. 12/943,085, now granted as U.S. Pat. No. 8,772,577, which is incorporated herein by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to naturally-occurring and recombinant nucleic acids obtained from novel  Bacillus thuringiensis  genes that encode pesticidal polypeptides characterized by pesticidal activity against insect pests. Compositions and methods of the invention utilize the disclosed nucleic acids, and their encoded pesticidal polypeptides, to control plant pests. 
     BACKGROUND OF THE INVENTION 
     Insect pests are a major factor in the loss of the world&#39;s agricultural crops. For example, armyworm feeding, black cutworm damage, or European corn borer damage can be economically devastating to agricultural producers. Insect pest-related crop loss from European corn borer attacks on field and sweet corn alone has reached about one billion dollars a year in damage and control expenses. 
     Traditionally, the primary method for impacting insect pest populations is the application of broad-spectrum chemical insecticides. However, consumers and government regulators alike are becoming increasingly concerned with the environmental hazards associated with the production and use of synthetic chemical pesticides. Because of such concerns, regulators have banned or limited the use of some of the more hazardous pesticides. Thus, there is substantial interest in developing alternative pesticides. 
     Biological control of insect pests of agricultural significance using a microbial agent, such as fungi, bacteria, or another species of insect affords an environmentally friendly and commercially attractive alternative to synthetic chemical pesticides. Generally speaking, the use of biopesticides presents a lower risk of pollution and environmental hazards, and biopesticides provide greater target specificity than is characteristic of traditional broad-spectrum chemical insecticides. In addition, biopesticides often cost less to produce and thus improve economic yield for a wide variety of crops. 
     Certain species of microorganisms of the genus  Bacillus  are known to possess pesticidal activity against a broad range of insect pests including Lepidoptera, Diptera, Coleoptera, Hemiptera, and others.  Bacillus thuringiensis  (Bt) and  Bacillus papilliae  are among the most successful biocontrol agents discovered to date. Insect pathogenicity has also been attributed to strains of  B. larvae, B. lentimorbus, B. sphaericus  (Harwook, ed., ((1989)  Bacillus  (Plenum Press), 306) and  B. cereus  (WO 96/10083). Pesticidal activity appears to be concentrated in parasporal crystalline protein inclusions, although pesticidal proteins have also been isolated from the vegetative growth stage of  Bacillus . Several genes encoding these pesticidal proteins have been isolated and characterized (see, for example, U.S. Pat. Nos. 5,366,892 and 5,840,868). 
     Microbial insecticides, particularly those obtained from  Bacillus  strains, have played an important role in agriculture as alternatives to chemical pest control. Recently, agricultural scientists have developed crop plants with enhanced insect resistance by genetically engineering crop plants to produce pesticidal proteins from  Bacillus . For example, corn and cotton plants have been genetically engineered to produce pesticidal proteins isolated from strains of Bt (see, e.g., Aronson (2002)  Cell Mol. Life Sci.  59(3):417-425; Schnepf et al. (1998)  Microbiol Mol Biol Rev.  62(3):775-806). These genetically engineered crops are now widely used in American agriculture and have provided the farmer with an environmentally friendly alternative to traditional insect-control methods. In addition, potatoes genetically engineered to contain pesticidal Cry toxins have been sold to the American farmer. While they have proven to be very successful commercially, these genetically engineered, insect-resistant crop plants provide resistance to only a narrow range of the economically important insect pests. 
     Accordingly, there remains a need for new Bt toxins with a broader range of insecticidal activity against insect pests, e.g., toxins which are active against a greater variety of insects from the order Lepidoptera. In addition, there remains a need for biopesticides having activity against a variety of insect pests and for biopesticides which have improved insecticidal activity. 
     SUMMARY OF THE INVENTION 
     Compositions and methods are provided for impacting insect pests. More specifically, the embodiments of the present invention relate to methods of impacting insects utilizing nucleotide sequences encoding insecticidal peptides to produce transformed microorganisms and plants that express an insecticidal polypeptide of the embodiments. In some embodiments, the nucleotide sequences encode polypeptides that are pesticidal for at least one insect belonging to the order Lepidoptera. 
     The embodiments provide a nucleic acid and fragments and variants thereof which encode polypeptides that possess pesticidal activity against insect pests (e.g. SEQ ID NO: 1 encoding SEQ ID NO: 2). The wild-type (e.g., naturally occurring) nucleotide sequence of the embodiments, which was obtained from Bt, encodes a novel insecticidal peptide. The embodiments further provide fragments and variants of the disclosed nucleotide sequence that encode biologically active (e.g., insecticidal) polypeptides. 
     The embodiments further provide isolated pesticidal (e.g., insecticidal) polypeptides encoded by either a naturally occurring, or a modified (e.g., mutagenized or manipulated) nucleic acid of the embodiments. In particular examples, pesticidal proteins of the embodiments include fragments of full-length proteins and polypeptides that are produced from mutagenized nucleic acids designed to introduce particular amino acid sequences into the polypeptides of the embodiments. In particular embodiments, the polypeptides have enhanced pesticidal activity relative to the activity of the naturally occurring polypeptide from which they are derived. 
     The nucleic acids of the embodiments can also be used to produce transgenic (e.g., transformed) monocot or dicot plants that are characterized by genomes that comprise at least one stably incorporated nucleotide construct comprising a coding sequence of the embodiments operably linked to a promoter that drives expression of the encoded pesticidal polypeptide. Accordingly, transformed plant cells, plant tissues, plants, and seeds thereof are also provided. 
     In a particular embodiment, a transformed plant can be produced using a nucleic acid that has been optimized for increased expression in a host plant. For example, one of the pesticidal polypeptides of the embodiments can be back-translated to produce a nucleic acid comprising codons optimized for expression in a particular host, for example a crop plant such as a corn ( Zea mays ) plant. Expression of a coding sequence by such a transformed plant (e.g., dicot or monocot) will result in the production of a pesticidal polypeptide and confer increased insect resistance to the plant. Some embodiments provide transgenic plants expressing pesticidal polypeptides that find use in methods for impacting various insect pests. 
     The embodiments further include pesticidal or insecticidal compositions containing the insecticidal polypeptides of the embodiments, and can optionally comprise further insecticidal peptides. The embodiments encompass the application of such compositions to the environment of insect pests in order to impact the insect pests. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The embodiments of the invention are drawn to compositions and methods for impacting insect pests, particularly plant pests. More specifically, the isolated nucleic acid of the embodiments, and fragments and variants thereof, comprise nucleotide sequences that encode pesticidal polypeptides (e.g., proteins). The disclosed pesticidal proteins are biologically active (e.g., pesticidal) against insect pests such as, but not limited to, insect pests of the order Lepidoptera. Insect pests of interest include, but are not limited to:  Ostrinia nubilalis  (European corn borer);  Diatraea grandiosella  (Southwestern corn borer);  Helicoverpa zea  (corn earworm);  Agrotis ipsilon  (black cutworm),  Spodoptera frugiperda  (fall armyworm) and  Pseudoplusia includens  (soybean looper). 
     The compositions of the embodiments comprise isolated nucleic acids, and fragments and variants thereof, which encode pesticidal polypeptides, expression cassettes comprising nucleotide sequences of the embodiments, isolated pesticidal proteins, and pesticidal compositions. Some embodiments provide modified pesticidal polypeptides characterized by improved insecticidal activity against Lepidopterans relative to the pesticidal activity of the corresponding wild-type protein. The embodiments further provide plants and microorganisms transformed with these novel nucleic acids, and methods involving the use of such nucleic acids, pesticidal compositions, transformed organisms, and products thereof in impacting insect pests. 
     The nucleic acids and nucleotide sequences of the embodiments may be used to transform any organism to produce the encoded pesticidal proteins. Methods are provided that involve the use of such transformed organisms to impact or control plant pests. The nucleic acids and nucleotide sequences of the embodiments may also be used to transform organelles such as chloroplasts (McBride et al. (1995)  Biotechnology  13: 362-365; and Kota et al. (1999)  Proc. Natl. Acad. Sci. USA  96: 1840-1845). 
     The embodiments further relate to the identification of fragments and variants of the naturally-occurring coding sequence that encode biologically active pesticidal proteins. The nucleotide sequences of the embodiments find direct use in methods for impacting pests, particularly insect pests such as pests of the order Lepidoptera. Accordingly, the embodiments provide new approaches for impacting insect pests that do not depend on the use of traditional, synthetic chemical insecticides. The embodiments involve the discovery of naturally-occurring, biodegradable pesticides and the genes that encode them. 
     The embodiments further provide fragments and variants of the naturally occurring coding sequence that also encode biologically active (e.g., pesticidal) polypeptides. The nucleic acids of the embodiments encompass nucleic acid or nucleotide sequences that have been optimized for expression by the cells of a particular organism, for example nucleic acid sequences that have been back-translated (i.e., reverse translated) using plant-preferred codons based on the amino acid sequence of a polypeptide having enhanced pesticidal activity. The embodiments further provide mutations which confer improved or altered properties on the polypeptides of the embodiments. See, e.g., copending U.S. application Ser. No. 10/606,320, filed Jun. 25, 2003, and Ser. No. 10/746,914, filed Dec. 24, 2003. 
     In the description that follows, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the embodiments. 
     Units, prefixes, and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The above-defined terms are more fully defined by reference to the specification as a whole. 
     As used herein, “nucleic acid” includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues (e.g., peptide nucleic acids) having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to that of naturally occurring nucleotides. 
     As used herein, the terms “encoding” or “encoded” when used in the context of a specified nucleic acid mean that the nucleic acid comprises the requisite information to direct translation of the nucleotide sequence into a specified protein. The information by which a protein is encoded is specified by the use of codons. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid or may lack such intervening non-translated sequences (e.g., as in cDNA). 
     As used herein, “full-length sequence” in reference to a specified polynucleotide or its encoded protein means having the entire nucleic acid sequence or the entire amino acid sequence of a native (non-synthetic), endogenous sequence. A full-length polynucleotide encodes the full-length, catalytically active form of the specified protein. 
     As used herein, the term “antisense” used in the context of orientation of a nucleotide sequence refers to a duplex polynucleotide sequence that is operably linked to a promoter in an orientation where the antisense strand is transcribed. The antisense strand is sufficiently complementary to an endogenous transcription product such that translation of the endogenous transcription product is often inhibited. Thus, where the term “antisense” is used in the context of a particular nucleotide sequence, the term refers to the complementary strand of the reference transcription product. 
     The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. 
     The terms “residue” or “amino acid residue” or “amino acid” are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively “protein”). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogues of natural amino acids that can function in a similar manner as naturally occurring amino acids. 
     Polypeptides of the embodiments can be produced either from a nucleic acid disclosed herein, or by the use of standard molecular biology techniques. For example, a protein of the embodiments can be produced by expression of a recombinant nucleic acid of the embodiments in an appropriate host cell, or alternatively by a combination of ex vivo procedures. 
     As used herein, the terms “isolated” and “purified” are used interchangeably to refer to nucleic acids or polypeptides or biologically active portions thereof that are substantially or essentially free from components that normally accompany or interact with the nucleic acid or polypeptide as found in its naturally occurring environment. Thus, an isolated or purified nucleic acid or polypeptide is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. 
     An “isolated” nucleic acid is generally free of sequences (such as, for example, protein-encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acids can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acids in genomic DNA of the cell from which the nucleic acid is derived. 
     As used herein, the term “isolated” or “purified” as it is used to refer to a polypeptide of the embodiments means that the isolated protein is substantially free of cellular material and includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of contaminating protein. When the protein of the embodiments or biologically active portion thereof is recombinantly produced, culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals. 
     Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. 
     As used herein, the term “impacting insect pests” refers to effecting changes in insect feeding, growth, and/or behavior at any stage of development, including but not limited to: killing the insect; retarding growth; preventing reproductive capability; antifeedant activity; and the like. 
     As used herein, the terms “pesticidal activity” and “insecticidal activity” are used synonymously to refer to activity of an organism or a substance (such as, for example, a protein) that can be measured by, but is not limited to, pest mortality, pest weight loss, pest repellency, and other behavioral and physical changes of a pest after feeding and exposure for an appropriate length of time. Thus, an organism or substance having pesticidal activity adversely impacts at least one measurable parameter of pest fitness. For example, “pesticidal proteins” are proteins that display pesticidal activity by themselves or in combination with other proteins. 
     As used herein, the term “pesticidally effective amount” connotes a quantity of a substance or organism that has pesticidal activity when present in the environment of a pest. For each substance or organism, the pesticidally effective amount is determined empirically for each pest affected in a specific environment. Similarly, an “insecticidally effective amount” may be used to refer to a “pesticidally effective amount” when the pest is an insect pest. 
     As used herein, the term “recombinantly engineered” or “engineered” connotes the utilization of recombinant DNA technology to introduce (e.g., engineer) a change in the protein structure based on an understanding of the protein&#39;s mechanism of action and a consideration of the amino acids being introduced, deleted, or substituted. 
     As used herein, the term “mutant nucleotide sequence” or “mutation” or “mutagenized nucleotide sequence” connotes a nucleotide sequence that has been mutagenized or altered to contain one or more nucleotide residues (e.g., base pair) that is not present in the corresponding wild-type sequence. Such mutagenesis or alteration consists of one or more additions, deletions, or substitutions or replacements of nucleic acid residues. When mutations are made by adding, removing, or replacing an amino acid of a proteolytic site, such addition, removal, or replacement may be within or adjacent to the proteolytic site motif, so long as the object of the mutation is accomplished (i.e., so long as proteolysis at the site is changed). 
     A mutant nucleotide sequence can encode a mutant insecticidal toxin showing improved or decreased insecticidal activity, or an amino acid sequence which confers improved or decreased insecticidal activity on a polypeptide containing it. As used herein, the term “mutant” or “mutation” in the context of a protein a polypeptide or amino acid sequence refers to a sequence which has been mutagenized or altered to contain one or more amino acid residues that are not present in the corresponding wild-type sequence. Such mutagenesis or alteration consists of one or more additions, deletions, or substitutions or replacements of amino acid residues. A mutant polypeptide shows improved or decreased insecticidal activity, or represents an amino acid sequence which confers improved insecticidal activity on a polypeptide containing it. Thus, the term “mutant” or “mutation” refers to either or both of the mutant nucleotide sequence and the encoded amino acids. Mutants may be used alone or in any compatible combination with other mutants of the embodiments or with other mutants. A “mutant polypeptide” may conversely show a decrease in insecticidal activity. Where more than one mutation is added to a particular nucleic acid or protein, the mutations may be added at the same time or sequentially; if sequentially, mutations may be added in any suitable order. 
