MXPA99001288A - Insecticidal protein toxins from xenorhabdus - Google Patents

Abstract

Proteins from the genus Xenorhabdus are toxic to insects upon exposure. These protein toxins can be applied to insect larvae food and plants for insect control.

Description

XENORHABDUS INSECTICIDES INSECTICIDES TOXI NAS Cross reference with related applications This patent application claims priority of a serial patent application serial number 60 / 045,641, filed on May 5, 1997.

FIELD OF THE INVENTION The present invention relates to toxins isolated from bacteria and the use of said toxins as insecticides.

BACKGROUND OF THE INVENTION In the past there has been interest in using biological agents as an agent as an option for pest management. One such method explored was the potential for insect control using certain genera of nematodes. Nematodes, such as those of the genera Steinernema and Heterorhabditis, can be used as biological agents due in part to their transmissible insecticidal bacterial symbionts of the genus Xenorhabdus and Photorhabdus, respectively. From the entry into the insect, the nematodes release their bacterial symbionts in the hemolymph, where the bacteria reproduce and eventually cause the death of the insect. Then the nematode develops and reproduces inside the corpse. Progeny of the nematode containing bacteria leave the corpse of the insect as infective young, which then invade additional larvae, thus repeating the cycle leading to the propagation of the nematode. While this cycle is easily performed on a micro scale, adaptation to the macro level in a laboratory setting, as needed to be effective as a general-purpose insecticide, is difficult, costly and inefficient to produce, maintain, distribute and apply. In addition to biological approaches to pest management, such as nematodes, there are currently commercially available pesticide control agents that are derived naturally. These naturally derived approaches can be as effective as synthetic chemical approaches. One such agent that occurs naturally is the crystal protein toxin produced by the bacterium Bacillus thuringiensis (Bt). These protein toxins have been formulated as sprayable insect control agents. A more recent application of Bt technology has been to isolate and transform into plants the genes that produce the toxins. The transgenic plants subsequently produce the Bt toxins, thereby providing insect control (see U.S. Patent Nos. 5,380,831, 5,567,600, and 5,567,862 issued to Mycogen in San Diego, CA). Transgenic Bt plants are quite effective and their use is predicted to be high in some crops and areas. This has raised a concern that resistance management consequences may arise more quickly than with traditional sprayable applications. In this way, it would be quite desirable to discover other bacterial sources other than Bt, which produce toxins that could be used in transgenic plant strategies, or could be combined with Bts to produce transgenic insect-controlling plants. It is known in the art that bacteria of the genus Xenorhabdus are symbiotically associated with the nematode Steinernema. Unfortunately, as reported in a number of articles, bacteria only had pesticidal activity when injected into insect larvae, and did not exhibit biological activity when delivered orally (see Jarosz J et al. "Involvement of Larvicidal Toxins! Pathogenesis of Insect Parasitism with the Rhabditoid Nematodes, Steinernema Feltiae and Heterorhabditis Bacteriophora "Entomophaga 36 (3) 1991 361 -368; Balcerzak, Malgorzata" Comparative studies on parasitism caused by entomogenous nematodes, Steinernema feltiae and Heterorhabditis bacteriophors I. The roles of the nematode-bacterial complex, and of the associated bacterium alone, in pathogenesis "Parasitológica Polonica Act 1991, 36 (4), 175-181). For the reasons stated above, it has been difficult to effectively exploit the insecticidal properties of the nematode or its bacterial symbionts. In this way, it would be quite convenient to discover protein agents derived from Xenorhabdus bacteria that have oral activity, so that the products produced from them could be either formulated as a sprayable insecticide or the bacterial genes encoding said protein agents could be isolated and used in the production of transgenic plants. Until the present inventors' invention, there was no known species or strain Xenorhabdus that produced protein toxin (s) demonstrated to have oral activity.

BRIEF DESCRIPTION OF THE INVENTION Natural toxins are protein complexes that are produced and secreted by growing bacterial cells of the genus Xenorhabdus. Protein complexes, with a natural molecular size ranging from about 800 to about 3300 kDa, can be separated by SDS-PAGE gel analysis into numerous component proteins. Toxins exhibit significant toxicity on exposure to a number of insects. Additionally, the activity of the toxins can be modified by altering environmental conditions. In addition, the toxins have characteristics of being protein, since the activity of them is heat-labile and sensitive to proteolysis. The present invention provides an insecticidal protein easily administered. The present invention also provides a method for delivering insecticidal toxins that are functionally active and effective against many orders of insects. The objects, advantages and characteristics of the present invention will become apparent from the following specification.

Brief description of the drawings Fig. 1 is a phenogram of Xenorhabdus strains as defined by rep-PCR using a specified set of primers. The upper axis of Fig. 1 measures the percentage of similarity of the strains based on the registration of rep-PCR products (ie 0.0 [no similarity] to 1.06 [100% similarity]). On the right axis, the numbers and letters indicate the various strains tested. The vertical lines that separate the horizontal lines indicate the degree of relationship (as read from the extrapolated intersection of the vertical line with the upper axis) between the strains or groups of strains at the base of the horizontal lines (for example, strain DEX1 is approximately 83% similar to strain X. Nem).

DETAILED DESCRIPTION OF THE INVENTION The present inventions are directed to the discovery of a unique class of insecticidal protein toxins produced by bacteria of the genus Xenorhabdus, said toxins having oral functional activity against insects, as defined herein. Species / strains of Xenorhabdus can be isolated from a variety of sources. One such source is in entomopathogenic nematodes, more particularly nematodes of the Steinernema genus or insect corpses infested by these nematodes. It is possible that other sources could host Xenorhabdus bacteria that produce insecticidal toxins having functional activity as defined herein. Such sources in the environment could be either water or ground based. The genus Xenorhabdus is defined taxonomically as a member of the Enterobacteriaceae Family, although it has certain atypical features of this family. For example, strains of this genus are normally negative nitrate reduction, and negative catalase. Approximately three years ago, the Xenorhabdus was subdivided to create a second genus; Photorhabdus, which is comprised of the simple species Photorhabdus luminescens (previously Xenorhabdus luminescens) (Boemare et al., 1993, Int. J. Syst. Bacteriol., 43, 249-255). This differentiation is based on several distinguishing features readily identifiable by the skilled artisan. These differences include the following: DNA-DNA hybridization studies; phenotypic presence (Photrhabdus) or absence (Xenorhabdus) of catalase activity; presence (Phtorhabdus) or absence (Xenorhabdus) of bioluminescence; the Host Nematode Family because Xenorhabdus is found in Steinernematidae and Photorhabdus is found in Heterorhabditidae); as well as comparative cellular fatty acid analysis (Janse et al., 1990, Lett.Appl Microbiol 10, 131-135, Suzuki et al., 1990, J. Gen. Appl. Microbiol., 36, 393-401). In addition, recent molecular studies focused on sequence analysis (Rainey et al., 1995, Int. J. Syst. Bacteriol., 45, 379-381) and restriction (Brunel et al., 1997, App. Environ. Micro. , 63, 574-580) of the 16S RNA genes also support the separation of these two genera. This change in the nomenclature is reflected in this specification, but shall not alter a future change in nomenclature in any way the scope of the inventions described herein. In order to establish that the strains described herein are comprised of Xenorhabdus strains, the strains were characterized based on recognized traits, which define the species / strains of Xenorhabdus and differ from other strains / species of Enterobacteriaceae and Photorhabdus. (Farmer, 1984 Bergey's Manual of Systemic Bacteriology Vol. 1, pp. 510-51 1; Akhurst and Boemare 1988, J. Gen. Microbiol. 134, pp. 1835-1845; Boemare et al. 1993 Int. J. Syst. Bacteriol. 43, pp. 249-255, which are incorporated herein by reference). The features expected for Xenorhabdus are the following: gram-negative bacilli, organism size 0.3-2 X 2-10 μm, pigmentation of white / yellowish colonies, presence of inclusion corpuscles, absence of catalase, inability to reduce nitrate , absence of bioluminescence, ability to take dye from the medium, positive gelatin hydrolysis, growth in Enterobacteriaceae selective media, growth temperature below 37 ° C, survival under anaerobic conditions, and mobility. Currently, the bacterial genus Xenorhabdus is comprised of four recognized species Xenorhabdus nematophilus, Xenorhabdus poinarii, Xenorhabdus bovienii and Xenorhabdus beddingii (Brunel et al., 1997, Environ. Micro., 63, 574-580). A variety of related strains have been described in the literature (eg, Akhurst and Boemare 1988 J. Gen. Microbiol., 134, 1835-1845; Boemare et al., 1993 Int. J. Syst. Bacteriol., 43, pp. 249 -255; Putz et al., 1990, Appl. Environ. Microbiol., 56, 181-186, Brunel et al., 1997, App. Environ. Micro., 63, 574-580, Rainey et al., 1995, Int. J. Syst. Bacteriol., 45, 379-381). Numerous Xenorhabdus strains have been characterized in the present. Such strains and the characteristics thereof are listed in Table 1 in the Examples. These strains have been deposited with the Agricultural Research Service Patent Culture Collection (NRRL) at 1815 North University Street Peoria, Illinois 61604 U.S.A. As you can see in Fig. 1, these strains are diverse. It is not unforeseen that in the future there may be other Xenorhabdus species that will have some or all of the attributes of the species described, as well as some different characteristics that are not currently defined as a trait (s) of Xenorhabdus. However, the scope of the invention herein is any species or strain of Xenorhabdus, which produces proteins as described herein that have functional activity as orally active insect control agents, without considering other features and characteristics. In addition, the strains specified herein and any mutant or phase variant thereof are included within the inventions. There are several terms that are used herein that have a particular meaning and are as follows: By "functional activity" it is meant that protein toxins function as orally active insect control agents, that proteins have a toxic effect, or they are capable of breaking or preventing the feeding of the insect, which may or may not cause the death of the insect. When an insect is contacted with an effective amount of the toxin derived from Xenorhabdus delivered via the expression of transgenic plant, composition (s) of formulated protein (s), composition (s) of sprayable protein (s), a matrix of bait or other delivery system, the results are usually the death of the insect, or insects do not feed on the source that makes the toxins available to insects.