     As used herein, the term “improved insecticidal activity” or “improved pesticidal activity” refers to an insecticidal polypeptide of the embodiments that has enhanced insecticidal activity relative to the activity of its corresponding wild-type protein, and/or an insecticidal polypeptide that is effective against a broader range of insects, and/or an insecticidal polypeptide having specificity for an insect that is not susceptible to the toxicity of the wild-type protein. A finding of improved or enhanced pesticidal activity requires a demonstration of an increase of pesticidal activity of at least 10%, against the insect target, or at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 100%, 150%, 200%, or 300% or greater increase of pesticidal activity relative to the pesticidal activity of the wild-type insecticidal polypeptide determined against the same insect. 
     For example, an improved pesticidal or insecticidal activity is provided where a wider or narrower range of insects is impacted by the polypeptide relative to the range of insects that is affected by a wild-type Bt toxin. A wider range of impact may be desirable where versatility is desired, while a narrower range of impact may be desirable where, for example, beneficial insects might otherwise be impacted by use or presence of the toxin. While the embodiments are not bound by any particular mechanism of action, an improved pesticidal activity may also be provided by changes in one or more characteristics of a polypeptide; for example, the stability or longevity of a polypeptide in an insect gut may be increased relative to the stability or longevity of a corresponding wild-type protein. 
     The term “toxin” as used herein refers to a polypeptide showing pesticidal activity or insecticidal activity or improved pesticidal activity or improved insecticidal activity. “Bt” or “ Bacillus thuringiensis ” toxin is intended to include the broader class of Cry toxins found in various strains of Bt, which includes such toxins as, for example, Cry1s, Cry2s, or Cry3s. 
     The terms “proteolytic site” or “cleavage site” refer to an amino acid sequence which confers sensitivity to a class of proteases or a particular protease such that a polypeptide containing the amino acid sequence is digested by the class of proteases or particular protease. A proteolytic site is said to be “sensitive” to the protease(s) that recognize that site. It is appreciated in the art that the efficiency of digestion will vary, and that a decrease in efficiency of digestion can lead to an increase in stability or longevity of the polypeptide in an insect gut. Thus, a proteolytic site may confer sensitivity to more than one protease or class of proteases, but the efficiency of digestion at that site by various proteases may vary. Proteolytic sites include, for example, trypsin sites, chymotrypsin sites, and elastase sites. 
     Research has shown that the insect gut proteases of Lepidopterans include trypsins, chymotrypsins, and elastases. See, e.g., Lenz et al. (1991)  Arch. Insect Biochem. Physiol.  16: 201-212; and Hedegus et al. (2003)  Arch. Insect Biochem. Physiol.  53: 30-47. For example, about 18 different trypsins have been found in the midgut of  Helicoverpa armigera  larvae (see Gatehouse et al. (1997)  Insect Biochem. Mol. Biol.  27: 929-944). The preferred proteolytic substrate sites of these proteases have been investigated. See, e.g., Peterson et al. (1995)  Insect Biochem. Mol. Biol.  25: 765-774. 
     Efforts have been made to understand the mechanism of action of Bt toxins and to engineer toxins with improved properties. It has been shown that insect gut proteases can affect the impact of Bt Cry proteins on the insect. Some proteases activate the Cry proteins by processing them from a “protoxin” form into a toxic form, or “toxin.” See, Oppert (1999)  Arch. Insect Biochem. Phys.  42: 1-12; and Carroll et al. (1997)  J. Invertebrate Pathology  70: 41-49. This activation of the toxin can include the removal of the N- and C-terminal peptides from the protein and can also include internal cleavage of the protein. Other proteases can degrade the Cry proteins. See Oppert, ibid. 
     A comparison of the amino acid sequences of Cry toxins of different specificities reveals five highly-conserved sequence blocks. Structurally, the toxins comprise three distinct domains which are, from the N- to C-terminus: a cluster of seven alpha-helices implicated in pore formation (referred to as “domain 1”), three anti-parallel beta sheets implicated in cell binding (referred to as “domain 2”), and a beta sandwich (referred to as “domain 3”). The location and properties of these domains are known to those of skill in the art. See, for example, Li et al. (1991)  Nature,  305:815-821 and Morse et al. (2001)  Structure,  9:409-417. When reference is made to a particular domain, such as domain 1, it is understood that the exact endpoints of the domain with regard to a particular sequence are not critical so long as the sequence or portion thereof includes sequence that provides at least some function attributed to the particular domain. Thus, for example, when referring to “domain 1,” it is intended that a particular sequence includes a cluster of seven alpha-helices, but the exact endpoints of the sequence used or referred to with regard to that cluster are not critical. One of skill in the art is familiar with the determination of such endpoints and the evaluation of such functions. 
     In an effort to better characterize and improve Bt toxins, strains of the bacterium Bt were studied. Crystal preparations prepared from cultures of the Bt strains were discovered to have pesticidal activity against numerous Lepidopteran pests (see, e.g., Experimental Example 1). An effort was undertaken to identify the nucleotide sequences encoding the crystal proteins from the selected strains, and the wild-type (i.e., naturally occurring) nucleic acids of the embodiments were isolated from these bacterial strains, cloned into an expression vector, and transformed into  E. coli . Depending upon the characteristics of a given preparation, it was recognized that the demonstration of pesticidal activity sometimes required trypsin pretreatment to activate the pesticidal proteins. Thus, it is understood that some pesticidal proteins require protease digestion (e.g., by trypsin, chymotrypsin, and the like) for activation, while other proteins are biologically active (e.g., pesticidal) in the absence of activation. 
     Such molecules may be altered by means described, for example, in U.S. application Ser. No. 10/606,320, filed Jun. 25, 2003, and Ser. No. 10/746,914, filed Dec. 24, 2003. In addition, nucleic acid sequences may be engineered to encode polypeptides that contain additional mutations that confer improved or altered pesticidal activity relative to the pesticidal activity of the naturally occurring polypeptide. The nucleotide sequences of such engineered nucleic acids comprise mutations not found in the wild type sequences. 
     The mutant polypeptides of the embodiments are generally prepared by a process that involves the steps of: obtaining a nucleic acid sequence encoding a Cry family polypeptide; analyzing the structure of the polypeptide to identify particular “target” sites for mutagenesis of the underlying gene sequence based on a consideration of the proposed function of the target domain in the mode of action of the toxin; introducing one or more mutations into the nucleic acid sequence to produce a desired change in one or more amino acid residues of the encoded polypeptide sequence; and assaying the polypeptide produced for pesticidal activity. 
     Many of the Bt insecticidal toxins are related to various degrees by similarities in their amino acid sequences and tertiary structure and means for obtaining the crystal structures of Bt toxins are well known. Exemplary high-resolution crystal structure solution of both the Cry3A and Cry3B polypeptides are available in the literature. The solved structure of the Cry3A gene (Li et al. (1991)  Nature  353:815-821) provides insight into the relationship between structure and function of the toxin. A combined consideration of the published structural analyses of Bt toxins and the reported function associated with particular structures, motifs, and the like indicates that specific regions of the toxin are correlated with particular functions and discrete steps of the mode of action of the protein. For example, many toxins isolated from Bt are generally described as comprising three domains: a seven-helix bundle that is involved in pore formation, a three-sheet domain that has been implicated in receptor binding, and a beta-sandwich motif (Li et al. (1991)  Nature  305: 815-821). 
     As reported in U.S. Pat. No. 7,105,332, and pending U.S. application Ser. No. 10/746,914, filed Dec. 24, 2003, the toxicity of Cry proteins can be improved by targeting the region located between alpha helices 3 and 4 of domain 1 of the toxin. This theory was premised on a body of knowledge concerning insecticidal toxins, including: 1) that alpha helices 4 and 5 of domain 1 of Cry3A toxins had been reported to insert into the lipid bilayer of cells lining the midgut of susceptible insects (Gazit et al. (1998)  Proc. Natl. Acad. Sci. USA  95: 12289-12294); 2) the inventors&#39; knowledge of the location of trypsin and chymotrypsin cleavage sites within the amino acid sequence of the wild-type protein; 3) the observation that the wild-type protein was more active against certain insects following in vitro activation by trypsin or chymotrypsin treatment; and 4) reports that digestion of toxins from the 3′ end resulted in decreased toxicity to insects. 
     A series of mutations may be created and placed in a variety of background sequences to create novel polypeptides having enhanced or altered pesticidal activity. See, e.g., U.S. application Ser. No. 10/606,320, filed Jun. 25, 2003, now abandoned, and Ser. No. 10/746,914, filed Dec. 24, 2003. These mutants include, but are not limited to: the addition of at least one more protease-sensitive site (e.g., trypsin cleavage site) in the region located between helices 3 and 4 of domain 1; the replacement of an original protease-sensitive site in the wild-type sequence with a different protease-sensitive site; the addition of multiple protease-sensitive sites in a particular location; the addition of amino acid residues near protease-sensitive site(s) to alter folding of the polypeptide and thus enhance digestion of the polypeptide at the protease-sensitive site(s); and adding mutations to protect the polypeptide from degradative digestion that reduces toxicity (e.g., making a series of mutations wherein the wild-type amino acid is replaced by valine to protect the polypeptide from digestion). Mutations may be used singly or in any combination to provide polypeptides of the embodiments. 
     In this manner, the embodiments provide sequences comprising a variety of mutations, such as, for example, a mutation that comprises an additional, or an alternative, protease-sensitive site located between alpha-helices 3 and 4 of domain 1 of the encoded polypeptide. A mutation which is an additional or alternative protease-sensitive site may be sensitive to several classes of proteases such as serine proteases, which include trypsin and chymotrypsin, or enzymes such as elastase. Thus, a mutation which is an additional or alternative protease-sensitive site may be designed so that the site is readily recognized and/or cleaved by a category of proteases, such as mammalian proteases or insect proteases. A protease-sensitive site may also be designed to be cleaved by a particular class of enzymes or a particular enzyme known to be produced in an organism, such as, for example, a chymotrypsin produced by the corn earworm  Heliothis zea  (Lenz et al. (1991)  Arch. Insect Biochem. Physiol.  16: 201-212). Mutations may also confer resistance to proteolytic digestion, for example, to digestion by chymotrypsin at the C-terminus of the peptide. 
     The presence of an additional and/or alternative protease-sensitive site in the amino acid sequence of the encoded polypeptide can improve the pesticidal activity and/or specificity of the polypeptide encoded by the nucleic acids of the embodiments. Accordingly, the nucleotide sequences of the embodiments can be recombinantly engineered or manipulated to produce polypeptides having improved or altered insecticidal activity and/or specificity compared to that of an unmodified wild-type toxin. In addition, the mutations disclosed herein may be placed in or used in conjunction with other nucleotide sequences to provide improved properties. For example, a protease-sensitive site that is readily cleaved by insect chymotrypsin, e.g., a chymotrypsin found in the bertha armyworm or the corn earworm (Hegedus et al. (2003)  Arch. Insect Biochem. Physiol.  53: 30-47; and Lenz et al. (1991)  Arch. Insect Biochem. Physiol.  16: 201-212), may be placed in a Cry background sequence to provide improved toxicity to that sequence. In this manner, the embodiments provide toxic polypeptides with improved properties. 
     For example, a mutagenized Cry nucleotide sequence can comprise additional mutants that comprise additional codons that introduce a second trypsin-sensitive amino acid sequence (in addition to the naturally occurring trypsin site) into the encoded polypeptide. An alternative addition mutant of the embodiments comprises additional codons designed to introduce at least one additional different protease-sensitive site into the polypeptide, for example, a chymotrypsin-sensitive site located immediately 5′ or 3′ of the naturally occurring trypsin site. Alternatively, substitution mutants may be created in which at least one codon of the nucleic acid that encodes the naturally occurring protease-sensitive site is destroyed and alternative codons are introduced into the nucleic acid sequence in order to provide a different (e.g., substitute) protease-sensitive site. A replacement mutant may also be added to a Cry sequence in which the naturally-occurring trypsin cleavage site present in the encoded polypeptide is destroyed and a chymotrypsin or elastase cleavage site is introduced in its place. 
     It is recognized that any nucleotide sequence encoding the amino acid sequences that are proteolytic sites or putative proteolytic sites (for example, sequences such as NGSR, RR, or LKM) can be used and that the exact identity of the codons used to introduce any of these cleavage sites into a variant polypeptide may vary depending on the use, i.e., expression in a particular plant species. It is also recognized that any of the disclosed mutations can be introduced into any polynucleotide sequence of the embodiments that comprises the codons for amino acid residues that provide the native trypsin cleavage site that is targeted for modification. Accordingly, variants of either full-length toxins or fragments thereof can be modified to contain additional or alternative cleavage sites, and these embodiments are intended to be encompassed by the scope of the embodiments disclosed herein. 
     It will be appreciated by those of skill in the art that any useful mutation may be added to the sequences of the embodiments so long as the encoded polypeptides retain pesticidal activity. Thus, sequences may also be mutated so that the encoded polypeptides are resistant to proteolytic digestion by chymotrypsin. More than one recognition site can be added in a particular location in any combination, and multiple recognition sites can be added to or removed from the toxin. Thus, additional mutations can comprise three, four, or more recognition sites. It is to be recognized that multiple mutations can be engineered in any suitable polynucleotide sequence; accordingly, either full-length sequences or fragments thereof can be modified to contain additional or alternative cleavage sites as well as to be resistant to proteolytic digestion. In this manner, the embodiments provide Cry toxins containing mutations that improve pesticidal activity as well as improved compositions and methods for impacting pests using other Bt toxins. 
     Mutations may protect the polypeptide from protease degradation, for example by removing putative proteolytic sites such as putative serine protease sites and elastase recognition sites from different areas. Some or all of such putative sites may be removed or altered so that proteolysis at the location of the original site is decreased. Changes in proteolysis may be assessed by comparing a mutant polypeptide with wild-type toxins or by comparing mutant toxins which differ in their amino acid sequence. Putative proteolytic sites and proteolytic sites include, but are not limited to, the following sequences: RR, a trypsin cleavage site; LKM, a chymotrypsin site; and NGSR, a trypsin site. These sites may be altered by the addition or deletion of any number and kind of amino acid residues, so long as the pesticidal activity of the polypeptide is increased. Thus, polypeptides encoded by nucleotide sequences comprising mutations will comprise at least one amino acid change or addition relative to the native or background sequence, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 35, 38, 40, 45, 47, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, or 280 or more amino acid changes or additions. Pesticidal activity of a polypeptide may also be improved by truncation of the native or full-length sequence, as is known in the art. 
     Compositions of the embodiments include nucleic acids, and fragments and variants thereof, that encode pesticidal polypeptides. In particular, the embodiments provide for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO: 2, or the nucleotide sequences encoding said amino acid sequence, for example the nucleotide sequence set forth in SEQ ID NO: 1, and fragments and variants thereof. 
     Also of interest are optimized nucleotide sequences encoding the pesticidal proteins of the embodiments. As used herein, the phrase “optimized nucleotide sequences” refers to nucleic acids that are optimized for expression in a particular organism, for example a plant. Optimized nucleotide sequences may be prepared for any organism of interest using methods known in the art. See, for example, U.S. application Ser. No. 10/606,320, filed Jun. 25, 2003, now abandoned, and Ser. No. 10/746,914, filed Dec. 24, 2003, which describe an optimized nucleotide sequence encoding a disclosed pesticidal protein. In this example, the nucleotide sequence was prepared by reverse-translating the amino acid sequence of the protein and changing the nucleotide sequence so as to comprise  maize -preferred codons while still encoding the same amino acid sequence. This procedure is described in more detail by Murray et al. (1989)  Nucleic Acids Res.  17:477-498. Optimized nucleotide sequences find use in increasing expression of a pesticidal protein in a plant, for example monocot plants of the Gramineae (Poaceae) family such as, for example, a  maize  or corn plant. 
     The embodiments further provide isolated pesticidal (e.g., insecticidal) polypeptides encoded by either a naturally-occurring or modified nucleic acid of the embodiments. More specifically, the embodiments provide polypeptides comprising an amino acid sequence set forth in SEQ ID NO: 2, and the polypeptides encoded by nucleic acids described herein, for example those set forth in SEQ ID NO: 1, and fragments and variants thereof. 
     In particular embodiments, pesticidal proteins of the embodiments provide full-length insecticidal polypeptides, fragments of full-length insecticidal polypeptides, and variant polypeptides that are produced from mutagenized nucleic acids designed to introduce particular amino acid sequences into polypeptides of the embodiments. In particular embodiments, the amino acid sequences that are introduced into the polypeptides comprise a sequence that provides a cleavage site for an enzyme such as a protease. 
     It is known in the art that the pesticidal activity of Bt toxins is typically activated by cleavage of the peptide in the insect gut by various proteases. Because peptides may not always be cleaved with complete efficiency in the insect gut, fragments of a full-length toxin may have enhanced pesticidal activity in comparison to the full-length toxin itself. Thus, some of the polypeptides of the embodiments include fragments of a full-length insecticidal polypeptide, and some of the polypeptide fragments, variants, and mutations will have enhanced pesticidal activity relative to the activity of the naturally occurring insecticidal polypeptide from which they are derived, particularly if the naturally occurring insecticidal polypeptide is not activated in vitro with a protease prior to screening for activity. Thus, the present application encompasses truncated versions or fragments of the sequences. 
     Mutations may be placed into any background sequence, including such truncated polypeptides, so long as the polypeptide retains pesticidal activity. One of skill in the art can readily compare two or more proteins with regard to pesticidal activity using assays known in the art or described elsewhere herein. It is to be understood that the polypeptides of the embodiments can be produced either by expression of a nucleic acid disclosed herein, or by the use of standard molecular biology techniques. 
     It is recognized that the pesticidal proteins may be oligomeric and will vary in molecular weight, number of residues, component peptides, activity against particular pests, and other characteristics. However, by the methods set forth herein, proteins active against a variety of pests may be isolated and characterized. The pesticidal proteins of the embodiments can be used in combination with other Bt toxins or other insecticidal proteins to increase insect target range. Furthermore, the use of the pesticidal proteins of the embodiments in combination with other Bt toxins or other insecticidal principles of a distinct nature has particular utility for the prevention and/or management of insect resistance. Other insecticidal agents include protease inhibitors (both serine and cysteine types), α-amylase, and peroxidase. 
     Fragments and variants of the nucleotide and amino acid sequences and the polypeptides encoded thereby are also encompassed by the embodiments. As used herein the term “fragment” refers to a portion of a nucleotide sequence of a polynucleotide or a portion of an amino acid sequence of a polypeptide of the embodiments. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native or corresponding full-length protein and hence possess pesticidal activity. Thus, it is acknowledged that some of the polynucleotide and amino acid sequences of the embodiments can correctly be referred to as both fragments and mutants. 
     It is to be understood that the term “fragment,” as it is used to refer to nucleic acid sequences of the embodiments, also encompasses sequences that are useful as hybridization probes. This class of nucleotide sequences generally does not encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding the proteins of the embodiments. 
     A fragment of a nucleotide sequence of the embodiments that encodes a biologically active portion of a pesticidal protein of the embodiments will encode at least 15, 25, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, or 1,200 contiguous amino acids, or up to the total number of amino acids present in a pesticidal polypeptide of the embodiments (for example, 737 amino acids for SEQ ID NO: 2). Thus, it is understood that the embodiments also encompass polypeptides that are fragments of the exemplary pesticidal proteins of the embodiments and having lengths of at least 15, 25, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, or 1,200 contiguous amino acids, or up to the total number of amino acids present in a pesticidal polypeptide of the embodiments (for example, 737 amino acids for SEQ ID NO: 2). Fragments of a nucleotide sequence of the embodiments that are useful as hybridization probes or PCR primers generally need not encode a biologically active portion of a pesticidal protein. Thus, a fragment of a nucleic acid of the embodiments may encode a biologically active portion of a pesticidal protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed herein. A biologically active portion of a pesticidal protein can be prepared by isolating a portion of one of the nucleotide sequences of the embodiments, expressing the encoded portion of the pesticidal protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the pesticidal protein. 
     Nucleic acids that are fragments of a nucleotide sequence of the embodiments comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 1,000, 1,200, 1,400, 1,600, 1,800, or 2,000 nucleotides, or up to the number of nucleotides present in a nucleotide sequence disclosed herein (for example, 2,214 nucleotides for SEQ ID NO: 1). Particular embodiments envision fragments derived from (e.g., produced from) a first nucleic acid of the embodiments, wherein the fragment encodes a truncated toxin characterized by pesticidal activity. Truncated polypeptides encoded by the polynucleotide fragments of the embodiments are characterized by pesticidal activity that is either equivalent to, or improved, relative to the activity of the corresponding full-length polypeptide encoded by the first nucleic acid from which the fragment is derived. It is envisioned that such nucleic acid fragments of the embodiments may be truncated at the 3′ end of the native or corresponding full-length coding sequence. Nucleic acid fragments may also be truncated at both the 5′ and 3′ end of the native or corresponding full-length coding sequence. 
     The term “variants” is used herein to refer to substantially similar sequences. For nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the pesticidal polypeptides of the embodiments. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, such as, for example, polymerase chain reaction (PCR) and hybridization techniques as outlined herein. 
     Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a pesticidal protein of the embodiments, such as a mutant toxin. Generally, variants of a particular nucleotide sequence of the embodiments will have at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters. A variant of a nucleotide sequence of the embodiments may differ from that sequence by as few as 1-15 nucleotides, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 nucleotide. 
     Variants of a particular nucleotide sequence of the embodiments (i.e., an exemplary nucleotide sequence) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant nucleotide sequence and the polypeptide encoded by the reference nucleotide sequence. Thus, for example, isolated nucleic acids that encode a polypeptide with a given percent sequence identity to the polypeptide of SEQ ID NO: 2 are disclosed. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs described elsewhere herein using default parameters. Where any given pair of polynucleotides of the embodiments is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or at least about 98%, 99% or more sequence identity. 
     As used herein, the term “variant protein” encompasses polypeptides that are derived from a native protein by: deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Accordingly, the term “variant protein” encompasses biologically active fragments of a native protein that comprise a sufficient number of contiguous amino acid residues to retain the biological activity of the native protein, i.e., to have pesticidal activity. Such pesticidal activity may be different or improved relative to the native protein or it may be unchanged, so long as pesticidal activity is retained. 
     Variant proteins encompassed by the embodiments are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, pesticidal activity as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native pesticidal protein of the embodiments will have at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters. A biologically active variant of a protein of the embodiments may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. 
     The embodiments further encompass a microorganism that is transformed with at least one nucleic acid of the embodiments, with an expression cassette comprising the nucleic acid, or with a vector comprising the expression cassette. In some embodiments, the microorganism is one that multiplies on plants. An embodiment of the invention relates to an encapsulated pesticidal protein which comprises a transformed microorganism capable of expressing at least one pesticidal protein of the embodiments. 
     The embodiments provide pesticidal compositions comprising a transformed microorganism of the embodiments. In such embodiments, the transformed microorganism is generally present in the pesticidal composition in a pesticidally effective amount, together with a suitable carrier. The embodiments also encompass pesticidal compositions comprising an isolated protein of the embodiments, alone or in combination with a transformed organism of the embodiments and/or an encapsulated pesticidal protein of the embodiments, in an insecticidally effective amount, together with a suitable carrier. 
     The embodiments further provide a method of increasing insect target range by using a pesticidal protein of the embodiments in combination with at least one other or “second” pesticidal protein. Any pesticidal protein known in the art can be employed in the methods of the embodiments. Such pesticidal proteins include, but are not limited to, Bt toxins, protease inhibitors, α-amylases, and peroxidases. 
     The embodiments also encompass transformed or transgenic plants comprising at least one nucleotide sequence of the embodiments. In some embodiments, the plant is stably transformed with a nucleotide construct comprising at least one nucleotide sequence of the embodiments operably linked to a promoter that drives expression in a plant cell. As used herein, the terms “transformed plant” and “transgenic plant” refer to a plant that comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome of a transgenic or transformed plant such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. 
     It is to be understood that as used herein the term “transgenic” includes any cell, cell line, callus, tissue, plant part, or plant the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term “transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation. 
     As used herein, the term “plant” includes whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny of same. Parts of transgenic plants are within the scope of the embodiments and comprise, for example, plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like, originating in transgenic plants or their progeny previously transformed with a DNA molecule of the embodiments and therefore consisting at least in part of transgenic cells. The class of plants that can be used in the methods of the embodiments is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants. 
     While the embodiments do not depend on a particular biological mechanism for increasing the resistance of a plant to a plant pest, expression of the nucleotide sequences of the embodiments in a plant can result in the production of the pesticidal proteins of the embodiments and in an increase in the resistance of the plant to a plant pest. The plants of the embodiments find use in agriculture in methods for impacting insect pests. Certain embodiments provide transformed crop plants, such as, for example,  maize  plants, which find use in methods for impacting insect pests of the plant, such as, for example, Lepidopteran pests. 
     A “subject plant or plant cell” is one in which genetic alteration, such as transformation, has been effected as to a gene of interest, or is a plant or plant cell which is descended from a plant or cell so altered and which comprises the alteration. A “control” or “control plant” or “control plant cell” provides a reference point for measuring changes in phenotype of the subject plant or plant cell. 
     A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimuli that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed. 
     One of skill in the art will readily acknowledge that advances in the field of molecular biology such as site-specific and random mutagenesis, polymerase chain reaction methodologies, and protein engineering techniques provide an extensive collection of tools and protocols suitable for use to alter or engineer both the amino acid sequence and underlying genetic sequences of proteins of agricultural interest. 
     Thus, the proteins of the embodiments may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the pesticidal proteins can be prepared by introducing mutations into a synthetic nucleic acid (e.g., DNA molecule). Methods for mutagenesis and nucleic acid alterations are well known in the art. For example, designed changes can be introduced using an oligonucleotide-mediated site-directed mutagenesis technique. See, for example, Kunkel (1985)  Proc. Natl. Acad. Sci. USA  82:488-492; Kunkel et al. (1987)  Methods in Enzymol.  154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983)  Techniques in Molecular Biology  (MacMillan Publishing Company, New York), and the references cited therein. 
     The mutagenized nucleotide sequences of the embodiments may be modified so as to change about 1, 2, 3, 4, 5, 6, 8, 10, 12 or more of the amino acids present in the primary sequence of the encoded polypeptide. Alternatively, even more changes from the native sequence may be introduced such that the encoded protein may have at least about 1% or 2%, or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or even about 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, 21%, 22%, 23%, 24%, or 25%, 30%, 35%, or 40% or more of the codons altered, or otherwise modified compared to the corresponding wild-type protein. In the same manner, the encoded protein may have at least about 1% or 2%, or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or even about 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, 21%, 22%, 23%, 24%, or 25%, 30%, 35%, or 40% or more additional codons compared to the corresponding wild-type protein. It should be understood that the mutagenized nucleotide sequences of the embodiments are intended to encompass biologically functional, equivalent peptides which have pesticidal activity, such as an improved pesticidal activity as determined by antifeedant properties against European corn borer larvae. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. 
     One of skill in the art would recognize that amino acid additions and/or substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, charge, size, and the like. Exemplary amino acid substitution groups that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. 
     Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978)  Atlas of Protein Sequence and Structure  (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be made. 
     Thus, the genes and nucleotide sequences of the embodiments include both the naturally occurring sequences and mutant forms. Likewise, the proteins of the embodiments encompass both naturally occurring proteins and variations (e.g., truncated polypeptides) and modified (e.g., mutant) forms thereof. Such variants will continue to possess the desired pesticidal activity. Obviously, the mutations that will be made in the nucleotide sequence encoding the variant must not place the sequence out of reading frame and generally will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444. 
     The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays, such as insect-feeding assays. See, for example, Marrone et al. (1985)  J. Econ. Entomol.  78: 290-293 and Czapla and Lang (1990)  J. Econ. Entomol.  83: 2480-2485, herein incorporated by reference. 
     Variant nucleotide sequences and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different coding sequences can be manipulated to create a new pesticidal protein possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, full-length coding sequences, sequence motifs encoding a domain of interest, or any fragment of a nucleotide sequence of the embodiments may be shuffled between the nucleotide sequences of the embodiments and corresponding portions of other known Cry nucleotide sequences to obtain a new gene coding for a protein with an improved property of interest. 
     Properties of interest include, but are not limited to, pesticidal activity per unit of pesticidal protein, protein stability, and toxicity to non-target species particularly humans, livestock, and plants and microbes that express the pesticidal polypeptides of the embodiments. The embodiments are not bound by a particular shuffling strategy, only that at least one nucleotide sequence of the embodiments, or part thereof, is involved in such a shuffling strategy. Shuffling may involve only nucleotide sequences disclosed herein or may additionally involve shuffling of other nucleotide sequences known in the art. Strategies for DNA shuffling are known in the art. See, for example, Stemmer (1994)  Proc. Natl. Acad. Sci. USA  91:10747-10751; Stemmer (1994)  Nature  370:389-391; Crameri et al. (1997)  Nature Biotech.  15:436-438; Moore et al. (1997)  J. Mol. Biol.  272:336-347; Zhang et al. (1997)  Proc. Natl. Acad. Sci. USA  94:4504-4509; Crameri et al. (1998)  Nature  391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458. 