By "natural size" is meant the non-denatured size of the protein toxin or protein toxin subunit produced by the Xenorhabdus strain of interest before any treatment or modification. The natural sizes of the proteins can be determined by a variety of methods available to the skilled artisan, including but not limited to, gel filtration chromatography, polyacrylamide and agarose gel electrophoresis, mass spectroscopy, sedimentation coefficients and the like. The treatment or modifications to alter the natural size of the protein can be done by proteolysis, mutagenesis, gene truncation, protein splitting and other such techniques available to the person skilled in the art of protein biochemistry and molecular biology. The protein toxins discussed herein are commonly referred to as "insecticides". By "insecticides" it is meant herein that protein toxins have a "functional activity", as defined herein further and used as insect control agents. The term "toxic" or "toxicity" as used herein, means transimitir that the toxins produced by Xenorhabdus have "functional activity" as defined herein. The term "Xenorhabdus toxin" means to include any protein produced by a strain of Xenorhabdus microorganism having functional activity against insects, where the Xenorhabdus toxin could be formulated as a sprayable composition, expressed by a transgenic plant, formulated as a matrix of bait, delivered via a baculovirus, a system based on RNA virus from the plant, or delivered by any other applicable host or delivery system. The fermentation broths from the selected strains reported in Table 1 were used to examine the following: extension of the production of insecticidal toxins by the genus Xenorhabdus, the insecticidal spectrum of these toxins, and the protein components of said toxins. It has been shown that the strains characterized herein have oral toxicity against a variety of insect orders. Such orders of insects include, but are not limited to, Coleoptera, Lepidoptera, Diptera, and Acariña. As with other bacterial toxins, the rate of mutation of bacteria in a population can result in variation in the sequence of toxin genes. Toxins of interest here are those that produce protein having functional activity against a variety of insects upon exposure, as described herein. Preferably, the toxins are active against Lepidoptera, Coleoptera, Diptera and Acariña. The inventions herein are intended to capture the protein toxin homologs for protein toxins produced by the strains herein and any strain derived therefrom, as well as any other protein toxin produced by Xenorhabdus having functional activity. These homologous proteins may differ in sequence, but do not differ in functional activity from those toxins described herein. The homologous toxins means that they include protein complexes between 100 kDa to 3200 kDa and are comprised of at least one subunit, where one subunit is a peptide, which may or may not be the same as the other subunit. Several protein subunits have been identified and are shown in the Examples herein. Normally, the protein subunits are between about 20 kDa to about 350 kDa; between about 130 kDa to about 300 kDa; 40 kDa up to 80 kDa; and about 20 kDa to about 40 kDa. The toxins described herein are quite unique since the toxins have functional activity, which is the key to developing an insect management strategy. To develop an insect management strategy, it is possible to delay or avoid the process of protein degradation by injecting a protein directly into an organism, avoiding its digestive tract. In such cases, the protein administered to the organism will retain its function until it is denatured, not specifically degraded, or eliminated by the immune system in higher organisms. Insect injection of an insecticidal toxin has a potential application only in the laboratory. The discovery that insecticidal protein toxins having functional activity as defined herein, exhibit their activity after oral ingestion or contact with toxins, allows the development of an insect management plan based solely on the ability to incorporate protein toxins in the diet of the insect. Such a plan could result in the production of insect baits.

Xenorhabdus toxins can be administered to insects in both a purified and non-purified form. The toxins may also be delivered in amounts of from about 1 to about 100 mg / liter of broth. This may vary depending on the condition of the formulation, conditions of the inoculum source, techniques for toxin isolation, and the like. The toxins found herein may be administered as a sprayable insecticide. The Xenorhabdus fermentation broth can be produced, diluted, or if necessary, concentrated approximately 100 to 1000 times using ultrafiltration or another technique available to the skilled artisan. The treatments can be applied with a syringe atomizer, a lane atomizer or any such equipment available to the skilled artisan, where the broth is applied to the plants. After the treatments, the broths can be tested by applying the insect of choice to said sprayed plant and can then be registered for damage to the leaves. If necessary, auxiliaries and photo-protectors can be added to increase the environmental life of the toxin. In a laboratory environment, broth, dilutions, or concentrates thereof can be applied using methods available to the skilled artisan. Subsequently, the material can be allowed to dry and the insects to be tested are applied directly to the appropriate plant tissue. After a week, plants can register for damage using a modified Guthrie Scale (Koziel, MG, Beland, GL, Bowman, C, Carozzi, NB, Crenshaw, R., Crossland, L., Dawson, J., Desai , N., Hill, M., Kadwell, S., Launis, K., Lewis, K., Maddox, D., McPherson, K., Meghji, MZ, Merlin, E., Rhodes, R., Warren, GW, Wright, M. and Evola, SV 1993). In this manner, the broth or other protein-containing fractions can confer protection against specific insect pests when delivered in a sprayable formulation or when the gene or derivative thereof, which encodes the protein or part thereof, is delivered via a plant. transgenic or microbe. The toxins can be administered as a secretion or cellular protein originally expressed in a heterologous eukaryotic or prokaryotic host. Bacteria are usually the hosts in which proteins are expressed. Eukaryotic hosts could include but are not limited to plants, insects and yeasts. Alternatively, the toxins can be produced in transgenic bacteria or plants in the field or in the insect by a baculovirus vector. Normally, an insect will be exposed to toxins by incorporating one or more of these toxins into the diet / food of the insect. Full lethality is preferred for feeding insects, but is not required to achieve functional activity. If an insect avoids the toxin or ceases feeding, such abstinence will be useful in some applications, even if the effects are sub-lethal or the lethality is delayed or indirect. For example, if transgenic plants resistant to insects are desired, a reluctance of insects to feed on plants is just as useful as lethal toxicity to insects, since the ultimate goal is the protection of insect-induced damage to the plant, in place of death of the insect.

There are many other ways in which toxins can be incorporated into an insect diet. For example, it is possible to adulterate the food source of the larvae with the toxic protein by atomizing the feed with a protein solution, as described herein. Alternatively, the purified protein could be genetically engineered into an otherwise harmless bacterium, which could then be grown in the culture, and either applied to the food source or allowed to reside in the soil in an area in the which eradication of the insect was desirable. Also, the protein could be genetically engineered directly into an insect food source. For example, the main source of food for many insect larvae is the material of the plant. One consideration associated with the commercial exploitation of transgenic plants is the management of resistance. This is of particular interest with Bacillus thuringiensis toxins. There are numerous companies commercially exploiting Bacillus thuringiensis, and there has been much interest in the development of resistance to Bt toxins. One strategy for managing insect resistance would be to combine the toxins produced by Xenorhabdus with toxins such as Bt, proteins vegetative insecticides of Bacillus strains (Ciba Geigy; WO 94/21795) or other insect toxins. The combinations could be formulated for a sprayable application or they could be molecular combinations. The plants could be transformed with Xenorhabdus genes that produce insect toxins and other insect toxin genes, such as Bt.

European Patent Application 0400246A1 describes a transformation of a plant with 2 Bts. This could be 2 genes, not just Bt genes. Another way to produce a transgenic plant that contains more than one insect-resistant gene would be to produce two plants, each plant containing a gene for insect resistance. These plants could then be backcrossed using traditional plant breeding techniques to produce a plant containing more than one insect resistance gene. In addition to producing a transformed plant, there are other delivery systems where it may be convenient to re-design the bacterial gene (s). Along the same lines, an easily isolated, genetically engineered protein toxin designed to fuse both an insect-attractive molecule and a food source and the functional activity of the toxin can be designed and expressed in bacteria or cells eukaryotic using standard techniques well known. After purification in the laboratory, such a toxic agent with "built-in" bait could be packaged within standard insect trap housings. Another delivery scheme is the incorporation of the genetic material of toxins into a baculovirus vector. Baculoviruses infect hosts of particular insects, including those that conveniently target Xenorhabdus toxins. Infectious baculovirus harboring an expression construct for Xenorhabdus toxins could be introduced into insect infestation areas to poison or poison infected insects.

The transfer of the functional properties requires nucleic acid sequences that encode the amino acid sequences for the Xenorhabdus toxins integrated in a protein expression vector appropriate for the host, in which the vector will reside. One way to obtain a nucleic acid sequence encoding a protein with functional properties is to isolate the natural genetic material which produces the Xenorhabdus toxins, using information deduced from the amino acid sequence of the toxin, of which large portions are exposed. ahead. As described below, methods for purifying the proteins responsible for toxin activity are also described. Insect viruses, or baculoviruses, are known to infect and adversely affect certain insects. The affectation of the viruses on the insects is slow, and the viruses do not stop the feeding of the insects immediately. In this way, viruses are not seen as optimal as insect pest control agents. However, combining the genes of Xenorhabdus toxins in a baculovirus vector could provide an efficient way to transmit toxins. In addition, since different baculoviruses are specific for different insects, it may be possible to use a particular toxin to selectively target particularly harmful insect pests. A particularly useful vector for toxin genes is the nuclear polyhedrosis virus. Transfer vectors using this virus have been described and are now the vectors of choice for transferring foreign genes into insects. The toxin gene of the recombinant virus can be constructed in an orally transmissible form. Baculoviruses usually infect insect victims through the intestinal mucosa of the midgut. The toxin gene inserted behind a strong viral coat protein promoter will be expressed and should rapidly kill the infected insect. In addition to a delivery system of transgenic plant or baculovirus or insect virus for the protein toxins of the present invention, the proteins can be encapsulated using Bacillus thuringiensis encapsulation technology, such as, but not limited to, US Pat. 4,695,455; 4,695,462; 4,861, 595, which are incorporated herein by reference. Another delivery system for the protein toxins of the present invention is the formulation of the protein in a bait matrix, which could then be used in insect bait stations above and below the ground. Examples of such technology include, but are not limited to, PCT patent application WO 93/23998, which is incorporated herein by reference. Molecular biology techniques and standards can be used to clone and sequence the toxins described herein. Additional information can be found in Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, which is incorporated herein by reference. The following abbreviations are used throughout the Examples: Tris = tris (hydroxymethyl) amino methane; SDS = sodium dodecyl sulfate; EDTA = ethylenediaminetetraacetic acid, IPTG = isopropylthio-B-galactoside, X-gal = 5-bromo-4-chloro-3-indolyl-B-D-galactoside, C = cetyltrimethylammonium bromide; kbp = pairs of kilobases; dATP, dCTP, dGTP, dTTP, I = 2'-deoxynucleoside adenine ducts, cytosine, guanine, thymine and inosine, respectively; ATP = adenosine 5 'triphosphate. The particular embodiments of this invention are further exemplified in the Examples. However, those skilled in the art will readily appreciate that specific detailed experiments are only illustrative of the invention, as described more fully in the claims, which follow subsequently.

EXAMPLE 1 CHARACTERIZATION OF XENORHABDUS CEPAS In order to establish that the collection described herein consisted of Xenorhabdus isolates, the strains were evaluated in terms of recognized microbiological traits that are characteristic of phase I variants of Xenorhabdus and which differentiate it of other Enterobacteriaceae and Photorhabdus spp. [Farmer, J.J. 1984. Bergey's Manual of Systemic Bacteriology, vol. 1 . pp 510-51 1. (ed. Kreig N. R. and Holt, J.G.). Williams & Wilkins, Baltimore.; Akhurst and Boemare, 1988, J. Gen. Microbiol. 134, 1835-1845; Forst and Nealson, 1996. Microbiol. Rev. 60, 21 -43]. These characteristic features were as follows: gram negative bacilli; organism size 0.3-2 μm wide and 2-10 μm long with occasional filaments (15-50 μm) and spheroplasts; pigmentation of white to yellow / brown colonies on nutrient agar; presence of crystalline inclusion corpuscles; absence of catalase; negative for oxidase; inability to reduce nitrate; absence of bioluminescence; ability to take dye from the growth medium; positive for protease production; growth temperature below 37 ° C; survival under anaerobic conditions, and positively mobile (Table 1). The methods were reviewed using strains of Escherichia coli, Xenorhabdus and Photorhabus as controls. The overall results shown in Table 1 were consistent with all species that are members of the Enterobacteriaceae family and the genus Xenorhabdus. A luminometer was used to establish the absence of bioluminescence associated with strains of Xenorhabdus. To measure the presence or absence of relative light emitting units, broth of each strain (cells and medium) was measured up to three time intervals after inoculation in liquid culture (24, 48 and / or 72 h), and compared with the support luminosity (medium without inoculation). Several strains of Photorhabdus were also tested as positive controls for luminosity. Before measuring light emission from selected broths, cell density was established by measuring ASSonm in a spectrophotometer from Gilford Systems (Oberlin, OH) using a test cell. The resulting light emitting units were then normalized to the density of the cell. Aliquots of broth were placed in 96-well microtiter plates (100 μl each) and read in a Luminunt Packard luminometer (Packard Instrument Co., Meriden CT). The integration period for each sample was 0.1 to 1.0 second. The samples were shaken in the luminometer for 10 seconds before taking the readings. A positive test was determined to be > 3 times the support luminescence (-1 -15 relative units of light). In addition, the absence of colony luminosity with some strains was confirmed with photographic film covers and visual analysis after visual adaptation in a dark room. The Gram spotting characteristics of each species were established with a commercial Gram-spot set (BBL, Cockeysville, MD) in conjunction with Gram spot control slides (Fisher Scientific, Pittsburgh, PA). The microscopic evaluation was then performed using a Zeiss microscope (Cari Zeiss, Germany) with 100X oil immersion objective lens (with 2X body magnification and 10X eyepiece). Microscopic examination of individual spots for organism size, cell description and inclusion corpuscles (the last two observations after logarithmic growth) was performed using wet mount slides (40X objective magnification and 2X body, 10X ocular) and contrast microscopy of phase with a micrometer (Akhurst, RJ and Boemare, NE 1990. Entomopathogenic Nematodes in Biological Control (Gaugler, R. and Kaya, H.), pp. 75-90, CRC Press, Boca Raton, USA., Baghdiguian S., Boyer-Giglio MH, Thaler, JO, Bonnot G., Boemare N. 1993. Biol. Cell 79, 177-185). Colony pigmentation was observed after inoculation on Bacto nutrient agar (Difco Laboratories, Detroit, Ml) prepared by the label instructions. Incubation occurred at 28 ° C and descriptions were recorded after 5-7 days. To test the presence of catalase activity, 1 ml of culture broth or a colony of the test organism was placed in a small plug of nutrient agar in a glass test tube. One ml of a solution of homemade hydrogen peroxide was gently added on the side of the tube. A positive reaction was recorded when gas bubbles (presumably oxygen) appeared immediately or within 5 seconds. Negative controls of uninoculated nutrient agar or culture broth and hydrogen peroxide solution were also examined. The oxidase reaction of each strain was determined by rubbing 24-h colonies on DrySIide Oxidase slides (Difco, Inc.; Detroit, Ml). Positive oxidase strains produce a dark purple color, indicative of cytochrome C oxidase, within 20 seconds after the organism was rubbed against the slide. The failure to produce a dark purple color indicated that the organism was negative oxidase. To test the nitrate reduction, each culture was inoculated in 10 ml of Bacto Nitrate Broth (Difco Laboratories, Detroit, M l). After 24 h of incubation at 28 ° C, the production of nitrite was tested by the addition of two drops of sulfanilic acid reagent and two drops of alpha-naphthylamine reagent (Difco Manual, 10th edition, Difco Laboratories, Detroit, Ml. , 1984). The generation of a different pink or red color indicated the formation of nitrate nitrite, while the lack of color formation indicated that the strain was negative to nitrate reduction. In the latter case, fine powder zinc was added to further confirm the presence of unreduced nitrate established by the formation of nitrite and the resulting red color.