     The nucleotide sequences of the embodiments can also be used to isolate corresponding sequences from other organisms, particularly other bacteria, and more particularly other  Bacillus  strains. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences that are selected based on their sequence identity to the entire sequences set forth herein or to fragments thereof are encompassed by the embodiments. Such sequences include sequences that are orthologs of the disclosed sequences. The term “orthologs” refers to genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share substantial identity as defined elsewhere herein. Functions of orthologs are often highly conserved among species. 
     In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989)  Molecular Cloning: A Laboratory Manual  (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.), hereinafter “Sambrook”. See also Innis et al., eds. (1990)  PCR Protocols: A Guide to Methods and Applications  (Academic Press, New York); Innis and Gelfand, eds. (1995)  PCR Strategies  (Academic Press, New York); and Innis and Gelfand, eds. (1999)  PCR Methods Manual  (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like. 
     In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as  32 P or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the sequences of the embodiments. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook. 
     For example, an entire sequence disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique to the sequences of the embodiments and are generally at least about 10 or 20 nucleotides in length. Such probes may be used to amplify corresponding Cry sequences from a chosen organism by PCR. This technique may be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of coding sequences in an organism. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook). 
     Hybridization of such sequences may be carried out under stringent conditions. The term “stringent conditions” or “stringent hybridization conditions” as used herein refers to conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold, 5-fold, or 10-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 or 500 nucleotides in length. 
     Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a final wash in 0.1×SSC at 60 to 65° C. for at least about 20 minutes. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. 
     Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T m  (thermal melting point) can be approximated from the equation of Meinkoth and Wahl (1984)  Anal. Biochem.  138:267-284: T m =81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, “% form” is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T m  is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Washes are typically performed at least until equilibrium is reached and a low background level of hybridization is achieved, such as for 2 hours, 1 hour, or 30 minutes. 
     T m  is reduced by about 1° C. for each 1% of mismatching; thus, T m , hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≧90% identity are sought, the T m  can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the T m  for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the T m ; moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the T m ; low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the T m . 
     Using the equation, hybridization and wash compositions, and desired T m , those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T m  of less than 45° C. (aqueous solution) or 32° C. (formamide solution), the SSC concentration can be increased so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993)  Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes , Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995)  Current Protocols in Molecular Biology , Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See also Sambrook. Thus, isolated sequences that encode a Cry protein of the embodiments and hybridize under stringent conditions to the Cry sequences disclosed herein, or to fragments thereof, are encompassed by the embodiments. 
     The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “percentage of sequence identity”, and (e) “substantial identity”. 
     (a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. 
     (b) As used herein, “comparison window” makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches. 
     Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988)  CABIOS  4:11-17; the local alignment algorithm of Smith et al. (1981)  Adv. Appl. Math.  2:482; the global alignment algorithm of Needleman and Wunsch (1970)  J. Mol. Biol.  48:443-453; the search-for-local alignment method of Pearson and Lipman (1988)  Proc. Natl. Acad. Sci.  85:2444-2448; the algorithm of Karlin and Altschul (1990)  Proc. Natl. Acad. Sci. USA  872264, as modified in Karlin and Altschul (1993)  Proc. Natl. Acad. Sci. USA  90:5873-5877. 
     Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989)  CABIOS  5:151-153; Corpet et al. (1988)  Nucleic Acids Res.  16:10881-90; Huang et al. (1992)  CABIOS  8:155-65; and Pearson et al. (1994)  Meth. Mol. Biol.  24:307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul et al (1990)  J. Mol. Biol.  215:403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the embodiments. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the embodiments. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997)  Nucleic Acids Res.  25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See the National Center for Biotechnology Information website on the world wide web at ncbi.hlm.nih.gov. Alignment may also be performed manually by inspection. 
     Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. The term “equivalent program” as used herein refers to any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10. 
     GAP uses the algorithm of Needleman and Wunsch (1970) supra, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater. 
     GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff (1989)  Proc. Natl. Acad. Sci. USA  89:10915). 
     (c) As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.). 
     (d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. 
     (e)(i) The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 80%, 90%, or 95% or more sequence identity when compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes generally means sequence identity of at least 60%, 70%, 80%, 90%, or 95% or more sequence identity. 
     Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5° C. lower than the T m  for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1° C. to about 20° C. lower than the T m , depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. 
     (e)(ii) The term “substantial identity” in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 80%, 85%, 90%, 95%, or more sequence identity to a reference sequence over a specified comparison window. Optimal alignment for these purposes can be conducted using the global alignment algorithm of Needleman and Wunsch (1970) supra. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Peptides that are “substantially similar” share sequences as noted above except that residue positions that are not identical may differ by conservative amino acid changes. 
     The use of the term “nucleotide constructs” herein is not intended to limit the embodiments to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, may also be employed in the methods disclosed herein. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments additionally encompass all complementary forms of such constructs, molecules, and sequences. Further, the nucleotide constructs, nucleotide molecules, and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules, and sequences which can be employed in the methods of the embodiments for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like. 
     A further embodiment relates to a transformed organism such as an organism selected from the group consisting of plant and insect cells, bacteria, yeast, baculoviruses, protozoa, nematodes, and algae. The transformed organism comprises: a DNA molecule of the embodiments, an expression cassette comprising the said DNA molecule, or a vector comprising the said expression cassette, which may be stably incorporated into the genome of the transformed organism. 
     The sequences of the embodiments are provided in DNA constructs for expression in the organism of interest. The construct will include 5′ and 3′ regulatory sequences operably linked to a sequence of the embodiments. The term “operably linked” as used herein refers to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. The construct may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple DNA constructs. 
     Such a DNA construct is provided with a plurality of restriction sites for insertion of the Cry toxin sequence to be under the transcriptional regulation of the regulatory regions. The DNA construct may additionally contain selectable marker genes. 
     The DNA construct will include in the 5′ to 3′ direction of transcription: a transcriptional and translational initiation region (i.e., a promoter), a DNA sequence of the embodiments, and a transcriptional and translational termination region (i.e., termination region) functional in the organism serving as a host. The transcriptional initiation region (i.e., the promoter) may be native, analogous, foreign or heterologous to the host organism and/or to the sequence of the embodiments. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. The term “foreign” as used herein indicates that the promoter is not found in the native organism into which the promoter is introduced. Where the promoter is “foreign” or “heterologous” to the sequence of the embodiments, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked sequence of the embodiments. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. Where the promoter is a native or natural sequence, the expression of the operably linked sequence is altered from the wild-type expression, which results in an alteration in phenotype. 
     The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the sequence of interest, the plant host, or any combination thereof). 
     Convenient termination regions are available from the Ti-plasmid of  A. tumefaciens , such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991)  Mol. Gen. Genet.  262:141-144; Proudfoot (1991)  Cell  64:671-674; Sanfacon et al. (1991)  Genes Dev.  5:141-149; Mogen et al. (1990)  Plant Cell  2:1261-1272; Munroe et al. (1990)  Gene  91:151-158; Ballas et al. (1989)  Nucleic Acids Res.  17:7891-7903; and Joshi et al. (1987)  Nucleic Acid Res.  15:9627-9639. 
     Where appropriate, a nucleic acid may be optimized for increased expression in the host organism. Thus, where the host organism is a plant, the synthetic nucleic acids can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990)  Plant Physiol.  92:1-11 for a discussion of host-preferred codon usage. For example, although nucleic acid sequences of the embodiments may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. (1989)  Nucleic Acids Res.  17:477-498). Thus, the  maize -preferred codon for a particular amino acid may be derived from known gene sequences from  maize. Maize  codon usage for 28 genes from  maize  plants is listed in Table 4 of Murray et al., supra. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989)  Nucleic Acids Res.  17:477-498, herein incorporated by reference. 
     Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other well-characterized sequences that may be deleterious to gene expression. The GC content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. The term “host cell” as used herein refers to a cell which contains a vector and supports the replication and/or expression of the expression vector is intended. Host cells may be prokaryotic cells such as  E. coli , or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells, or monocotyledonous or dicotyledonous plant cells. An example of a monocotyledonous host cell is a  maize  host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. 
     The expression cassettes may additionally contain 5′ leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)  Proc. Natl. Acad. Sci. USA  86: 6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995)  Gene  165(2): 233-238), MDMV leader ( Maize  Dwarf Mosaic Virus), human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991)  Nature  353: 90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987)  Nature  325: 622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in  Molecular Biology of RNA , ed. Cech (Liss, New York), pp. 237-256); and  maize  chlorotic mottle virus leader (MCMV) (Lommel et al. (1991)  Virology  81: 382-385). See also, Della-Cioppa et al. (1987)  Plant Physiol.  84: 965-968. 
     In preparing the expression cassette, the various DNA fragments may be manipulated so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved. 
     A number of promoters can be used in the practice of the embodiments. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, inducible, or other promoters for expression in the host organism. Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985)  Nature  313: 810-812); rice actin (McElroy et al. (1990)  Plant Cell  2: 163-171); ubiquitin (Christensen et al. (1989)  Plant Mol. Biol.  12: 619-632 and Christensen et al. (1992)  Plant Mol. Biol.  18: 675-689); pEMU (Last et al. (1991)  Theor. Appl. Genet.  81: 581-588); MAS (Velten et al. (1984)  EMBO J.  3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, those discussed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611. 
     Depending on the desired outcome, it may be beneficial to express the gene from an inducible promoter. Of particular interest for regulating the expression of the nucleotide sequences of the embodiments in plants are wound-inducible promoters. Such wound-inducible promoters, may respond to damage caused by insect feeding, and include potato proteinase inhibitor (pin II) gene (Ryan (1990)  Ann. Rev. Phytopath.  28: 425-449; Duan et al. (1996)  Nature Biotechnology  14: 494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford et al. (1989)  Mol. Gen. Genet.  215: 200-208); systemin (McGurl et al. (1992)  Science  225: 1570-1573); WIP1 (Rohmeier et al. (1993)  Plant Mol. Biol.  22: 783-792; Eckelkamp et al. (1993)  FEBS Letters  323: 73-76); MPI gene (Corderok et al. (1994)  Plant J.  6(2): 141-150); and the like, herein incorporated by reference. 
     Additionally, pathogen-inducible promoters may be employed in the methods and nucleotide constructs of the embodiments. Such pathogen-inducible promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983)  Neth. J. Plant Pathol.  89: 245-254; Uknes et al. (1992)  Plant Cell  4: 645-656; and Van Loon (1985)  Plant Mol. Virol.  4: 111-116. See also WO 99/43819, herein incorporated by reference. 
     Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al. (1987)  Plant Mol. Biol.  9:335-342; Matton et al. (1989)  Molecular Plant - Microbe Interactions  2:325-331; Somsisch et al. (1986)  Proc. Natl. Acad. Sci. USA  83:2427-2430; Somsisch et al. (1988)  Mol. Gen. Genet.  2:93-98; and Yang (1996)  Proc. Natl. Acad. Sci. USA  93:14972-14977. See also, Chen et al. (1996)  Plant J.  10:955-966; Zhang et al. (1994)  Proc. Natl. Acad. Sci. USA  91:2507-2511; Warner et al. (1993)  Plant J.  3:191-201; Siebertz et al. (1989)  Plant Cell  1:961-968; U.S. Pat. No. 5,750,386 (nematode-inducible); and the references cited therein. Of particular interest is the inducible promoter for the  maize  PRms gene, whose expression is induced by the pathogen  Fusarium moniliforme  (see, for example, Cordero et al. (1992)  Physiol. Mol. Plant Path.  41:189-200). 
     Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the  maize  In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the  maize  GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991)  Proc. Natl. Acad. Sci. USA  88:10421-10425 and McNellis et al. (1998)  Plant J.  14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991)  Mol. Gen. Genet.  227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference. 
     Tissue-preferred promoters can be utilized to target enhanced pesticidal protein expression within a particular plant tissue. Tissue-preferred promoters include those discussed in Yamamoto et al. (1997)  Plant J.  12(2)255-265; Kawamata et al. (1997)  Plant Cell Physiol.  38(7):792-803; Hansen et al. (1997)  Mol. Gen Genet.  254(3):337-343; Russell et al. (1997)  Transgenic Res.  6(2):157-168; Rinehart et al. (1996)  Plant Physiol.  112(3):1331-1341; Van Camp et al. (1996)  Plant Physiol.  112(2):525-535; Canevascini et al. (1996)  Plant Physiol.  112(2):513-524; Yamamoto et al. (1994)  Plant Cell Physiol.  35(5):773-778; Lam (1994)  Results Probl. Cell Differ.  20:181-196; Orozco et al. (1993)  Plant Mol Biol.  23(6):1129-1138; Matsuoka et al. (1993)  Proc Natl. Acad. Sci. USA  90(20):9586-9590; and Guevara-Garcia et al. (1993)  Plant J.  4(3):495-505. Such promoters can be modified, if necessary, for weak expression. 
     Leaf-preferred promoters are known in the art. See, for example, Yamamoto et al. (1997)  Plant J.  12(2):255-265; Kwon et al. (1994)  Plant Physiol.  105:357-67; Yamamoto et al. (1994)  Plant Cell Physiol.  35(5):773-778; Gotor et al. (1993)  Plant J.  3:509-18; Orozco et al. (1993)  Plant Mol. Biol.  23(6):1129-1138; and Matsuoka et al. (1993)  Proc. Natl. Acad. Sci. USA  90(20):9586-9590. 
     Root-preferred or root-specific promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire et al. (1992)  Plant Mol. Biol.  20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991)  Plant Cell  3(10):1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990)  Plant Mol. Biol.  14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of  Agrobacterium tumefaciens ); and Miao et al. (1991)  Plant Cell  3(1):11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also Bogusz et al. (1990)  Plant Cell  2(7):633-641, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume  Parasponia andersonii  and the related non-nitrogen-fixing nonlegume  Trema tomentosa  are described. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the nonlegume  Nicotiana tabacum  and the legume  Lotus corniculatus , and in both instances root-specific promoter activity was preserved. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed roIC and roID root-inducing genes of  Agrobacterium rhizogenes  (see  Plant Science  (Limerick) 79(1):69-76). They concluded that enhancer and tissue-preferred DNA determinants are dissociated in those promoters. Teeri et al. (1989) used gene fusion to lacZ to show that the  Agrobacterium  T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2′ gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see  EMBO J.  8(2):343-350). The TR1′ gene fused to nptII (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995)  Plant Mol. Biol.  29(4):759-772); and rolB promoter (Capana et al. (1994)  Plant Mol. Biol.  25(4):681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179. 