The ability of each strain to take dye from the growth medium was tested with Bacto MacConkey agar containing the neutral red dye; Bacto Tergitol-7 agar containing the bromotimol blue dye and Bacto EMB agar containing the blue dyes of methylene and eosin-Y (agars formulated from Difco Laboratories, Detroit, Ml, all prepared according to the instructions on the label). After inoculation in these media, the dye uptake was recorded on incubation at 28 ° C for 5 days. Growth in Bacto MacConkey and Bacto Tergitol-7 media is characteristic for members of the Enterobacteriaceae family. The mobility of each strain was tested using a Bacto Motility Test Medium solution (Difco Laboratories, Detroit, Ml) prepared with the instructions on the label. Mango test inoculation was performed with each strain and positive mobility was judged after incubation at 28 ° C by macroscopic observation of a diffuse zone of growth that spreads from the inoculation line. The protease production was tested by observing the hydrolysis of gelatin using Bacto gelatin plates (Difco Laboratories, Detroit, Ml) made with the label instructions. The cultures were inoculated and the plates were incubated at 22 ° C for 3-5 days before the evaluation of the gelatin hydrolysis. To assess growth at different temperatures, agar plates [2% protease peptone # 3 with two percent Bacto-Agar (Difco, Detroit, Ml) in deionized water] were labeled from a common source of inoculum. Plates were incubated at 20, 28 and 37 ° C for 5 days. The temperatures of the incubator were checked with an electronic thermocouple and measured to ensure that the temperature settings are valid. The oxygen requirements for strains of Xenorhabdus were tested in the following manner. A mango test inoculation was made in the middle of fluid thioglycollate broth (Difco, Detroit, Ml). The tubes were incubated at room temperature for one week and the cultures were then examined by type and degree of growth. Resazurin indicator was used to indicate the presence of oxygenation of the medium or the aerobiosis zone (Difco Manual, 10th edition, Difco Laboratories, Detroit, Ml). In the case of unclear results, the final concentration of fluid thioglycollate broth medium agar was raised to 0.75% and the growth characteristics were revised again. The diversity of strains of Xenorhabdus was measured by PCR-mediated genomic finger print analysis (Polymerase Chain Reaction) using genomic DNA from each strain. This technique is based on the families of repetitive DNA sequences present throughout the genome of various bacterial species (reviewed by Veersalovic, J., Schneider, M., DE Bruijn, FJ and Lupski, JR 1994. Methods Mol. Cell Biol., 5, 25-40). It is thought that three of these, the repetitive extragenic palindromic sequence (REP), repetitive enterobacterial intergenic consensus (ERIC) and the BOX element, play an important role in the organization of the bacterial genome. It is believed that genomic organization is shaped by selection and the differential dispersion of these elements within the genome of closely related bacterial strains can be used to discriminate between strains (eg, Louws, FJ, Fulbright, DW, Stephens, CT and DE Bruijn, FJ 1994. Appl. Environ. Micro 60, 2286-2295). The Rep-PCR uses oligonucleotide primers complementary to these repetitive sequences to amplify the DNA fragments of varying size that lie between them. The resulting products are separated by electrophoresis to establish the DNA "footprint" for each strain. To isolate genomic DNA from the strains, cell pellets were resuspended in TE buffer (10 or 50 mM Tris-HCl, EDTA 1 or 50 mM, pH 8.0) to a final volume of 10 ml and 12 ml were then added. of NaCI 5 M. This mixture was centrifuged 20 min at 15, 000 xg. The resulting pellet was resuspended in 5.7 ml of TE and 300 μl of 10% SDS and 60 μl of 20 mg / ml K proteinases were added (Gibco BRL Products, Grand Island, NY). This mixture was incubated at 37 ° C for 1 h, approximately 10 mg of lysozyme was added, and the mixture was then incubated for an additional 30 to 45 minutes. One ml of 5M NaCl and 800 μl of C / NaCl solution (10% w / v of C , 0.7 M NaCI) were then added and the mixture was incubated 10 to 20 min at 65 ° C, and in some cases, stirred gently, then incubated and agitated for an additional 20 min to assist in the clarification of the cellular material. An equal volume of chloroform / isoamyl alcohol solution (24: 1, v / v) was added, mixed gently and then centrifuged. Two extractions were performed with an equal volume of phenol / chloroform / isoamyl alcohol (PCI); 50: 49: 1). Genomic DNA was precipitated with 0.6 volume of isopropanol. The precipitated DNA was removed with a sterile plastic circuit *: A = Gram manc, B = crystalline inclusion corpuscles, C = Bioluminescence, D = Cell form, E = Motility, N = Reduction of nitrate, G = Presence of catalase, H = Gelatin hydrolysis, l = taking dye, J = Pigmentation in nutrient agar, K = Growth in agar EMB, L = Growth in MacConkey agar, M = Growth in agar Tergitol-7 , N = facultative anaerobe, 0 = Growth at 20 ° C, P = Growth at 28 ° C, Q = growth at 37 ° C, = oxidase. T- + = positive for trait, -negative for trait; rd = bacillus, S = dimensioned within descriptors of the Gender, ND = not determined §: W = white, C = cream, Y = yellow or glass bar, washed twice with 70% ethanol, dried and dissolved in 2 ml of STE (10mM Tris-HCl pH 8.0, 10mM NaCl, 1mM EDTA). The DNA was then quantified at A260nm. In a second method, 0.01 volume of RNAase A (final 50 μg / ml) was added and incubated at 37 ° C for 2 h. The sample was then extracted with an equal volume of PCI. The samples were then precipitated with 2 volumes of 100% ethanol and collected as described above. The samples were then air dried and resuspended in 250-1000 μl of TE. To perform the Xenorhabdus genomic DNA rep-PCR analysis, the following primers were used: REP1-I; 5'-I I ICGICGICATCIGGC-3 'and REP2-I; 5'-ICGICTTATCIGGCCTAC-3 \ PCR was performed using the following reaction of 25 μl: 7.75 μl of H2O, 2.5 μl of LA buffer 10X (PanVera Corp., Madison Wl), 16 μl of dNTP mixture (2.5 mM each) , 1 μl of each primer at 50 pM / μl, 1 μl of DMSO, 1.5 μl of genomic DNA (concentrations ranging from 0.075-0,480 μg / μl) and 0.25 μl of TaKaRa EX Taq (PanVera Corp., Madison, Wl). PCR amplification was performed on a Perkin Elmer DNA Thermal Cycler (Norwalk, CT) using the following conditions: 95 ° C for 7 min, then [94 ° C for 1 min, 44 ° C for 1 min, 65 ° C for 8 min] for 35 cycles; followed by 65 ° C for 15 min. After the cycles, 25 μl of reaction was added to 5 μl of 6X gel loading buffer (0.25% bromophenol blue, 404 p / v sucrose in H2O). Then, 1% gel agarose 15x20cm in TBE buffer (0.09 M Tris-borate, 0.002 M EDTA) was run using 8 μl of each reaction. The gel was run for approximately 16 h at 45 V. The gels were then stained in 20 μg / ml ethidium bromide for 1 h and stained in TBE buffer for about 3 h. Then Polaroid® photographs of the gels were taken under UV illumination. The presence or absence of bands at specific sizes for each spot was recorded from the photographs using RFLP software program Plus (Scanalytics, Billerica, MA) and entered as a similarity matrix in the numerical taxonomy computation program, NTSYS -pc (Exeter Software, Setauket, NY). The controls of the strain of E. Coli HB101 and Xanthomonas orvzae pv. Orvzae were evaluated under the same conditions produced by PCR fingerprints corresponding to the published reports (Versalovic, J., Koeuth, T. and Lupski, JR 1991. Nucleic Acids Res. 19, 6823-6831; Vera Cruz, CM, Halda- Alija, L., Louws, F., Skinner, DZ, George, ML, Nelson, RJ., DE Bruijn, FJ, Rice, C. and Leach, JE 1995, Int. Rice Res. Notes, 20, 23-24 Vera Cruz, CM, Ardales, EY, Skinner, DZ, Talag, J., Nelson, RJ, Louws, FJ, Leung, H., Mew, TW and Leach, JE 1996. Phytopathology 86, 1352-1359). The Xenorhabdus strains data were then analyzed with a series of programs within NTSYS-pc; SIMQUAL (Similarity for Qualitative data) to generate a matrix of coefficients of similarity (using the Jaccard coefficient) and grouping SAHN (Sequential, Agglomerative, Hierarchical and Nested) using the U PGMA method (non-weighted pair-group method with arithmetic averages) ), which groups related strains and can be expressed as a phenogram (Figure 1). The COPH (cofeetic values) and MXCOMP (matrix comparison) programs were used to generate a cofeetic value matrix and compare the correlation between it and the original matrix on which the grouping is based. A resulting standardized Mantel statistic was generated, which was a measure of the virtue of good fit for a cluster analysis (r = 0.8-0.9 representing a very good fit). In our case, r = 0.9, indicated an excellent adjustment. Therefore, it was determined that the spots described herein were comprised of a diverse group of readily distinguishable strains representative of the genus Xenorhabdus. The strains described herein were deposited prior to the submission of the application with the following International Depositary Authority: Agricultural Research Service Patent Culture Collection (NRRL), National Center for Agricultural Utilization Research, ARS-USDA, 1815 North University St., Peoria, IL 61604. The following strains with N RRL designations were deposited on April 29, 1997: S. Carp (NRRL-B-21732); X. Wi (NRRL-B-21733); X. nem (NRRL-B-21734); X. NH3 (NRRL-B-21735); X. riobravis (NRRL-B-21736); GL 133B (NRRL-B-21737); DEX1 (NRRL-B-21738) DEX2 (NRRL-B-21739), DEX3 (NRRL-B-21740); DEX4 (NRRL-B-21741); DEX 5 (NRRL-B-21742); and DEX 6 (NRRL-B-21743). The remaining strains described herein were deposited with NRRL on April 30, 1998. In total, thirty-nine (39) strains were deposited.

EXAMPLE 2 NON-TOXIC UTILITY OF TOXI NA (S) PRODUCED BY SEVERAL XENORHABDUS STRAINS "Storage" cultures of the various strains of Xenorhabdus were produced by inoculating 175 ml of 2% liquid peptone # 3 protease medium (PP3) (Difco Laboratories, Detroit, Ml) with a variant phase I colony in a flask with three deflectors with a Delong neck covered with a Kaput closure. After inoculation, flasks were incubated for 24-72 h at 28 ° C on a rotary shaker at 150 rpm. The cultures were then transferred to a sterile bottle containing a sterile magnetic stir bar and then overlaid with sterile mineral oil to limit exposure to air. The storage cultures were kept in the dark at room temperature. These cultures were then used as sources of inoculum for the fermentation of each strain. Phase I variant colonies were also stored frozen at -70 ° C to be used as a source of inoculum. Single phase I colonies were selected from PPS plates containing bromothymol blue (0.0025%) and placed in 3.0 ml of PP3 and grown overnight on a rotary shaker (150 rpm) at 28 ° C. Glycerol (diluted in PP3) was then added to achieve a final concentration of 20% and the cultures were frozen in aliquots at -70 ° C. For culture inoculation, a portion of the frozen aliquot was removed aseptically and labeled on PP3 containing bromothymol blue for the reselection of phase I colonies. Cultures or pre-production "seed" flasks were produced either by inoculating 2 ml of an oil over-layered storage culture or by transferring a phase I variant colony in a sterile 175 ml medium into a flask with three 500 ml baffles covered with a Kaput closure. Normally, following 16 h of incubation at 28 ° C on a rotary shaker at 150 rpm, seed cultures were transferred into production flasks. The production flasks were normally inoculated by adding -1% of the seed culture actively growing to tryptic soy broth or sterile PP3 (TSB, Difco Laboratories, Detroit, Ml). For small-scale productions, the flasks were inoculated directly with a variant phase I colony. The production of broths occurred in flasks with three 500 ml baffles covered with a Kaput closure. The production flasks were shaken at 28 ° C on a rotary shaker at 150 rpm. The production fermentations were finished after 24-72 h. Following the appropriate incubation, the broths were dispensed in sterile 1 .0 I polyethylene bottles, shaken vigorously at 2600 xg for 1 h at 10 ° C and decanted from the waste pellet and cells. Then the broths were filter sterilized or the clarification of the additional broth was achieved with a tangential flow microfiltration device (Pall filtron, Northborough, MA) using a 0.5 μM open channel polyether sulfone membrane (PES) filter. The resulting broths were then concentrated (up to 10 times) using a 10,000 or 100,000 MW cutoff membrane, M12 ultrafiltration device (Amicon, Beverly MA) or centrifugal concentrators (Millipore, Bedford, MA and Pall Filtron, Northborough, MA) with a pore size of 10,000 or 100,000 MW. In the case of centrifugal concentrators, the broths were stirred vigorously at 200 xg for about 2 h. The membrane permeate was added for the corresponding retentate to achieve the desired concentration of the components greater than the pore size used. Following these procedures, the broths were used for biochemical analysis or biological evaluation. The heat inactivation of samples of processed broth was achieved by heating samples of 1 ml at 100 ° C in a heat block filled with sand for 10-29 min. The broth (s) and the toxin complex (s) from different strains of Xenorhabdus were useful to reduce insect populations and were used in a method to inhibit a population of insects, which included applying to an insect site an inactivating amount of effective insect of the described active. A demonstration of the observed functional activity extent of the broths from a selected group of fermented Xenorhadus strains is shown as described above in Table 2. It is possible that additional or improved functional activities with these strains could be detected through the concentration Increased broth or by using different fermentation methods as described herein. Consistent with the activity that is associated with a protein, the functional activity showed heat lability and / or was present in the high molecular weight retentate (greater than 10 kDa and predominantly greater than 100 kDa) after the concentration of the broth. The culture broth (s) of various Xenorhabdus strains showed differential functional activity (mortality and / or growth inhibition) against a number of insects. More specifically, the activity was seen against corn root worm larvae and pod weevil larvae, which are members of the Coleoptera insect order. Other members of the Coleoptera include centipedes, pollen beetles, beetles, Colorado beetles and Colorado potato beetles. The broths and purified toxin complex (s) were also active against tobacco budworm, tobacco sphincter larvae, corn earworm, beet armed worm, autumn worm and European corn borer, which they are members of the order Lepidoptera. Other typical members of this order are cabbage measuring caterpillar, black night caterpillar, apple worm, clothing moth, cornmeal moth, oak beetle, cabbage worm, bag worm, eastern lizard, and earthworm of Earth. Activity was also seen against mosquito larvae which are members of the Diptera order. Other members of the Diptera order are pea ghee, carrot fly, cabbage root fly, turnip root fly, onion fly, typula and common fly and several mosquito species. Activity was also seen with the broth or wines against the two-spotted red cress, which is a member of the Acariña order, which includes red strawberry crests, mites, red citrus mite, European red mite, reddish pear mite and red berry of the tomato. The activity against corn rootworm larvae was tested as follows. Culture broth (s) of Xenorhabdus (10X concentrate, sterilized by filter), PP3 or TSB (10X concentrate), complex (s) of purified toxins or 10 mM sodium phosphate buffer, pH 7.0, were applied directly to the surface (approximately 1.5 cm2) of artificial diet (Rose, R. I. and McCabe, JM 1973, J. Econ.Estomol.66, 398-400) in aliquots of 40 .mu.l. The toxin complex was diluted in 10 mM sodium phosphate buffer, pH 7.0. The diet plates were allowed to air dry in a sterile flow hood and the cavities were infested with neonatal, simple Diabrotica undecimpunctata howardi (southern corn rootworm, SCR) incubated from the sterilized eggs on the surface. The plates were sealed, placed in a humidified growth chamber and maintained at 27 ° C for the appropriate period (3-5 days). Then determinations of larval weight and mortality were recorded. In general, 8-16 insects were used per treatment in all the studies. The control mortality was generally less than 5%. The activity against pod scab (Anthomonas grandis) was tested as follows. Concentrated Xenorhabdus broths (10X) or control media (PP3) were applied in 60 μl aliquots to the surface of 0.35 g of artificial diet (Stoneville Yellow lepidopterous diet) and allowed to dry. A 12-24 hr. Simple pod larva larva was placed in the diet, the cavities were sealed and held at 25 ° C, 505 relative humidity (RH) for 5 days. The weights of the larvae and mortality were then evaluated. The control mortality varied between 0-25%. The activity against mosquito larvae was tested as follows. The assay was conducted on a 96-well microtiter plate. Each well contained 200 μl of aqueous solution (10X concentrated Xenorhabdus culture broth (s)), control medium (2% of PP3) and approximately 20, 1-day larvae (Aedes aegypti). There were 6 cavities per treatment. The results were read 24 h after the infestation. No control mortality was observed.

The activity against lepidopteran larvae was tested as follows. Culture broth (s) of concentrated Xenorhabdus (s) (10X), control medium (PP3 or TSB), purified toxin complex (s) or 10 mM sodium phosphate buffer, pH 7.0, were applied directly. to the surface (~ 1.5 cm2) of the standard artificial Lepidoptera diet (Stoneville Yellow diet) in 40 μl aliquots. The diet plates were allowed to air dry in a sterile flow hood and each cavity was infested with a neonatal, simple larva. European corn borer eggs (ostrinia nubilalis), autumn worm (Spodoptera frugiperda), corn ear worm (Helicoverpa zea) and tobacco sphincter larvae (Manduca sexta) were obtained from commercial sources and incubated in-house while the larvae of the tobacco budworm (Heliothis virescens) and the armed beetworm (Spodoptera exigua) were supplied internally. Following the infestation with the larvae, the diet plates were sealed, placed in a humidified growth chamber and kept in the dark at 27 ° C for the appropriate period. The determinations of weight and mortality were recorded on day 5. Generally, 16 insects were used per treatment in all the studies. Control mortality generally varied from 0-12.5%. Activity against the two-spotted red spot (Tetranychus urticae) was determined as follows. Squash plants were cut to a simple cotyledon and atomized until finished with 10X concentrated broth (s) or control medium (PP3). After drying, the plants were infested with a mixed population of red crests and held at room temperature and humidity for 72 h. Live mites were then counted to determine the control levels.