     “Seed-preferred” promoters include both “seed-specific” promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as “seed-germinating” promoters (those promoters active during seed germination). See Thompson et al. (1989)  BioEssays  10:108, herein incorporated by reference. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 ( maize  19 kDa zein); and milps (myo-inositol-1-phosphate synthase) (see U.S. Pat. No. 6,225,529, herein incorporated by reference). Gamma-zein and Glob-1 are endosperm-specific promoters. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to,  maize  15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00/12733, where seed-preferred promoters from end1 and end2 genes are disclosed; herein incorporated by reference. A promoter that has “preferred” expression in a particular tissue is expressed in that tissue to a greater degree than in at least one other plant tissue. Some tissue-preferred promoters show expression almost exclusively in the particular tissue. 
     Where low level expression is desired, weak promoters will be used. Generally, the term “weak promoter” as used herein refers to a promoter that drives expression of a coding sequence at a low level. By low level expression at levels of about 1/1000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts is intended. Alternatively, it is recognized that the term “weak promoters” also encompasses promoters that drive expression in only a few cells and not in others to give a total low level of expression. Where a promoter drives expression at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease expression levels. 
     Such weak constitutive promoters include, for example the core promoter of the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), the core 35S CaMV promoter, and the like. Other constitutive promoters include, for example, those disclosed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611; herein incorporated by reference. 
     Generally, the expression cassette will comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella et al. (1983)  EMBO J.  2:987-992); methotrexate (Herrera Estrella et al. (1983)  Nature  303:209-213; and Meijer et al. (1991)  Plant Mol. Biol.  16:807-820); streptomycin (Jones et al. (1987)  Mol. Gen. Genet.  210:86-91); spectinomycin (Bretagne-Sagnard et al. (1996)  Transgenic Res.  5:131-137); bleomycin (Hille et al. (1990)  Plant Mol. Biol.  7:171-176); sulfonamide (Guerineau et al. (1990)  Plant Mol. Biol.  15:127-136); bromoxynil (Stalker et al. (1988)  Science  242:419-423); glyphosate (Shaw et al. (1986)  Science  233:478-481; and U.S. application Ser. No. 10/004,357; and Ser. No. 10/427,692); phosphinothricin (DeBlock et al. (1987)  EMBO J.  6:2513-2518). See generally, Yarranton (1992)  Curr. Opin. Biotech.  3: 506-511; Christopherson et al. (1992)  Proc. Natl. Acad. Sci. USA  89: 6314-6318; Yao et al. (1992)  Cell  71: 63-72; Reznikoff (1992)  Mol. Microbiol.  6: 2419-2422; Barkley et al. (1980) in  The Operon , pp. 177-220; Hu et al. (1987)  Cell  48: 555-566; Brown et al. (1987)  Cell  49: 603-612; Figge et al. (1988)  Cell  52: 713-722; Deuschle et al. (1989)  Proc. Natl. Acad. Sci. USA  86: 5400-5404; Fuerst et al. (1989)  Proc. Natl. Acad. Sci. USA  86: 2549-2553; Deuschle et al. (1990)  Science  248: 480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993)  Proc. Natl. Acad. Sci. USA  90: 1917-1921; Labow et al. (1990)  Mol. Cell. Biol.  10: 3343-3356; Zambretti et al. (1992)  Proc. Natl. Acad. Sci. USA  89: 3952-3956; Bairn et al. (1991)  Proc. Natl. Acad. Sci. USA  88: 5072-5076; Wyborski et al. (1991)  Nucleic Acids Res.  19: 4647-4653; Hillenand-Wissman (1989)  Topics Mol. Struc. Biol.  10: 143-162; Degenkolb et al. (1991)  Antimicrob. Agents Chemother.  35: 1591-1595; Kleinschnidt et al. (1988)  Biochemistry  27: 1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992)  Proc. Natl. Acad. Sci. USA  89: 5547-5551; Oliva et al. (1992)  Antimicrob. Agents Chemother.  36: 913-919; Hlavka et al. (1985)  Handbook of Experimental Pharmacology , Vol. 78 (Springer-Verlag, Berlin); and Gill et al. (1988)  Nature  334: 721-724. Such disclosures are herein incorporated by reference. 
     The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the embodiments. 
     The methods of the embodiments involve introducing a polypeptide or polynucleotide into a plant. “Introducing” is intended to mean presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the embodiments do not depend on a particular method for introducing a polynucleotide or polypeptide into a plant, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. 
     “Stable transformation” is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. “Transient transformation” is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant. 
     Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986)  Biotechniques  4: 320-334), electroporation (Riggs et al. (1986)  Proc. Natl. Acad. Sci. USA  83: 5602-5606),  Agrobacterium -mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al. (1984)  EMBO J.  3: 2717-2722), and ballistic particle acceleration (see, for example, U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; and U.S. Pat. No. 5,932,782; Tomes et al. (1995) in  Plant Cell, Tissue, and Organ Culture: Fundamental Methods , ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988)  Biotechnology  6: 923-926); and Lecl transformation (WO 00/28058). For potato transformation see Tu et al. (1998)  Plant Molecular Biology  37: 829-838 and Chong et al. (2000)  Transgenic Research  9: 71-78. Additional transformation procedures can be found in Weissinger et al. (1988)  Ann. Rev. Genet.  22: 421-477; Sanford et al. (1987)  Particulate Science and Technology  5: 27-37 (onion); Christou et al. (1988)  Plant Physiol.  87: 671-674 (soybean); McCabe et al. (1988)  Bio/Technology  6: 923-926 (soybean); Finer and McMullen (1991) In  Vitro Cell Dev. Biol.  27P: 175-182 (soybean); Singh et al. (1998)  Theor. Appl. Genet.  96: 319-324 (soybean); Datta et al. (1990)  Biotechnology  8: 736-740 (rice); Klein et al. (1988)  Proc. Natl. Acad. Sci. USA  85: 4305-4309 ( maize ); Klein et al. (1988)  Biotechnology  6:559-563 ( maize ); U.S. Pat. Nos. 5,240,855; 5,322,783 and U.S. Pat. No. 5,324,646; Klein et al. (1988)  Plant Physiol.  91: 440-444 ( maize ); Fromm et al. (1990)  Biotechnology  8: 833-839 ( maize ); Hooykaas-Van Slogteren et al. (1984)  Nature  ( London ) 311: 763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987)  Proc. Natl. Acad. Sci. USA  84: 5345-5349 (Liliaceae); De Wet et al. (1985) in  The Experimental Manipulation of Ovule Tissues , ed. Chapman et al. ( Longman , New York), pp. 197-209 (pollen); Kaeppler et al. (1990)  Plant Cell Reports  9: 415-418 and Kaeppler et al. (1992)  Theor. Appl. Genet.  84: 560-566 (whisker-mediated transformation); D&#39;Halluin et al. (1992)  Plant Cell  4: 1495-1505 (electroporation); Li et al. (1993)  Plant Cell Reports  12: 250-255 and Christou and Ford (1995)  Annals of Botany  75: 407-413 (rice); Osjoda et al. (1996)  Nature Biotechnology  14: 745-750 ( maize  via  Agrobacterium tumefaciens ); all of which are herein incorporated by reference. 
     In specific embodiments, the sequences of the embodiments can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the Cry toxin protein or variants and fragments thereof directly into the plant or the introduction of the Cry toxin transcript into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986)  Mol Gen. Genet.  202: 179-185; Nomura et al. (1986)  Plant Sci.  44: 53-58; Hepler et al. (1994)  Proc. Natl. Acad. Sci.  91: 2176-2180 and Hush et al. (1994)  The Journal of Cell Science  107: 775-784, all of which are herein incorporated by reference. Alternatively, the Cry toxin polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use of particles coated with polyethylimine (PEI; Sigma #P3143). 
     Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, and WO99/25853, all of which are herein incorporated by reference. Briefly, the polynucleotide of the embodiments can be contained in transfer cassette flanked by two non-identical recombination sites. The transfer cassette is introduced into a plant have stably incorporated into its genome a target site which is flanked by two non-identical recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome. 
     The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986)  Plant Cell Reports  5: 81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive or inducible expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure that expression of the desired phenotypic characteristic has been achieved. 
     The nucleotide sequences of the embodiments may be provided to the plant by contacting the plant with a virus or viral nucleic acids. Generally, such methods involve incorporating the nucleotide construct of interest within a viral DNA or RNA molecule. It is recognized that the recombinant proteins of the embodiments may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired pesticidal protein. It is also recognized that such a viral polyprotein, comprising at least a portion of the amino acid sequence of a pesticidal protein of the embodiments, may have the desired pesticidal activity. Such viral polyproteins and the nucleotide sequences that encode for them are encompassed by the embodiments. Methods for providing plants with nucleotide constructs and producing the encoded proteins in the plants, which involve viral DNA or RNA molecules are known in the art. See, for example, U.S. Pat. Nos. 5,889,191; 5,889,190; 5,866,785; 5,589,367; and 5,316,931; herein incorporated by reference. 
     The embodiments further relate to plant-propagating material of a transformed plant of the embodiments including, but not limited to, seeds, tubers, corms, bulbs, leaves, and cuttings of roots and shoots. 
     The embodiments may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn ( Zea mays ),  Brassica  sp. (e.g.,  B. napus, B. rapa, B. juncea ), particularly those  Brassica  species useful as sources of seed oil, alfalfa ( Medicago sativa ), rice ( Oryza sativa ), rye ( Secale cereale ),  sorghum  ( Sorghum bicolor, Sorghum vulgare ), millet (e.g., pearl millet ( Pennisetum glaucum ), proso millet ( Panicum miliaceum ), foxtail millet ( Setaria italica ), finger millet ( Eleusine coracana )), sunflower ( Helianthus annuus ), safflower ( Carthamus tinctorius ), wheat ( Triticum aestivum ), soybean ( Glycine max ), tobacco ( Nicotiana tabacum ), potato ( Solanum tuberosum ), peanuts ( Arachis hypogaea ), cotton ( Gossypium barbadense, Gossypium hirsutum ), sweet potato ( Ipomoea batatus ), cassava ( Manihot esculenta ), coffee ( Coffea  spp.), coconut ( Cocos nucifera ), pineapple ( Ananas comosus ),  citrus  trees ( Citrus  spp.), cocoa ( Theobroma cacao ), tea ( Camellia sinensis ), banana ( Musa  spp.), avocado ( Persea americana ), fig ( Ficus casica ), guava ( Psidium guajava ), mango ( Mangifera indica ), olive ( Olea europaea ),  papaya  ( Carica papaya ), cashew ( Anacardium occidentale ), macadamia ( Macadamia integrifolia ), almond ( Prunus amygdalus ), sugar beets ( Beta vulgaris ), sugarcane ( Saccharum  spp.), oats, barley, vegetables, ornamentals, and conifers. 
     Vegetables include tomatoes ( Lycopersicon esculentum ), lettuce (e.g.,  Lactuca sativa ), green beans ( Phaseolus vulgaris ), lima beans ( Phaseolus limensis ), peas ( Lathyrus  spp.), and members of the genus  Cucumis  such as cucumber ( C. sativus ), cantaloupe ( C. cantalupensis ), and musk melon ( C. melo ). Ornamentals include azalea ( Rhododendron  spp.),  hydrangea  ( Macrophylla hydrangea ),  hibiscus  ( Hibiscus rosasanensis ), roses ( Rosa  spp.), tulips ( Tulipa  spp.), daffodils ( Narcissus  spp.), petunias ( Petunia hybrida ), carnation ( Dianthus caryophyllus ), poinsettia ( Euphorbia pulcherrima ), and chrysanthemum. Conifers that may be employed in practicing the embodiments include, for example, pines such as loblolly pine ( Pinus taeda ), slash pine ( Pinus elliotii ),  ponderosa  pine ( Pinus ponderosa ), lodgepole pine ( Pinus contorta ), and Monterey pine ( Pinus radiata ); Douglas fir ( Pseudotsuga menziesii ); Western hemlock ( Tsuga canadensis ); Sitka spruce ( Picea glauca ); redwood ( Sequoia sempervirens ); true firs such as silver fir ( Abies amabilis ) and balsam fir ( Abies balsamea ); and cedars such as Western red cedar ( Thuja plicata ) and Alaska yellow-cedar ( Chamaecyparis nootkatensis ). Plants of the embodiments include crop plants (for example, corn, alfalfa, sunflower,  Brassica , soybean, cotton, safflower, peanut,  sorghum , wheat, millet, tobacco, etc.), such as corn and soybean plants. 
     Turfgrasses include, but are not limited to: annual bluegrass ( Poa annua ); annual ryegrass ( Lolium multiflorum ); Canada bluegrass ( Poa compressa ); Chewings fescue ( Festuca rubra ); colonial bentgrass ( Agrostis tenuis ); creeping bentgrass ( Agrostis palustris ); crested wheatgrass ( Agropyron desertorum ); fairway wheatgrass ( Agropyron cristatum ); hard fescue ( Festuca longifolia ); Kentucky bluegrass ( Poa pratensis ); orchardgrass ( Dactylis glomerata ); perennial ryegrass ( Lolium perenne ); red fescue ( Festuca rubra ); redtop ( Agrostis alba ); rough bluegrass ( Poa trivialis ); sheep fescue ( Festuca ovina ); smooth bromegrass ( Bromus inermis ); tall fescue ( Festuca arundinacea ); timothy ( Phleum pratense ); velvet bentgrass ( Agrostis canina ); weeping alkaligrass ( Puccinellia distans ); western wheatgrass ( Agropyron smithii ); Bermuda grass ( Cynodon  spp.); St. Augustine grass ( Stenotaphrum secundatum );  zoysia  grass ( Zoysia  spp.); Bahia grass ( Paspalum notatum ); carpet grass ( Axonopus affinis ); centipede grass ( Eremochloa ophiuroides ); kikuyu grass ( Pennisetum clandesinum ); seashore paspalum ( Paspalum vaginatum ); blue gramma ( Bouteloua gracilis ); buffalo grass ( Buchloe dactyloids ); sideoats gramma ( Bouteloua curtipendula ). 
     Plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice,  sorghum , rye, millet, etc. Oil-seed plants include cotton, soybean, safflower, sunflower,  Brassica, maize , alfalfa, palm, coconut, flax, castor, olive etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc. 
     In certain embodiments the nucleic acid sequences of the embodiments can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired phenotype. For example, the polynucleotides of the embodiments may be stacked with any other polynucleotides encoding polypeptides having pesticidal and/or insecticidal activity, such as other Bt toxic proteins (described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986)  Gene  48:109), pentin (described in U.S. Pat. No. 5,981,722) and the like. The combinations generated can also include multiple copies of any one of the polynucleotides of interest. The polynucleotides of the embodiments can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including but not limited to traits desirable for animal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g. hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,049); barley high lysine (Williamson et al. (1987)  Eur. J. Biochem.  165: 99-106; and WO 98/20122) and high methionine proteins (Pedersen et al. (1986)  J. Biol. Chem.  261: 6279; Kirihara et al. (1988)  Gene  71: 359; and Musumura et al. (1989)  Plant Mol. Biol.  12: 123)); increased digestibility (e.g., modified storage proteins (U.S. application Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins (U.S. application Ser. No. 10/005,429, filed Dec. 3, 2001)), the disclosures of which are herein incorporated by reference. 
     The polynucleotides of the embodiments can also be stacked with traits desirable for disease or herbicide resistance (e.g., fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence and disease resistance genes (Jones et al. (1994)  Science  266:789; Martin et al. (1993)  Science  262: 1432; and Mindrinos et al. (1994)  Cell  78:1089); acetolactate synthase (ALS) mutants that lead to herbicide resistance such as the S4 and/or Hra mutations; inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., bar gene); and glyphosate resistance (EPSPS gene and GAT gene as disclosed in U.S. application Ser. No. 10/004,357; and Ser. No. 10/427,692); and traits desirable for processing or process products such as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert et al. (1988)  J. Bacteriol.  170: 5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs)), the disclosures of which are herein incorporated by reference. One could also combine the polynucleotides of the embodiments with polynucleotides providing agronomic traits such as male sterility (e.g., see U.S. Pat. No. 5,583,210), stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (e.g. WO 99/61619; WO 00/17364; WO 99/25821), the disclosures of which are herein incorporated by reference. 
     These stacked combinations can be created by any method including but not limited to cross breeding plants by any conventional or TOPCROSS® methodology, or genetic transformation. If the traits are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, and WO99/25853, all of which are herein incorporated by reference. 
     Compositions of the embodiments find use in protecting plants, seeds, and plant products in a variety of ways. For example, the compositions can be used in a method that involves placing an effective amount of the pesticidal composition in the environment of the pest by a procedure selected from the group consisting of spraying, dusting, broadcasting, or seed coating. 
     Before plant propagation material (fruit, tuber, bulb, corm, grains, seed), but especially seed, is sold as a commercial product, it is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures of several of these preparations, if desired together with further carriers, surfactants, or application-promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal, or animal pests. In order to treat the seed, the protectant coating may be applied to the seeds either by impregnating the tubers or grains with a liquid formulation or by coating them with a combined wet or dry formulation. In addition, in special cases, other methods of application to plants are possible, e.g., treatment directed at the buds or the fruit. 
     The plant seed of the embodiments comprising a nucleotide sequence encoding a pesticidal protein of the embodiments may be treated with a seed protectant coating comprising a seed treatment compound, such as, for example, captan, carboxin, thiram, methalaxyl, pirimiphos-methyl, and others that are commonly used in seed treatment. In one embodiment, a seed protectant coating comprising a pesticidal composition of the embodiments is used alone or in combination with one of the seed protectant coatings customarily used in seed treatment. 
     It is recognized that the genes encoding the pesticidal proteins can be used to transform insect pathogenic organisms. Such organisms include baculoviruses, fungi, protozoa, bacteria, and nematodes. 