EXAMPLE 3 NON-FUNCTIONAL ACTIVITY OF TOXIN PROTEINS HIGHLY PURI FICADAS FROM THE XENORHABDUS X. riobravis strain The functional toxin protein was purified from the fermentation broth of the Xenorhabdus X. riobravis strain as described herein. This toxin was tested against neonate larvae of five insect species, southern corn rootworm, European corn borer, tobacco sphincter larva, corn earworm and tobacco budworm following the methods described in Example 2 The results are shown in Table 3. All species showed lethal and / or inhibitory effects of growth after five days, when they were presented with toxin at a dose of 440 ng of toxin diet / cm2.

Table 3. Effect of highly purified X. riobravis toxin on several insect species * - Values are% mortality /% growth inhibition corrected for control effects.

EXAMPLE 4 EFFECT OF DIFFERENT CULTURE MEDIA ON THE FUNCTIONAL ACTIVITY OF SELECTED XENORHABDUS CEPAS FERMENTATION FLOWS Several different culture media were used to further optimize conditions for the detection of functional activity in the fermentation broths of various strains of Xenorhabdus. GL133B, X. riobravis, X. Wi, DEX8 and DEX1 were grown in PP3, TSB and PP3 plus 1.25% NaCl (PP3S) as described herein. The broths were then prepared as described herein and evaluated against larvae of the tobacco sphincters to determine any change in functional activity. In both experimental cases (condition A, which is PP3 vs. TSB, and condition b, which is PP3 vs. PP3S), the functional activity of the fermentations in PP3S and / or TSB was improved as compared to PP3 fermentations simultaneous (Table 4). In certain cases, the activity was discovered, which was not apparent with PP3 fermentations. The functional activity produced under condition A and condition B, was shown to be heat labile and was retained by high molecular weight membranes (> 100,000 kDa). The addition of NaCl to broth after bacterial growth was complete, the activity of the toxin was not increased, indicating that the increased functional activity observed was not due to increasing the concentration of NaCl in the media, but was due to the increased toxin. = > 25% mortality and / or growth inhibition vs. Control ** = 1; tobacco budworm; 2; European corn borer; 3; larva of the sphincidae of tobacco, 4; southern corn rootworm, 5; pod weevil, 6; Mosquito, 7; red cresa of two spots, 8; corn earworn, 9; armed worm of autumn, 10; armed worm of the beet.

The increased activity observed with X. riobravis fermented in PP3S was further investigated by partial purification of the toxin (s) from the fermentations in PP3 and PP3S as described herein. Consistent with the observations using culture broth, the active fraction or fractions of PP3S broth (obtained from size exclusion chromatography and anion exchange as described herein) contained increased biological activity, protein concentration and a protein pattern more complex as determined by SDS-PAGE analysis.

Table 4. The effect of different culture media on the functional potency of selected Xenorhabdus fermentation broths * J + - - = 2 '5-50% mortality, ++ = 51 -75% mortality, +++ = > 76% mortality, - = < 25% mortality EXAMPLE 5 CEPAS OF XENORHABDUS X. nem, X. riobravis, and X. Wi: PURIFICATION, CHARACTERIZATION AND ACTIVITY The protocol, as follows, was established based on purifying those fractions that have the highest activity against larvae of the tobacco sphincters (Manduca sixth), hereinafter THW, as determined in bioassays (see Example 2). Typically, 4- 20 I of Xenorhabdus culture that had been grown in PP3 broth was received and concentrated by filtering, as described herein, using an Amicon spiral filtering cartridge type S1 Y100 attached to a filtration device. Amicon M-12 (Amicon Inc., Beverly, MA). The retentate contained natural proteins, where the majority consisted of those having molecular sizes greater than 100 kDa, while the flow through the material contained natural proteins less than 100 kDa in size. The majority of the activity against THW was contained in the retention of 100 kDa. The retentate was continuously diafiltered with 10 mM sodium phosphate (pH = 7.0) until the filtrate reached an A2β. 0.100 Unless stated otherwise, all procedures of this point were performed in buffer defined as 10 mM sodium phosphate (pH 7.0). The retentate was then concentrated to a final volume of about 0. 20 I and then filtered using a sterile 0.45 μm filtration unit (Corning, Corning, NY). The filtrate was loaded at 7.5 m / min on a H $ 16/10 column from Pharmacia, which had been packed with strong anion exchange matrix POROS 50 HQ from PerSeptive Biosystem balanced in buffer using a SPRINT system from PerSeptive Biosystem (PerSeptive Biosystems, Framingham, MA). After loading, the column was washed with buffer until an A2sonm 0.100 was achieved. The proteins were then levigated from the column at 2.5 ml / min using buffer with 0.4 M NaCI for 20 min for a total volume of 50 ml. The column was then washed using a buffer with 0.1 M NaCl at the same flow rate for an additional 20 min (final volume = 50 ml). Proteins levigated with 0.4 M NaCl and 1.0 M were placed in separate dialysis bags (SPECTRA / POR Membrane MWCO: 2,000; Spectrum, Houston, TX) and dialyzed overnight at 4 ° C in 12 I of shock absorber. In some cases, the 0.4 M fraction was not dialyzed but instead was immediately desalted by gel filtration (see below). The majority of the activity against THW was contained in the 0.4 M fraction. The 0.4 M fraction was further purified by application of ml to a XK 26/100 column from Pharmacia that had been pre-packed with Sepharose CL4B (Pharmacia) using a flow rate of 0.75 ml / min. Fractionation of the 0.4 M fraction on the Sepharose CL4B column produced four to five distinct peaks when X. nem and X. Wi. The proteins of the strain X. riobravis, while having a different peak equivalent to the empty volume, also had a low, very wide absorbance region, varying from ca. 280 min at ca. 448 min of the run of 800 min. Normally, two peaks of higher absorbance were observed after 450 min and before 800 min. The active fractions of X. Wi and X. nem were normally levigated at approximately 256 min up to 416 min from a run of 800. The fractions were deposited based on a peak profile of A280nm and concentrated to a final volume of 0.75 ml using a Biomax-50K membrane from ULTRAFREE-15 centrifugal filter device from Millipore (Millipore Inc., Bedford, MA) or were concentrated by binding to a MonoQ HR10 / 10 column from Pharmacia, as described herein. Protein concentrations were determined using a BioRad Protein Assay Kit (BioRad, Hercules, CA) with bovine gamma globulin as a standard. The natural molecular weight of the THW toxin complex was determined using a HR 16/50 column from Pharmacia that had been pre-packed with Sepharose CL4B in said phosphate buffer. The column was then calibrated using proteins of known molecular size thereby allowing calculation of the approximate natural molecular size of the toxin complex. As shown in Table 5, the molecular size of the toxin complex was as follows: 1500 + 530 kDa for strain X. nem; 100 + 350 kDa for strain X. riobravis; 3290 kDa + 1 150 kDa for strain X. Wi; 980 ± 245 for strain ILM078; 1013 ± 185 for strain DEX6; and 956 ± 307 for strain ILM080. A highly purified fraction was then analyzed via ion exchange, gel filtration, ion exchange, hydrophobic interaction chromatography and ion exchange chromatography, as described herein, for size using quantitative gel filtration. It was found that this material has a natural molecular size of 1049 ± 402 kDa (Table 5).

The proteins found in the toxin complex were examined for individual polypeptide size using SDS-PAGE analysis. Typically, 20 μg of toxin complex protein from each strain was loaded onto a 2-15% polyacrylamide gel (Integrated Separation Systems, Natick, MA) and electrophoresed at 20 mA in SDS-PAGE buffer (BioRad ). After completion of the electrophoresis, the gels were stained overnight in Coomassie blue of BioRad R-250 (0.2% in methanol: acetic acid: water, 40: 10:40 v / v / v). Subsequently, the gels were peeled off in acetic methanohacid: water; 40: 10:40 (v / v / v). The gels were then rinsed with water for 15 min and screened using a PERSONAL LASER DENSITOMETER from Molecular Dynamics (Sunnyvale, CA). The traces were quantified and the molecular sizes were calculated by comparing BioRad's high molecular weight standards, which ranged from 200-45 kDa. The sizes of the individual polypeptides comprising the THW toxin complex for each strain are listed in Table 6. The sizes of the individual polypeptides ranged from 32 kDa to 330 kDa. Each of the strains of X. Wi, X. nem. X. riobravis, ILM080, ILM078, and DEX6 had polypeptides comprising the toxin complex that were in the range of 160-330 kDa, the range of 100-160 kDa, and the range of 50-80 kDa. These data indicate that the toxin complex may vary in the composition of the peptide and components of strain to strain; however, in all cases, the toxin attributes appear to consist of a complex of oligomeric proteins with subunits ranging from 23 kDa to 330 kDa.

EXAMPLE 5 SUB-FRACTIONAMENT OF XENORHABDUS TOXIN COMPLEX OF X. riobravis AND X. Wi For subfractionation, approximately 10 mg of Xenorhabdus protein toxin complex from X. riobravis was isolated, as described above, and applied to a MonoQ HR 10/10 column from Pharmacia equilibrated with 10 mM phosphate buffer, pH 7.0 at a flow rate of 2 ml / min. The column was washed with said buffer until the absorbance at 280 nm returned to the baseline. The proteins bound to the column were levigated with a linear gradient of 0 to 1.0 M NaCl in said buffer at 2 ml / min for 1 h. Two fractions of ml were collected and subjected to analysis by THW bioassay as described herein. Peak activity was determined by examining a 2-fold dilution of each fraction in the THW bioassays. A peak of activity against THW was observed which was levitated to approximately NaCl 0.3-0.4 M. Fractions having activity against THW were extracted and analyzed by SDS-PAGE gel electrophoresis.

It was observed that there were four predominant peptides having the approximate sizes of 220 kDa, 190 kDa, 130 kDa, and 54 kDa. The peptides described above were subjected to electrophoresis in a 4-20% SDS-PAGE (Integrated Separation systems) and "transblotted" to PROBLOTT PVDF membranes (Applied Biosystems, Foster City, CA). The spots were sent for amino acid analysis and N-terminal amino acid sequencing to Harvard MicroChem and Cambridge ProChem, respectively. For sub-fractionation experiments with X. Wi, ca. 10 mg of toxin were applied to a MonoQ HR 10/10 column equilibrated with 10 mM phosphate buffer, pH 7.0 at a flow rate of 2 ml / min. The column was washed with said buffer until the A2sonm returned to the baseline. The proteins bound to the column were levigated with a linear gradient of 0 to 1.0 M NaCl in said buffer at 2 ml / min for 1 h. Two fractions of ml were collected and subjected to analysis by TWH bioassay as described herein. At least two main detectable peaks were observed at A2sonm. The majority of the functional activity of THW that was observed was levigated to approximately NaCI 0.10-0.25 M. The fractions having activity against THW were extracted and analyzed by gel electrophoresis. By means of SDS-PAGE it was observed that there were up to eight predominant peptides having approximate sizes of 330 kDa, 320 kDa, 270 kDa, 220 kDa, 200 kDa, 190 kDa, 170 kDa, 130 kDa, 91 kDa, 76 kDa, 55 kDa and 36 kDa. The peak fraction of activity extracted from THW was applied to a column of phenyl-sepharose HR 5/5. Solid (N H 4) 2 SO 4 was added to a final concentration of 1.7 m. The solution was then applied onto the column equilibrated with 1.7 M (NH) 2 SO in 50 mM potassium phosphate buffer, pH 7, at 1 ml / min. The proteins bound to the column were then levigated with a linear gradient of 1 .7 M (NH) 2 SO 4, 50 mM potassium phosphate, pH 7.0 to 10 mM potassium phosphate, pH 7.0 at 0.5 ml / min for 60 min. After THW bioassays, it was determined that the peak activity was levigated at an A280nm between 40 min at ca. 50 min The fractions were dialyzed overnight against 10 mM sodium phosphate buffer, pH 7.0. SDS-PAGE showed that there were up to six predominant peptides having approximate sizes of 270 kDa, 220 kDa, 170 kDa, 130 kDa, and 76 kDa. Peptides from the active THW fractions from either the 5/5 or 10/10 phenyl sepharose column were electrophoresed on 4-20% SDS-PAGE gel (Integrated Separation Systems) and transblotted to PROBLOTT membranes PVDF (Applied Biosystems, Foster City, CA). The spots were sent for amino acid analysis and N-terminal amino acid sequencing to Harvard MicroChem and Cambridge ProChem, respectively. The N-terminal amino acid sequences for the peptides of 130 kDa (SEQ ID NO: 1), 76 kDa (SEQ ID NO: 2), and 38 kDa (SEQ ID NO: 3) are included herein. The insect bioassays were performed using either toxin complex or purified phenyl-sepharose fractions of THW. Functional activity (at least 205 mortality) and / or growth inhibition (at least 40%) was observed for autumn armed worm, armed beetworm, tobacco sphingulate larvae, tobacco budworm, corn borer European and southern corn rootworm. In the toxin complex preparations tested, greater activity against tobacco sphincter larvae and tobacco budworm was observed than against southern corn rootworm. The insect activity of the X. Wi toxin complex and any further purified fraction showed to be sensitive to heat.

EXAMPLE 6 PRODUCTION, ISOLATION AND CHARACTERIZATION OF XENORHABDUS X. carpocapsae strain 15 inoculum of an overnight culture of X. carpocapsae, also known as X. carp, was added to a 125 ml flask containing 25 ml of PP3 and it was incubated for 72 to 28 ° C on a rotary shaker at 250 rpm. Subsequently, the cultures were centrifuged for 20 min at 10,000 xg as described herein followed by filtration of the supernatant using a 0.2 μm membrane filter. A 15 ml sample of the supernatant was then added to a UItrafree-15 100,000 NMWL centrifugal filter device (Millipore, MA) and centrifuged at 200 xg. The retentate was washed 2x with 100 mM KPO, pH 6.9, and then resuspended in 1.0 ml thereof. The proteins were analyzed by SDS-PAGE as described herein, using a 10% resolving gel and 4% stacking gel with calibrated sizes using pre-stained BioRad standards (Hercules, CA). The gels were electrophoresed at 40V for 16 h at 15 ° C, and then stained with Colloidal Blue from Novex, Inc., (San Diego, CA).

Table 5. Characterization of a toxin complex of Xenorhabdus strains For further separations, samples were applied to a BIO-SEP S4000 column (Phenomenex, Torrance, CA), 7.5 mm I.D., 60 cm CML under a Socratic system using 100 mM KPO, pH 6.9. The total amount charged per sample was 250-500 μg of protein. Fractions were collected in 3 groups depending on the size of the protein (size exclusion chromatography) as follows: proteins greater than 1,000 kDa; proteins being 800-1, 00 kDa; and proteins less than 800,000 kDa. The fraction of 800,000-1,000,000 Da was selected for further analysis.

Table 6. Molecular sizes of peptides in toxin complex of strains of Xenorhabdus in kDa.

The 800-1000 kDa fractions, which had the most functional activity, were extracted and concentrated using a centrifugal filter device of 100,000 NMWL (Millipore, Bedford, MA). Each fraction of extracted retentate was washed 2x and resuspended in 300 μl of 100 mM KP, pH 6.9. Protein concentrations were determined using the bicinchoninic acid protein assay reagent set (Pierce, Rockford, IL). The proteins in this fraction were analyzed by SDS-PAGE as described herein and found to have many proteins of different sizes. This material was then further separated into a DEAE column, whereby the proteins were levigated with increasing salt concentrations. Those fractions having the highest activity were then examined again via SDS-PAGE and found to be comprised of 4 predominant proteins having sizes as follows: 200, 190, 175 and 45 kDa. The active fraction of the DEAE step was passed through an HPLC gel filtration column as described above (BioSep S4000), and the toxic activity against Manduca sexta was found to be contained within a fraction having natural proteins > 800 kDa. Bioassays were performed as follows. Eggs were purchased from M. Sexta from Carolina Biological Supply Co. The eggs were incubated and cultured on a fresh wheat germ diet (ICN, CA) while being incubated at 25 ° C in a 16-hour light cycle incubator. / 8 hours of darkness. The oral toxicity data were determined by placing twelve M. sexta larvae on a piece of insect feed containing 300 μg of ultrafiltration retentate obtained as described above. Observations were made over 5 days. For the HPLC size exclusion chromatography fractions, 20 μg of total protein was applied to the wheat germ diet. The experiment was repeated in duplicate.