     A gene encoding a pesticidal protein of the embodiments may be introduced via a suitable vector into a microbial host, and said host applied to the environment, or to plants or animals. The term “introduced” in the context of inserting a nucleic acid into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA). 
     Microorganism hosts that are known to occupy the “phytosphere” (phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops of interest may be selected. These microorganisms are selected so as to be capable of successfully competing in the particular environment with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the pesticidal protein, and desirably, provide for improved protection of the pesticide from environmental degradation and inactivation. 
     Such microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms such as bacteria, e.g.,  Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc , and  Alcaligenes , fungi, particularly yeast, e.g.,  Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula , and  Aureobasidium . Of particular interest are such phytosphere bacterial species as  Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli  and  Azotobacter vinelandii  and phytosphere yeast species such as  Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae , and  Aureobasidium pollulans . Of particular interest are the pigmented microorganisms. 
     A number of ways are available for introducing a gene expressing the pesticidal protein into the microorganism host under conditions that allow for stable maintenance and expression of the gene. For example, expression cassettes can be constructed which include the nucleotide constructs of interest operably linked with the transcriptional and translational regulatory signals for expression of the nucleotide constructs, and a nucleotide sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system that is functional in the host, whereby integration or stable maintenance will occur. 
     Transcriptional and translational regulatory signals include, but are not limited to, promoters, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See, for example, U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (1992)  Molecular Cloning: A Laboratory Manual , ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), hereinafter “Sambrook II”; Davis et al., eds. (1980)  Advanced Bacterial Genetics  (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, N.Y.; and the references cited therein. 
     Suitable host cells, where the pesticidal protein-containing cells will be treated to prolong the activity of the pesticidal proteins in the cell when the treated cell is applied to the environment of the target pest(s), may include either prokaryotes or eukaryotes, normally being limited to those cells that do not produce substances toxic to higher organisms, such as mammals. However, organisms that produce substances toxic to higher organisms could be used, where the toxin is unstable or the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. Illustrative prokaryotes, both Gram-negative and gram-positive, include Enterobacteriaceae, such as  Escherichia, Erwinia, Shigella, Salmonella , and  Proteus ; Bacillaceae; Rhizobiaceae, such as  Rhizobium ; Spirillaceae, such as photobacterium,  Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum ; Lactobacillaceae; Pseudomonadaceae, such as  Pseudomonas  and  Acetobacter ; Azotobacteraceae and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such as  Saccharomyces  and  Schizosaccharomyces ; and  Basidiomycetes  yeast, such as  Rhodotorula, Aureobasidium, Sporobolomyces , and the like. 
     Characteristics of particular interest in selecting a host cell for purposes of pesticidal protein production include ease of introducing the pesticidal protein gene into the host, availability of expression systems, efficiency of expression, stability of the protein in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like. 
     Host organisms of particular interest include yeast, such as  Rhodotorula  spp.,  Aureobasidium  spp.,  Saccharomyces  spp. (such as  S. cerevisiae ),  Sporobolomyces  spp., phylloplane organisms such as  Pseudomonas  spp. (such as  P. aeruginosa, P. fluorescens ),  Erwinia  spp., and  Flavobacterium  spp., and other such organisms, including Bt,  E. coli, Bacillus subtilis , and the like. 
     Genes encoding the pesticidal proteins of the embodiments can be introduced into microorganisms that multiply on plants (epiphytes) to deliver pesticidal proteins to potential target pests. Epiphytes, for example, can be gram-positive or gram-negative bacteria. 
     Root-colonizing bacteria, for example, can be isolated from the plant of interest by methods known in the art. Specifically, a  Bacillus cereus  strain that colonizes roots can be isolated from roots of a plant (see, for example, Handelsman et al. (1991)  Appl. Environ. Microbiol.  56:713-718). Genes encoding the pesticidal proteins of the embodiments can be introduced into a root-colonizing  Bacillus cereus  by standard methods known in the art. 
     Genes encoding pesticidal proteins can be introduced, for example, into the root-colonizing  Bacillus  by means of electrotransformation. Specifically, genes encoding the pesticidal proteins can be cloned into a shuttle vector, for example, pHT3101 (Lerecius et al. (1989)  FEMS Microbiol. Letts.  60: 211-218. The shuttle vector pHT3101 containing the coding sequence for the particular pesticidal protein gene can, for example, be transformed into the root-colonizing  Bacillus  by means of electroporation (Lerecius et al. (1989)  FEMS Microbiol. Letts.  60: 211-218). 
     Expression systems can be designed so that pesticidal proteins are secreted outside the cytoplasm of gram-negative bacteria, such as  E. coli , for example. Advantages of having pesticidal proteins secreted are: (1) avoidance of potential cytotoxic effects of the pesticidal protein expressed; and (2) improvement in the efficiency of purification of the pesticidal protein, including, but not limited to, increased efficiency in the recovery and purification of the protein per volume cell broth and decreased time and/or costs of recovery and purification per unit protein. 
     Pesticidal proteins can be made to be secreted in  E. coli , for example, by fusing an appropriate  E. coli  signal peptide to the amino-terminal end of the pesticidal protein. Signal peptides recognized by  E. coli  can be found in proteins already known to be secreted in  E. coli , for example the OmpA protein (Ghrayeb et al. (1984)  EMBO J,  3:2437-2442). OmpA is a major protein of the  E. coli  outer membrane, and thus its signal peptide is thought to be efficient in the translocation process. Also, the OmpA signal peptide does not need to be modified before processing as may be the case for other signal peptides, for example lipoprotein signal peptide (Duffaud et al. (1987)  Meth. Enzymol.  153: 492). 
     Pesticidal proteins of the embodiments can be fermented in a bacterial host and the resulting bacteria processed and used as a microbial spray in the same manner that Bt strains have been used as insecticidal sprays. In the case of a pesticidal protein(s) that is secreted from  Bacillus , the secretion signal is removed or mutated using procedures known in the art. Such mutations and/or deletions prevent secretion of the pesticidal protein(s) into the growth medium during the fermentation process. The pesticidal proteins are retained within the cell, and the cells are then processed to yield the encapsulated pesticidal proteins. Any suitable microorganism can be used for this purpose.  Pseudomonas  has been used to express Bt toxins as encapsulated proteins and the resulting cells processed and sprayed as an insecticide (Gaertner et al. (1993), in:  Advanced Engineered Pesticides , ed. Kim). 
     Alternatively, the pesticidal proteins are produced by introducing a heterologous gene into a cellular host. Expression of the heterologous gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. These cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s). The resulting product retains the toxicity of the toxin. These naturally encapsulated pesticidal proteins may then be formulated in accordance with conventional techniques for application to the environment hosting a target pest, e.g., soil, water, and foliage of plants. See, for example EPA 0192319, and the references cited therein. 
     In the embodiments, a transformed microorganism (which includes whole organisms, cells, spore(s), pesticidal protein(s), pesticidal component(s), pest-impacting component(s), mutant(s), living or dead cells and cell components, including mixtures of living and dead cells and cell components, and including broken cells and cell components) or an isolated pesticidal protein can be formulated with an acceptable carrier into a pesticidal composition(s) that is, for example, a suspension, a solution, an emulsion, a dusting powder, a dispersible granule or pellet, a wettable powder, and an emulsifiable concentrate, an aerosol or spray, an impregnated granule, an adjuvant, a coatable paste, a colloid, and also encapsulations in, for example, polymer substances. Such formulated compositions may be prepared by such conventional means as desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of cells comprising the polypeptide. 
     Such compositions disclosed above may be obtained by the addition of a surface-active agent, an inert carrier, a preservative, a humectant, a feeding stimulant, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, a UV protectant, a buffer, a flow agent or fertilizers, micronutrient donors, or other preparations that influence plant growth. One or more agrochemicals including, but not limited to, herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, acaricides, plant growth regulators, harvest aids, and fertilizers, can be combined with carriers, surfactants or adjuvants customarily employed in the art of formulation or other components to facilitate product handling and application for particular target pests. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g., natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders, or fertilizers. The active ingredients of the embodiments are normally applied in the form of compositions and can be applied to the crop area, plant, or seed to be treated. For example, the compositions of the embodiments may be applied to grain in preparation for or during storage in a grain bin or silo, etc. The compositions of the embodiments may be applied simultaneously or in succession with other compounds. Methods of applying an active ingredient of the embodiments or an agrochemical composition of the embodiments that contains at least one of the pesticidal proteins produced by the bacterial strains of the embodiments include, but are not limited to, foliar application, seed coating, and soil application. The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest. 
     Suitable surface-active agents include, but are not limited to, anionic compounds such as a carboxylate of, for example, a metal; a carboxylate of a long chain fatty acid; an N-acylsarcosinate; mono or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such esters; fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecyl sulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated alkylphenol sulfates; lignin sulfonates; petroleum sulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates or lower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate; salts of sulfonated naphthalene-formaldehyde condensates; salts of sulfonated phenol-formaldehyde condensates; more complex sulfonates such as the amide sulfonates, e.g., the sulfonated condensation product of oleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g., the sodium sulfonate of dioctyl succinate. Non-ionic agents include condensation products of fatty acid esters, fatty alcohols, fatty acid amides or fatty-alkyl- or alkenyl-substituted phenols with ethylene oxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fatty acid esters, condensation products of such esters with ethylene oxide, e.g., polyoxyethylene sorbitar fatty acid esters, block copolymers of ethylene oxide and propylene oxide, acetylenic glycols such as 2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols. Examples of a cationic surface-active agent include, for instance, an aliphatic mono-, di-, or polyamine such as an acetate, naphthenate or oleate; or oxygen-containing amine such as an amine oxide of polyoxyethylene alkylamine; an amide-linked amine prepared by the condensation of a carboxylic acid with a di- or polyamine; or a quaternary ammonium salt. 
     Examples of inert materials include but are not limited to inorganic minerals such as kaolin, phyllosilicates, carbonates, sulfates, phosphates, or botanical materials such as cork, powdered corncobs, peanut hulls, rice hulls, and walnut shells. 
     The compositions of the embodiments can be in a suitable form for direct application or as a concentrate of primary composition that requires dilution with a suitable quantity of water or other diluent before application. The pesticidal concentration will vary depending upon the nature of the particular formulation, specifically, whether it is a concentrate or to be used directly. The composition contains 1 to 98% of a solid or liquid inert carrier, and 0 to 50% or 0.1 to 50% of a surfactant. These compositions will be administered at the labeled rate for the commercial product, for example, about 0.01 lb-5.0 lb. per acre when in dry form and at about 0.01 pts.-10 pts. per acre when in liquid form. 
     In a further embodiment, the compositions, as well as the transformed microorganisms and pesticidal proteins of the embodiments, can be treated prior to formulation to prolong the pesticidal activity when applied to the environment of a target pest as long as the pretreatment is not deleterious to the pesticidal activity. Such treatment can be by chemical and/or physical means as long as the treatment does not deleteriously affect the properties of the composition(s). Examples of chemical reagents include but are not limited to halogenating agents; aldehydes such as formaldehyde and glutaraldehyde; anti-infectives, such as zephiran chloride; alcohols, such as isopropanol and ethanol; and histological fixatives, such as Bouin&#39;s fixative and Helly&#39;s fixative (see, for example, Humason (1967)  Animal Tissue Techniques  (W.H. Freeman and Co.). 
     In other embodiments, it may be advantageous to treat the Cry toxin polypeptides with a protease, for example trypsin, to activate the protein prior to application of a pesticidal protein composition of the embodiments to the environment of the target pest. Methods for the activation of protoxin by a serine protease are well known in the art. See, for example, Cooksey (1968)  Biochem. J.  6:445-454 and Carroll and Ellar (1989)  Biochem. J.  261:99-105, the teachings of which are herein incorporated by reference. For example, a suitable activation protocol includes, but is not limited to, combining a polypeptide to be activated, for example a purified novel Cry polypeptide (e.g., having the amino acid sequence set forth in SEQ ID NO:2), and trypsin at a 1/100 weight ratio of protein/trypsin in 20 nM NaHCO 3 , pH 8 and digesting the sample at 36° C. for 3 hours. 
     The compositions (including the transformed microorganisms and pesticidal proteins of the embodiments) can be applied to the environment of an insect pest by, for example, spraying, atomizing, dusting, scattering, coating or pouring, introducing into or on the soil, introducing into irrigation water, by seed treatment or general application or dusting at the time when the pest has begun to appear or before the appearance of pests as a protective measure. For example, the pesticidal protein and/or transformed microorganisms of the embodiments may be mixed with grain to protect the grain during storage. It is generally important to obtain good control of pests in the early stages of plant growth, as this is the time when the plant can be most severely damaged. The compositions of the embodiments can conveniently contain another insecticide if this is thought necessary. In one embodiment, the composition is applied directly to the soil, at a time of planting, in granular form of a composition of a carrier and dead cells of a  Bacillus  strain or transformed microorganism of the embodiments. Another embodiment is a granular form of a composition comprising an agrochemical such as, for example, an herbicide, an insecticide, a fertilizer, an inert carrier, and dead cells of a  Bacillus  strain or transformed microorganism of the embodiments. 
     Those skilled in the art will recognize that not all compounds are equally effective against all pests. Compounds of the embodiments display activity against insect pests, which may include economically important agronomic, forest, greenhouse, nursery, ornamentals, food and fiber, public and animal health, domestic and commercial structure, household, and stored product pests. Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera and Lepidoptera. 
     Insects of the order Lepidoptera include, but are not limited to, armyworms, cutworms, loopers, and heliothines in the family Noctuidae  Agrotis ipsilon  Hufnagel (black cutworm);  A. orthogonia  Morrison (western cutworm);  A. segetum  Denis &amp; Schiffermüller (turnip moth);  A. subterranea  Fabricius (granulate cutworm);  Alabama argillacea  Hübner (cotton leaf worm);  Anticarsia gemmatalis  Hübner (velvetbean caterpillar);  Athetis mindara  Barnes and McDunnough (rough skinned cutworm);  Earias insulana  Boisduval (spiny bollworm);  E. vittella  Fabricius (spotted bollworm);  Egira  ( Xylomyges )  curialis  Grote ( citrus  cutworm);  Euxoa messoria  Harris (darksided cutworm);  Helicoverpa armigera  Hübner (American bollworm);  H. zea  Boddie (corn earworm or cotton bollworm);  Heliothis virescens  Fabricius (tobacco budworm);  Hypena scabra  Fabricius (green cloverworm);  Mamestra configurata  Walker (bertha armyworm);  M. brassicae  Linnaeus (cabbage moth);  Melanchra picta  Harris (zebra caterpillar);  Pseudaletia unipuncta  Haworth (armyworm);  Pseudoplusia includens  Walker (soybean looper);  Richia albicosta  Smith (Western bean cutworm);  Spodoptera frugiperda  J E Smith (fall armyworm);  S. exigua  Hübner (beet armyworm);  S. litura  Fabricius (tobacco cutworm, cluster caterpillar);  Trichoplusia ni  Hübner (cabbage looper); borers, casebearers, webworms, coneworms, and skeletonizers from the families Pyralidae and Crambidae such as Achroia grisella Fabricius (lesser wax moth);  Amyelois transitella  Walker (naval orangeworm);  Anagasta kuehniella  Zeller (Mediterranean flour moth);  Cadra cautella  Walker (almond moth);  Chilo partellus  Swinhoe (spotted stalk borer);  C. suppressalis  Walker (striped stem/rice borer);  C. terrenellus  Pagenstecher (sugarcane stemp borer);  Corcyra cephalonica  Stainton (rice moth);  Crambus caliginosellus  Clemens (corn root webworm);  C. teterrellus  Zincken (bluegrass webworm);  Cnaphalocrocis medinalis  Guenée (rice leaf roller);  Desmia funeralis  Hübner (grape leaffolder);  Diaphania hyalinata  Linnaeus (melon worm);  D. nitidalis  Stoll (pickleworm);  Diatraea grandiosella  Dyar (southwestern corn borer),  D. saccharalis  Fabricius (surgarcane borer);  Elasmopalpus lignosellus  Zeller (lesser cornstalk borer);  Eoreuma loftini  Dyar (Mexican rice borer);  Ephestia elutella  Hübner (tobacco (cacao) moth);  Galleria mellonella  Linnaeus (greater wax moth);  Hedylepta accepta  Butler (sugarcane leafroller);  Herpetogramma licarsisalis  Walker (sod webworm);  Homoeosoma electellum  Hulst (sunflower moth);  Loxostege sticticalis  Linnaeus (beet webworm);  Maruca testulalis  Geyer (bean pod borer);  Orthaga thyrisalis  Walker (tea tree web moth);  Ostrinia nubilalis  Hübner (European corn borer);  Plodia interpunctella  Hübner (Indian meal moth);  Scirpophaga incertulas  Walker (yellow stem borer);  Udea rubigalis  Guenée (celery leaftier); and leafrollers, budworms, seed worms, and fruit worms in the family Tortricidae  Acleris gloverana  Walsingham (Western blackheaded budworm);  A. variana  Fernald (Eastern blackheaded budworm);  Adoxophyes orana  Fischer von Rosslerstamm (summer fruit tortrix moth);  Archips  spp. including  A. argyrospila  Walker (fruit tree leaf roller) and  A. rosana  Linnaeus (European leaf roller);  Argyrotaenia  spp.;  Bonagota salubricola Meyrick  (Brazilian apple leafroller);  Choristoneura  spp.;  Cochylis hospes  Walsingham (banded sunflower moth);  Cydia latiferreana  Walsingham (filbertworm);  C. pomonella  Linnaeus (codling moth);  Endopiza viteana  Clemens (grape berry moth);  Eupoecilia ambiguella  Hübner (vine moth);  Grapholita molesta  Busck (oriental fruit moth);  Lobesia botrana  Denis &amp; Schiffermüller (European grape vine moth);  Platynota flavedana Clemens  (variegated leafroller);  P. stultana  Walsingham (omnivorous leafroller);  Spilonota ocellana  Denis &amp; Schiffermüller (eyespotted bud moth); and  Suleima helianthana  Riley (sunflower bud moth). 
     Selected other agronomic pests in the order Lepidoptera include, but are not limited to,  Alsophila pometaria  Harris (fall cankerworm);  Anarsia lineatella  Zeller (peach twig borer);  Anisota senatoria  J. E. Smith (orange striped oakworm);  Antheraea pernyi  Guérin-Meneville (Chinese Oak Silkmoth);  Bombyx mori  Linnaeus (Silkworm);  Bucculatrix thurberiella  Busck (cotton leaf  perforator );  Collas eurytheme  Boisduval (alfalfa caterpillar);  Datana integerrima  Grote &amp; Robinson (walnut caterpillar);  Dendrolimus sibiricus  Tschetwerikov (Siberian silk moth),  Ennomos subsignaria  Hübner (elm spanworm);  Erannis tiliaria  Harris (linden looper);  Erechthias flavistriata Walsingham  (sugarcane bud moth);  Euproctis chrysorrhoea  Linnaeus (browntail moth);  Harrisina americana  Guérin-Méneville (grapeleaf skeletonizer);  Heliothis subflexa  Guenée; Hemileuca oliviae Cockrell (range caterpillar);  Hyphantria cunea  Drury (fall webworm);  Keiferia lycopersicella  Walsingham (tomato pinworm); Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock looper);  L. fiscellaria  lugubrosa Hulst (Western hemlock looper);  Leucoma salicis  Linnaeus (satin moth);  Lymantria dispar  Linnaeus (gypsy moth);  Malacosoma  spp.;  Manduca quinquemaculata  Haworth (five spotted hawk moth, tomato hornworm);  M. sexta  Haworth (tomato hornworm, tobacco hornworm);  Operophtera brumata  Linnaeus (winter moth);  Orgyia  spp.;  Paleacrita vemata  Peck (spring cankerworm);  Papilio cresphontes  Cramer (giant swallowtail, orange dog);  Phryganidia californica  Packard (California oakworm);  Phyllocnistis citrella  Stainton ( citrus  leafminer);  Phyllonorycter blancardella  Fabricius (spotted tentiform leafminer);  Pieris brassicae  Linnaeus (large white butterfly);  P. rapae  Linnaeus (small white butterfly);  P. napi  Linnaeus (green veined white butterfly);  Platyptilia carduidactyla  Riley (artichoke plume moth);  Plutella xylostella  Linnaeus (diamondback moth);  Pectinophora gossypiella  Saunders (pink bollworm);  Pontia protodice  Boisduval &amp; Leconte (Southern cabbageworm);  Sabulodes aegrotata  Guenée (omnivorous looper); Schizura  concinna  J. E. Smith (red humped caterpillar);  Sitotroga cerealella  Olivier (Angoumois grain moth);  Thaumetopoea pityocampa  Schiffermuller (pine processionary caterpillar);  Tineola bisselliella  Hummel (webbing clothesmoth);  Tuta absoluta  Meyrick (tomato leafminer) and  Yponomeuta padella  Linnaeus (ermine moth). 
     Of interest are larvae and adults of the order Coleoptera including weevils from the families Anthribidae, Bruchidae, and Curculionidae including, but not limited to:  Anthonomus grandis  Boheman (boll weevil);  Cylindrocopturus adspersus  LeConte (sunflower stem weevil);  Diaprepes abbreviatus  Linnaeus (Diaprepes root weevil);  Hypera punctata  Fabricius (clover leaf weevil);  Lissorhoptrus oryzophilus  Kuschel (rice water weevil);  Metamasius hemipterus hemipterus  Linnaeus (West Indian cane weevil);  M. hemipterus  sericeus Olivier (silky cane weevil);  Sitophilus granarius  Linnaeus (granary weevil);  S. oryzae  Linnaeus (rice weevil);  Smicronyx fulvus  LeConte (red sunflower seed weevil);  S. sordidus  LeConte (gray sunflower seed weevil);  Sphenophorus maidis  Chittenden ( maize  billbug);  Rhabdoscelus obscurus  Boisduval (New Guinea sugarcane weevil); flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetles, and leafminers in the family Chrysomelidae including, but not limited to:  Chaetocnema ectypa  Horn (desert corn flea beetle);  C. pulicaria  Melsheimer (corn flea beetle);  Colaspis brunnea  Fabricius (grape colaspis);  Diabrotica barberi  Smith &amp; Lawrence (northern corn rootworm);  D. undecimpunctata howardi  Barber (southern corn rootworm);  D. virgifera virgifera  LeConte (western corn rootworm);  Leptinotarsa decemlineata  Say (Colorado potato beetle);  Oulema melanopus  Linnaeus (cereal leaf beetle);  Phyllotreta cruciferae  Goeze (corn flea beetle);  Zygogramma exclamationis  Fabricius (sunflower beetle); beetles from the family Coccinellidae including, but not limited to:  Epilachna varivestis  Mulsant (Mexican bean beetle); chafers and other beetles from the family Scarabaeidae including, but not limited to:  Antitrogus parvulus  Britton (Childers cane grub);  Cyclocephala borealis  Arrow (northern masked chafer, white grub);  C. immaculata  Olivier (southern masked chafer, white grub);  Dermolepida albohirtum  Waterhouse (Greyback cane beetle);  Euetheola humilis  rugiceps LeConte (sugarcane beetle);  Lepidiota frenchi  Blackburn (French&#39;s cane grub);  Tomarus gibbosus  De Geer (carrot beetle);  T. subtropicus  Blatchley (sugarcane grub);  Phyllophaga crinita  Burmeister (white grub);  P. latifrons  LeConte (June beetle);  Popillia japonica  Newman (Japanese beetle);  Rhizotrogus majalis  Razoumowsky (European chafer); carpet beetles from the family Dermestidae; wireworms from the family Elateridae,  Eleodes  spp.,  Melanotus  spp. including  M. communis  Gyllenhal (wireworm); Conoderus spp.;  Limonius  spp.;  Agriotes  spp.;  Ctenicera  spp.;  Aeolus  spp.; bark beetles from the family Scolytidae; beetles from the family Tenebrionidae; beetles from the family Cerambycidae such as, but not limited to,  Migdolus fryanus  Westwood (longhorn beetle); and beetles from the Buprestidae family including, but not limited to,  Aphanisticus cochinchinae seminulum  Obenberger (leaf-mining buprestid beetle). 
     Adults and immatures of the order Diptera are of interest, including leafminers  Agromyza parvicornis  Loew (corn blotch leafminer); midges including, but not limited to:  Contarinia sorghicola  Coquillett ( sorghum  midge);  Mayetiola destructor  Say (Hessian fly); Neolasioptera murtfeldtiana Felt, (sunflower seed midge);  Sitodiplosis mosellana  Géhin (wheat midge); fruit flies (Tephritidae),  Oscinella frit  Linnaeus (frit flies); maggots including, but not limited to:  Delia  spp. including  Delia platura  Meigen (seedcorn maggot);  D. coarctata  Fallen (wheat bulb fly);  Fannia canicularis  Linnaeus,  F. femoralis  Stein (lesser house flies);  Meromyza americana  Fitch (wheat stem maggot);  Musca domestica  Linnaeus (house flies);  Stomoxys calcitrans  Linnaeus (stable flies)); face flies, horn flies, blow flies,  Chrysomya  spp.; Phormia spp.; and other muscoid fly pests, horse flies  Tabanus  spp.; bot flies  Gastrophilus  spp.;  Oestrus  spp.; cattle grubs  Hypoderma  spp.; deer flies  Chrysops  spp.;  Melophagus ovinus  Linnaeus (keds); and other Brachycera, mosquitoes  Aedes  spp.;  Anopheles  spp.;  Culex  spp.; black flies  Prosimulium  spp.;  Simulium  spp.; biting midges, sand flies, sciarids, and other Nematocera. 
     Included as insects of interest are those of the order Hemiptera such as, but not limited to, the following families: Adelgidae, Aleyrodidae, Aphididae, Asterolecaniidae, Cercopidae, Cicadellidae, Cicadidae, Cixiidae, Coccidae, Coreidae, Dactylopiidae, Delphacidae, Diaspididae, Eriococcidae, Flatidae, Fulgoridae, Issidae, Lygaeidae, Margarodidae, Membracidae, Miridae, Ortheziidae, Pentatomidae, Phoenicococcidae, Phylloxeridae, Pseudococcidae, Psyllidae, Pyrrhocoridae and Tingidae. 
     Agronomically important members from the order Hemiptera include, but are not limited to:  Acrosternum hilare  Say (green stink bug);  Acyrthisiphon pisum  Harris (pea aphid);  Adelges  spp. (adelgids); Adelphocoris rapidus Say (rapid plant bug);  Anasa tristis  De Geer (squash bug);  Aphis craccivora  Koch (cowpea aphid);  A. fabae  Scopoli (black bean aphid);  A. gossypii  Glover (cotton aphid, melon aphid);  A. maidiradicis  Forbes (corn root aphid);  A. pomi  De Geer (apple aphid);  A. spiraecola  Patch (spirea aphid);  Aulacaspis tegalensis  Zehntner (sugarcane scale);  Aulacorthum solani  Kaltenbach (foxglove aphid);  Bemisia tabaci  Gennadius (tobacco whitefly, sweetpotato whitefly);  B. argentifolii  Bellows &amp; Perring (silverleaf whitefly);  Blissus leucopterus leucopterus  Say (chinch bug);  Blostomatidae  spp.;  Brevicoryne brassicae  Linnaeus (cabbage aphid);  Cacopsylla pyricola  Foerster (pear psylla); Calocoris  norvegicus  Gmelin (potato capsid bug);  Chaetosiphon fragaefolii  Cockerell (strawberry aphid);  Cimicidae  spp.;  Coreidae  spp.;  Corythuca gossypii  Fabricius (cotton lace bug);  Cyrtopeltis modesta  Distant (tomato bug);  C. notatus  Distant (suckfly); Deois flavopicta Stål (spittlebug);  Dialeurodes citri  Ashmead ( citrus  whitefly);  Diaphnocoris chlorionis  Say (honeylocust plant bug);  Diuraphis noxia  Kurdjumov/Mordvilko (Russian wheat aphid); Duplachionaspis  divergens  Green (armored scale);  Dysaphis plantaginea  Paaserini (rosy apple aphid);  Dysdercus suturellus  Herrich-Schaffer (cotton stainer);  Dysmicoccus boninsis  Kuwana (gray sugarcane mealybug);  Empoasca fabae  Harris (potato leafhopper);  Eriosoma lanigerum  Hausmann (woolly apple aphid);  Erythroneoura  spp. (grape leafhoppers);  Eumetopina flavipes  Muir (Island sugarcane planthopper);  Eurygaster  spp.;  Euschistus servus  Say (brown stink bug);  E. variolarius  Palisot de Beauvois (one-spotted stink bug); Graptostethus spp. (complex of seed bugs); and  Hyalopterus pruni  Geoffroy (mealy plum aphid);  Icerya purchasi  Maskell (cottony cushion scale);  Labopidicola allii  Knight (onion plant bug);  Laodelphax striatellus  Fallen (smaller brown planthopper);  Leptoglossus corculus  Say (leaf-footed pine seed bug);  Leptodictya tabida  Herrich-Schaeffer (sugarcane lace bug);  Lipaphis erysimi  Kaltenbach (turnip aphid);  Lygocoris pabulinus  Linnaeus (common green capsid);  Lygus lineolaris  Palisot de Beauvois (tarnished plant bug);  L. Hesperus  Knight (Western tarnished plant bug);  L. pratensis  Linnaeus (common meadow bug);  L. rugulipennis  Poppius (European tarnished plant bug);  Macrosiphum euphorbiae  Thomas (potato aphid);  Macrosteles quadrilineatus  Forbes (aster leafhopper);  Magicicada septendecim  Linnaeus (periodical cicada);  Mahanarva fimbriolata  Stål (sugarcane spittlebug);  Melanaphis sacchari  Zehntner (sugarcane aphid);  Melanaspis glomerata  Green (black scale);  Metopolophium dirhodum  Walker (rose grain aphid);  Myzus persicae  Sulzer (peach-potato aphid, green peach aphid);  Nasonovia ribisnigri  Mosley (lettuce aphid);  Nephotettix cinticeps  Uhler (green leafhopper);  N. nigropictus  Stål (rice leafhopper);  Nezara viridula  Linnaeus (southern green stink bug);  Nilaparvata lugens  Stål (brown planthopper);  Nysius ericae  Schilling (false chinch bug);  Nysius raphanus  Howard (false chinch bug);  Oebalus pugnax  Fabricius (rice stink bug);  Oncopeltus fasciatus  Dallas (large milkweed bug);  Orthops campestris  Linnaeus;  Pemphigus  spp. (root aphids and gall aphids);  Peregrinus maidis  Ashmead (corn planthopper);  Perkinsiella saccharicida  Kirkaldy (sugarcane delphacid);  Phylloxera devastatrix  Pergande (pecan phylloxera);  Planococcus citri  Risso ( citrus  mealybug);  Plesiocoris rugicollis  Fallen (apple capsid);  Poecilocapsus lineatus  Fabricius (four-lined plant bug);  Pseudatomoscelis seriatus  Reuter (cotton fleahopper);  Pseudococcus  spp. (other mealybug complex);  Pulvinaria elongata  Newstead (cottony grass scale);  Pyrilla perpusilla  Walker (sugarcane leafhopper);  Pyrrhocoridae  spp.;  Quadraspidiotus perniciosus  Comstock (San Jose scale);  Reduviidae  spp.;  Rhopalosiphum maidis  Fitch (corn leaf aphid);  R. padi  Linnaeus (bird cherry-oat aphid);  Saccharicoccus sacchari  Cockerell (pink sugarcane mealybug);  Schizaphis graminum  Rondani (greenbug);  Sipha flava  Forbes (yellow sugarcane aphid);  Sitobion avenae  Fabricius (English grain aphid);  Sogatella furcifera  Horvath (white-backed planthopper);  Sogatodes oryzicola  Muir (rice delphacid);  Spanagonicus albofasciatus  Reuter (whitemarked fleahopper);  Therioaphis maculata  Buckton (spotted alfalfa aphid);  Tinidae  spp.;  Toxoptera aurantii  Boyer de Fonscolombe (black  citrus  aphid); and  T. citricida  Kirkaldy (brown  citrus  aphid);  Trialeurodes abutiloneus  (bandedwinged whitefly) and  T. vaporariorum  Westwood (greenhouse whitefly);  Trioza diospyri  Ashmead (persimmon psylla); and  Typhlocyba pomaria  McAtee (white apple leafhopper). 
     Also included are adults and larvae of the order Acari (mites) such as  Aceria tosichella  Keifer (wheat curl mite);  Panonychus ulmi  Koch (European red mite);  Petrobia latens  Müller (brown wheat mite);  Steneotarsonemus bancrofti  Michael (sugarcane stalk mite); spider mites and red mites in the family  Tetranychidae, Oligonychus  grypus Baker &amp; Pritchard,  O. indicus  Hirst (sugarcane leaf mite),  O. pratensis  Banks (Banks grass mite),  O. stickneyi  McGregor (sugarcane spider mite);  Tetranychus urticae  Koch (two spotted spider mite);  T. mcdanieli  McGregor (McDaniel mite);  T. cinnabarinus  Boisduval (carmine spider mite);  T. turkestani  Ugarov &amp; Nikolski (strawberry spider mite), flat mites in the family  Tenuipalpidae, Brevipalpus lewisi  McGregor ( citrus  flat mite); rust and bud mites in the family  Eriophyidae  and other foliar feeding mites and mites important in human and animal health, i.e. dust mites in the family Epidermoptidae, follicle mites in the family Demodicidae, grain mites in the family Glycyphagidae, ticks in the order Ixodidae.  Ixodes scapularis  Say (deer tick);  I. holocyclus  Neumann (Australian paralysis tick);  Dermacentor variabilis  Say (American dog tick);  Amblyomma americanum  Linnaeus (lone star tick); and scab and itch mites in the families Psoroptidae, Pyemotidae, and Sarcoptidae. 
     Insect pests of the order Thysanura are of interest, such as  Lepisma saccharina  Linnaeus (silverfish);  Thermobia domestica  Packard (firebrat). 
     Additional arthropod pests covered include: spiders in the order Araneae such as  Loxosceles reclusa  Gertsch &amp; Mulaik (brown recluse spider); and the  Latrodectus mactans  Fabricius (black widow spider); and centipedes in the order Scutigeromorpha such as  Scutigera coleoptrata  Linnaeus (house centipede). In addition, insect pests of the order Isoptera are of interest, including those of the termitidae family, such as, but not limited to,  Cylindrotermes nordenskioeldi  Holmgren and  Pseudacanthotermes militaris  Hagen (sugarcane termite). Insects of the order Thysanoptera are also of interest, including but not limited to thrips, such as  Stenchaetothrips minutus  van Deventer (sugarcane thrips). 
     Insect pests may be tested for pesticidal activity of compositions of the embodiments in early developmental stages, e.g., as larvae or other immature forms. The insects may be reared in total darkness at from about 20° C. to about 30° C. and from about 30% to about 70% relative humidity. Bioassays may be performed as described in Czapla and Lang (1990)  J. Econ. Entomol.  83(6): 2480-2485. Methods of rearing insect larvae and performing bioassays are well known to one of ordinary skill in the art. 
     A wide variety of bioassay techniques are known to one skilled in the art. General procedures include addition of the experimental compound or organism to the diet source in an enclosed container. Pesticidal activity can be measured by, but is not limited to, changes in mortality, weight loss, attraction, repellency and other behavioral and physical changes after feeding and exposure for an appropriate length of time. Bioassays described herein can be used with any feeding insect pest in the larval or adult stage. 
     The following examples are presented by way of illustration, not by way of limitation. 
     EXPERIMENTAL 
     Example 1 
     Bioassay for Testing the Pesticidal Activity of the  B. thuringiensis  Toxin Against Selected Insects 
     Bioassays were conducted to evaluate the effects of the Bt insecticidal toxin peptide, set forth in SEQ ID NO: 2, on a variety of lepidopteran insect pests as set forth in Table 1. Feeding assays were conducted on an artificial diet containing the insecticidal protein. The insecticidal protein was topically applied using a lepidopteran-specific artificial diet. The toxin was applied at a rate of 0.3 μg per 25 μL sample per well and allowed to dry. The protein is in 10 mM carbonate buffer at a pH of 10. One neonate larva was placed in each well to feed ad libitum for 5 days. Results were expressed as positive for larvae reactions such as stunting and or mortality. Results were expressed as negative if the larvae were similar to the negative control that is feeding diet to which the above buffer only has been applied. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Results of feeding bioassay for SEQ ID NO: 2 
               