SEQUENCE LISTING (1) IN GENERAL TRAINING: (i) APPLICANT: Ensígn, Jerald C. Bowen, David J. Tenor, Jennifer L. Ciche, Todd A. Petell, James K. Strickland, James A. Orr, Gregory L. Fatig, Raymond Bintrim, Scott B (ii) TITLE OF THE INVENTION: Xenorhabdus Insecticidal Protein Toxins (iii) NUMBER OF SEQUENCES: 3 (iv) DIRECTION FOR CORRESPONDENCE : (A) DESTINY: Dow AgroSciences, LLC (B) STREET: 9330 Zionsville Road (C) CIU DAD: Indianapolis (D) STATE: IN (D) COUNTRY: EU (E) ZIP CODE: 46268 (v) LEGIBLE FORM OF COMPUTER: (A) TYPE OF MEDIA: FLEXIBLE DISK (B) COMPUTER: IBM (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PACKAGE: patent in release # 1 .0, Version # 1 .30 (vi) CURRENT REQUEST DATA: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (viii) LAWYER / AGENT INFORMATION: (A) NAME: Borucki, Andrea T. (B) REGISTRATION NUMBER: 33651 ( C) NUMBER OF REFERENCE / CASE: 50612P1 (2) IN FORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LONGITU D: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TI FRAGMENT PO: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 Asn Gln Asn Val Glu Pro Be Ala Gly Asp lie Val 1 5 10 (2) IN FORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 Ser Gln Asn Val Tyr Arg Tyr Pro 1 5 (2) IN FORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 Met Thr Lys Gln Glu Tyr Leu 1 5

Claims (28)

1. Claims 1. A composition, comprising an effective amount of a Xenorhabdus protein toxin having functional activity against an insect, said protein toxin being derived from a protein having a natural molecular size of at least 100 kDa. The composition of claim 1, wherein the Xenorhabdus toxin having oral functional activity against an insect is produced by a purified culture of Xenorhabdus nematophilus, Xenorhabdus poinarii, Xenorhabdus bovienii, Xenorhabdus beddingii or Xenorhabdus species. The composition of claim 2, wherein said purified Xenorhabdus culture was selected from the group consisting of S. Carp, x. Wi, X. nem. X. NH3, X. riobravis, GL 133B, DEX1, DEX2, DEX3, DEX4, DEX5, DEX6, DEX7, DEX8, ILM037, ILMO039, ILM070, ILM078, ILM079, ILM080, ILM081, ILM082, ILM083, ILM084, ILM102, ILM103 , ILM104, ILM129, ILM133, ILM 135, ILM138, ILM142, ILM 143, GLX26, GLX40, GLX166, SEX20, SEX76, and SEX180. The composition of claim 1, wherein the toxin having functional activity against an insect is produced by a purified culture of Xenorhabdus strain designated S. Carp, X. Wi, X. nem, X. NH3, x. riobravis, GL 133B, DEX1, DEX2, DEX3, DEX4, DEX5, DEX6, DEX7, DEX8, ILM037, ILM039, ILM070, ILM078, ILM079, ILM080, ILM081, ILM082, ILM083, ILM084, ILM 102, I LM103, ILM104, ILM129 , ILM133, ILM135, ILM138, ILM142, ILM143, GLX26, GLX40, GLX166, SEX20, SEX76, and SEX180. The composition of claim 4, wherein the toxin having functional activity against an insect is a mixture of one or more toxins produced from purified cultures of Xenorhabdus. The composition of claim 3, wherein the toxin having functional activity against an insect, said toxin being a mixture of one or more toxins, is produced from said purified cultures of Xenorhabdus, said purified cultures being selected from the group that consists of s. Carp, x. Wi, X. nem, X. NH3, X. riobravis, GL 133B, DEX1, DEX2, DEX3, DEX4, DEX5, DEX6, DEX7, DEX8, ILM037, I LM039, ILM070, I LM078, ILM079, ILM080, ILM081, ILM082 , ILM083, I LM084, ILM102, ILM103, ILM104, ILM129, ILM133, ILM135, ILM138, ILM142, ILM143, GLX26, GLX40, GLX166, SEX20, SEX76, and SEX180. The composition of claim 1, wherein the insect is of the order Coleoptera, Lepidoptera, Diptera or Acariña. The composition of claim 7, wherein the insect species of the Coleoptera order are selected from the group consisting of corn rootworm, pod weevil, centipede, pollen beetle, beetle, seed beetle and potato beetle. Coloring 9. The composition of claim 7, wherein the insect species of the order Lepidoptera are selected from the group consisting of armed beetworm, European corn borer, tobacco sphingulate larva, tobacco budworm, measuring caterpillar of cabbage, black night caterpillar, corn ear worm, apple worm, clothing moth, cornmeal moth, oak pitcher, cabbage worm, cotton worm, bag worm, eastern lizard, earthworm and armed worm of autumn. The composition of claim 7, wherein the insect species of the order Diptera are selected from the group consisting of pea ghee, carrot fly, cabbage root fly, turnip root fly, fly of the Onion, typical, common fly, and several species of mosquitoes. eleven . The composition of claim 7, wherein the species of insects of the order Acariña are selected from the group consisting of two-spotted red crests, red strawberry crests, mites, red citrus mite, European red mite, reddish pear mite and red berry of the tomato. 12. A substantially pure microorganism culture comprised of Xenorhabdus strain selected from the group consisting of S. carp, x. Wi, x. nem, X. NH3, X. riobravís, GL 133B, ILM037, ILM039, ILM070, ILM078, ILM079, ILM080, ILM081, ILM082, ILM083, ILM084, ILM102, ILM103, ILM104, ILM129, ILM133, ILM135, ILM138, ILM142, ILM143 , GLX26, GLX40, GLX166, SEX20, SEX76, and SEX180. 13. A purified protein preparation comprising, a Xenorhabdus protein with at least one subunit having an approximate molecular weight between about 20 kDa to about 350 kDa; between about 130 kDa to about 350 kDa; about 80 kDa to about 130 kDa; about 40 kDa to about 80 kDa; or about 20 kDa to about 40 kDa. The purified protein preparation of claim 13 comprising, a natural Xenorhabdus protein with at least one subunit having a molecular weight of at least 100 kDa or greater. 15. A purified protein preparation comprising a protein containing an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. 16. A method for controlling an insect comprising, delivering to an insect an effective amount of a protein toxin having functional activity against an insect, wherein the protein is produced by a purified bacterial culture of the genus Xenorhabdus and has a natural molecular weight of at least 100 kDa. The method of claim 16, wherein the Xenorhabdus toxin having functional activity against an insect is produced by a purified culture of Xenorhabdus nematophilus, Xenorhadus poinarii, Xenorhabdus bovienii, Xenorhabdus beddingii or Xenorhabdus species. 18. The method of claim 17, wherein said purified Xenorhabdus culture was selected from the group consisting of S. carp. X. Wi, x. nem, x. NH3, X. riobravis, GL 133B, DEX1, DEX2, DEX3, DEX4, DEX5, DEX6, DEX7, DEX8, I LM037, ILM039, ILM070, ILM078, ILM079, ILM080, ILM081, ILM082, ILM083, ILM084, ILM102, ILM103, ILM 104, ILM129, ILM133, ILM135, ILM138, ILM142, ILM143, GLX26, GLX40, GLX166, SEX20, SEX76, and SEX180. 19. The method of claim 16, wherein the toxin having functional activity against an insect is produced by a purified culture of Xenorhabdus strain designated S. carp, x. Wi, x. nem. X. NH3, X. riobravis, GL133B, DEX1, DEX2, DEX3, DEX4, DEX5, DEX6, DEX7, DEX8, ILM037, I LM039, ILM070, ILM078, ILM079, ILM080, I LM081, ILM082, ILM083, ILM084, ILM102, ILM103, ILM104, ILM129, 1LM133, ILM135, ILM138, ILM142, ILM143, GLX26, GLX40, GLX166, SEX20, SEX76, and SEX180. The method of claim 16, wherein the toxin having functional activity against an insect is a mixture of one or more toxins produced from purified cultures of Xenorhabdus. twenty-one . The method of claim 16, wherein the insect is of the order Coleoptera, Lepidoptera, Diptera, or Acariña. The method of claim 21, wherein the insect species of the Coleoptera order are selected from the group consisting of corn rootworm, pod weevil, centipede, pollen beetles, beetles, seed beetles and potato beetles. Colorado. 23. The method of claim 21, wherein the insect species of the order Lepidoptera are selected from the group consisting of armed beetworm, European corn borer, tobacco sphincter larva, tobacco budworm, measuring caterpillar of cabbage, black night caterpillar, corn ear worm, apple worm, clothing moth, cornmeal moth, oak pitcher, cabbage worm, cotton worm, bag worm, eastern lizard, earthworm and armed worm of autumn. 24. The method of claim 21, wherein the insect species of the order Diptera are selected from the group consisting of pea ghee, carrot fly, cabbage root fly, turnip root fly, onion fly, Typically, common fly, and several species of mosquitoes. 25. The method of claim 21, wherein the species of insects of the order Acariña are selected from the group consisting of two-spotted red crests, red strawberry crests, mites, red citrus mite, European red mite, reddish the pear and red berry of the tomato. 26. A method for altering the level of toxin or toxin composition produced by strains of Xenorhabdus comprising, modifying the composition of the media. The method of claim 26, wherein said media composition is modified by fermenting said Xenorhabdus in tryptic soy broth. The method of claim 26, wherein said media composition is modified by increasing the ionic strength of said media.