            
           
           
               
               
               
            
               
                   
                 Insect Tested 
                 Result 
               
               
                   
                   
               
               
                   
                 European corn borer ( Ostrinia nubilalis ) 
                 + 
               
               
                   
                 Corn earworm ( Helicoverpa zea ) 
                 + 
               
               
                   
                 Black cutworm ( Agrotis ipsilon ) 
                 + 
               
               
                   
                 Fall armyworm ( Spodoptera frugiperda ) 
                 + 
               
               
                   
                 Southwestern corn borer ( Diatraea grandiosella ) 
                 + 
               
               
                   
                 Soybean looper ( Pseudoplusia includens ) 
                 + 
               
               
                   
                   
               
            
           
         
       
     
     Example 2 
     Determination of LC 50  and IC 50    
     Bioassays were conducted to determine an LC 50  and IC 50  of the insecticidal toxin peptide, set forth in SEQ ID NO: 2, on European corn borer (ECB), Southwestern corn borer (SWCB), soybean looper (SBL) and velvet bean caterpillar (VBC). Feeding assays were conducted on an artificial diet containing the insecticidal protein. The insecticidal protein was diluted with 10 mM carbonate buffer at pH 10 and with insect diet to give a series of final toxin concentrations at 3.16-fold dilutions (0.1, 0.316, 1.0, 3.16, 10 etc.). One neonate larva was placed in each well to feed ad libitum for 4 days for SBL and VBC or 6 days for ECB and SWCB. Each bioassay was done with eight duplicates at each dose and was replicated four times. Results are expressed as LC 50  for mortality and/or IC 50  by both mortality and growth inhibition (still at first instar after four or six days) (Table 2a). For three other insect species—corn earworm (CEW), fall armyworm (FAW) and black cutworm (BCW)—only a top concentration (250 ppm in diet) of the toxin peptide plus the buffer control was used. The mortality and effect (mortality plus growth inhibition) responses were scored after 4 days (Table 2b). 
     
       
         
           
               
             
               
                 TABLE 2a 
               
             
            
               
                   
               
               
                 GS128 activity on ECB, SWCB, SBL and VBC 
               
            
           
           
               
               
               
               
            
               
                   
                 Insect 
                 LC 50  (95% FL), ppm 
                 IC 50  (95% FL), ppm 
               
               
                   
                   
               
               
                   
                 ECB 
                 0.6440 (0.4870-0.8588) 
                 0.2927 (0.2290-0.3635) 
               
               
                   
                 SWCB 
                 2.206 (1.594-2.945)  
                 0.1259 (0.0879-0.1708) 
               
               
                   
                 SBL 
                 0.3816 (0.2906-0.4951) 
                 0.1642 (0.1302-0.2068) 
               
               
                   
                 VBC 
                 138.7 (91.00-244.2)  
                 0.1459 (0.0812-0.2484) 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2b 
               
             
            
               
                   
               
               
                 GS128 activity on CEW, FAW and BCW 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Insect 
                 Treatment 
                 % mortality 
                 % affected 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 CEW 
                 250 ppm 
                 4.35 
                 34.8 
               
               
                   
                 FAW 
                 250 ppm 
                 8.33 
                 12.5 
               
               
                   
                 BCW 
                 250 ppm 
                 0.0 
                 17.4 
               
               
                   
                   
               
            
           
         
       
     
     Example 3 
     Transformation of  Maize  by Particle Bombardment and Regeneration of Transgenic Plants 
     Immature  maize  embryos from greenhouse donor plants are bombarded with a DNA molecule containing the toxin nucleotide sequence (e.g., SEQ ID NO: 1) operably linked to a ubiquitin promoter and the selectable marker gene PAT (Wohlleben et al. (1988)  Gene  70: 25-37), which confers resistance to the herbicide Bialaphos. Alternatively, the selectable marker gene is provided on a separate DNA molecule. Transformation is performed as follows. Media recipes follow below. 
     Preparation of Target Tissue 
     The ears are husked and surface sterilized in 30% CLOROX™ bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5 cm target zone in preparation for bombardment. 
     Preparation of DNA 
     A plasmid vector comprising a toxin nucleotide sequence (e.g., SEQ ID NO: 1) operably linked to a ubiquitin promoter is made. For example, a suitable transformation vector comprises a UBI1 promoter from  Zea mays , a 5′ UTR from UBI1 and a UBI1 intron, in combination with a PinII terminator. The vector additionally contains a PAT selectable marker gene driven by a CAMV35S promoter and includes a CAMV35S terminator. Optionally, the selectable marker can reside on a separate plasmid. A DNA molecule comprising a toxin nucleotide sequence as well as a PAT selectable marker is precipitated onto 1.1 μm (average diameter) tungsten pellets using a CaCl 2  precipitation procedure as follows:
         100 μL prepared tungsten particles in water   10 μL (1 μg) DNA in Tris EDTA buffer (1 μg total DNA)   100 μL 2.5 M CaCl 2      10 μL 0.1 M spermidine       

     Each reagent is added sequentially to a tungsten particle suspension, while maintained on the multitube vortexer. The final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes. After the precipitation period, the tubes are centrifuged briefly, liquid removed, washed with 500 mL 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 μL 100% ethanol is added to the final tungsten particle pellet. For particle gun bombardment, the tungsten/DNA particles are briefly sonicated and 10 μL spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment. 
     Particle Gun Treatment 
     The sample plates are bombarded at level #4 in particle gun #HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA. 
     Subsequent Treatment 
     Following bombardment, the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5″ pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored and scored for expression of the toxin by assays known in the art or as described above. 
     Bombardment and Culture Media 
     Bombardment medium (560Y) comprises 4.0 g/L N6 basal salts (SIGMA C-1416), 1.0 mL/L Eriksson&#39;s Vitamin Mix (1000×SIGMA-1511), 0.5 mg/L thiamine HCl, 120.0 g/L sucrose, 1.0 mg/L 2,4-D, and 2.88 g/L L-proline (brought to volume with dl H 2 0 following adjustment to pH 5.8 with KOH); 2.0 g/L Gelrite™ (added after bringing to volume with dI H 2 0); and 8.5 mg/L silver nitrate (added after sterilizing the medium and cooling to room temperature). Selection medium (560R) comprises 4.0 g/L N6 basal salts (SIGMA C-1416), 1.0 mL/L Eriksson&#39;s Vitamin Mix (1000×SIGMA-1511), 0.5 mg/L thiamine HCl, 30.0 g/L sucrose, and 2.0 mg/L 2,4-D (brought to volume with dI H 2 0 following adjustment to pH 5.8 with KOH); 3.0 g/L Gelrite™ (added after bringing to volume with dI H 2 0); and 0.85 mg/L silver nitrate and 3.0 mg/L Bialaphos (both added after sterilizing the medium and cooling to room temperature). 
     Plant regeneration medium (288J) comprises 4.3 g/L MS salts (GIBCO 11117-074), 5.0 mL/L MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/L thiamine HCl, 0.10 g/L pyridoxine HCl, and 0.40 g/L Glycine brought to volume with polished D-I H 2 0) (Murashige and Skoog (1962)  Physiol. Plant.  15: 473), 100 mg/L myo-inositol, 0.5 mg/L zeatin, 60 g/L sucrose, and 1.0 mL/L of 0.1 mM abscisic acid (brought to volume with polished dI H 2 0 after adjusting to pH 5.6); 3.0 g/L Gelrite™ (added after bringing to volume with dI H 2 0); and 1.0 mg/L indoleacetic acid and 3.0 mg/L Bialaphos (added after sterilizing the medium and cooling to 60° C.). 
     Hormone-free medium (272V) comprises 4.3 g/L MS salts (GIBCO 11117-074), 5.0 mL/L MS vitamins stock solution (0.100 g/L nicotinic acid, 0.02 g/L thiamine HCl, 0.10 g/L pyridoxine HCl, and 0.40 g/L Glycine brought to volume with polished dI H 2 0), 0.1 g/L myo-inositol, and 40.0 g/L sucrose (brought to volume with polished dI H 2 0 after adjusting pH to 5.6); and 6 g/L Bacto-agar (added after bringing to volume with polished dI H 2 0), sterilized and cooled to 60° C. 
     Example 4 
       Agrobacterium -Mediated Transformation of  Maize  and Regeneration of Transgenic Plants 
     For  Agrobacterium -mediated transformation of  maize  with a toxin nucleotide sequence (e.g., SEQ ID NO: 1), the method of Zhao can be used (U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326; the contents of which are hereby incorporated by reference). Briefly, immature embryos are isolated from  maize  and the embryos contacted with a suspension of  Agrobacterium  under conditions whereby the bacteria are capable of transferring the toxin nucleotide sequence (SEQ ID NO: 1) to at least one cell of at least one of the immature embryos (step 1: the infection step). In this step the immature embryos can be immersed in an  Agrobacterium  suspension for the initiation of inoculation. The embryos are co-cultured for a time with the  Agrobacterium  (step 2: the co-cultivation step). The immature embryos can be cultured on solid medium following the infection step. Following this co-cultivation period an optional “resting” step is contemplated. In this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of  Agrobacterium  without the addition of a selective agent for plant transformants (step 3: resting step). The immature embryos can be cultured on solid medium with antibiotic, but without a selecting agent, for elimination of  Agrobacterium  and for a resting phase for the infected cells. Next, inoculated embryos are cultured on medium containing a selective agent and growing transformed callus is recovered (step 4: the selection step). The immature embryos are cultured on solid medium with a selective agent resulting in the selective growth of transformed cells. The callus is then regenerated into plants (step 5: the regeneration step), and calli grown on selective medium can be cultured on solid medium to regenerate the plants. 
     Example 5 
     Transformation of Soybean Embryos 
     Soybean embryos are bombarded with a plasmid containing the toxin nucleotide sequence of SEQ ID NO: 1 operably linked to a pinII promoter as follows. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface-sterilized, immature seeds of an appropriate soybean cultivar are cultured in the light or dark at 26° C. on an appropriate agar medium for six to ten weeks. Somatic embryos producing secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos that multiplied as early, globular-staged embryos, the suspensions are maintained as described below. 
     Soybean embryogenic suspension cultures can maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium. 
     Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987)  Nature  ( London ) 327: 70-73, U.S. Pat. No. 4,945,050). A Du Pont Biolistic PDS1000/HE instrument (helium retrofit) can be used for these transformations. 
     A selectable marker gene that can be used to facilitate soybean transformation is a transgene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985)  Nature  313: 810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from  E. coli ; Gritz et al. (1983)  Gene  25: 179-188), and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of  Agrobacterium tumefaciens . The expression cassette comprising a toxin nucleotide sequence (e.g., SEQ ID NO: 1) operably linked to the pinII promoter can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene. 
     To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (in order): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl 2  (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μL 70% ethanol and resuspended in 40 μL of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five microliters of the DNA-coated gold particles are then loaded on each macro carrier disk. 
     Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60×15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi, and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above. 
     Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post-bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post-bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos. 
     All publications, patents and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications, patents and patent applications are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. 
     Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the embodiments.