Patent Grant
7071386
Insects and other pests cost farmers billions of dollars annually in crop losses and in the expense of keeping these pests under control. The losses caused by insect pests in agricultural production environments include decreases in crop yield, reduced crop quality, and increased harvesting costs. Insect pests are also a burden to vegetable and fruit growers, to producers of ornamental flowers, and to home gardeners and homeowners.
Cultivation methods, such as crop rotation and the application of high levels of nitrogen fertilizers, have partially addressed problems caused by agricultural pests. However, various demands on the utilization of farmland restrict the use of crop rotation. In addition, overwintering traits of some insects are disrupting crop rotations in some areas.
Thus, synthetic chemical insecticides are relied upon most heavily to achieve a sufficient level of control. However, the use of synthetic chemical insecticides has several drawbacks. For example, the use of these chemicals can adversely affect many beneficial insects. Target insects have also developed resistance to some chemical pesticides. Furthermore, rain and improper calibration of insecticide application equipment can result in poor control. The use of insecticides often raises environmental concerns such as contamination of soil and water supplies when not used properly, and residues can also remain on treated fruits and vegetables. Working with some insecticides can also pose hazards to the persons applying them. Stringent new restrictions on the use of pesticides and the elimination of some effective pesticides could limit effective options for controlling damaging and costly pests.
The replacement of synthetic chemical pesticides, or combination of these agents with biological pesticides, could reduce the levels of toxic chemicals in the environment. Some biological pesticidal agents that are now being used with some success are derived from the soil microbe Bacillus thuringiensis (B.t.). While most B.t. strains do not exhibit pesticidal activity, some B.t. strains produce proteins that are highly toxic to pests, such as insects, and are specific in their toxic activity. Genes that encode δ-endotoxin proteins have been isolated. Other species of Bacillus also produce pesticidal proteins.
Höfte and Whiteley classified B.t. crystal proteins into four major classes (Höfte, H., H. R. Whiteley [1989]Microbiological Reviews 52(2):242–255). The classes were CryI (Lepidoptera-specific), CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported. For example, CryV and CryVI have been proposed to designate a class of toxin genes that are nematode-specific.
The 1989 nomenclature and classification scheme of Höfte and Whiteley for crystal proteins was based on both the deduced amino acid sequence and the activity spectrum of the toxin. That system was adapted to cover 14 different types of toxin genes divided into five major classes. The 1989 nomenclature scheme became unworkable as more and more genes were discovered that encoded proteins with varying spectrums of pesticidal activity. Thus, a revised nomenclature scheme was adopted, which is based solely on amino acid identity (Crickmore et al., 1998, Microbiology and Molecular Biology Reviews 62:807–813).
Recombinant DNA-based B.t. products have been produced and approved for use. In addition, with the use of genetic engineering techniques, various approaches for delivering these toxins to agricultural environments are being perfected. These include the use of plants genetically engineered with toxin genes for insect resistance and the use of stabilized intact microbial cells as toxin delivery vehicles. Thus, isolated Bacillus toxin genes are becoming commercially valuable.
B.t. protein toxins were initially formulated as sprayable insect control agents. A relatively more recent application of B.t. technology has been to isolate and transform plants with genes that encode these toxins. Transgenic plants subsequently produce the toxins, thereby providing insect control. See U.S. Pat. Nos. 5,380,831; 5,567,600; and 5,567,862 to Mycogen Corporation. Transgenic B.t. plants are quite efficacious, and usage is predicted to be high in some crops and areas.
There are some obstacles to the successful agricultural use of Bacillus (and other biological) pesticidal proteins. Certain insects can be refractory to the effects of Bacillus toxins. Insects such as boll weevils, black cutworm, and Helicoverpa zea, as well as adult insects of most species, heretofore have demonstrated no significant sensitivity to many B.t. δ-endotoxins.
Another potential obstacle is the development of resistance to B.t. toxins by insects. The potential for wide-spread use of B.t. plants has caused some concern that resistance management issues may arise more quickly than with traditional sprayable applications. While a number of insects have been selected for resistance to B.t. toxins in the laboratory, only the diamondback moth (Plutella xylostella) has demonstrated resistance in a field setting (Ferre, J. and Van Rie, J., Annu. Rev. Entomol. 47:501–533, 2002).
Resistance management strategies in B.t. transgene plant technology have become of great interest. Several strategies have been suggested for preserving the ability to effectively use B. thuringiensis toxins. These strategies include high dose with refuge, and alternation with, or co-deployment of, different toxins (McGaughey et al. (1998), “B.t. Resistance Management,” Nature Biotechnol. 16:144–146), as in a natural bacterium, for example.
Thus, there remains a great need for developing additional genes that can be expressed in plants in order to effectively control various insects. In addition to continually trying to discover new B.t. toxins (which is becoming increasingly difficult due to the numerous B.t. toxins that have already been discovered), it would be quite desirable to discover other bacterial sources (distinct from B.t.) that produce toxins that could be used in transgenic plant strategies.
The relatively more recent efforts to clone insecticidal toxin genes from the Photorhabdus/Xenorhabdus group of bacteria present potential alternatives to toxins derived from B. thuringiensis. The genus Xenorhabdus is taxonomically defined as a member of the Family Enterobacteriaceae, although it has certain traits atypical of this family. For example, strains of this genus are typically nitrate reduction negative and catalase negative. Xenorhabdus has only recently been subdivided to create a second genus, Photorhabdus, which is comprised of the single species Photorhabdus luminescens (previously Xenorhabdus luminescens) (Boemare et al., 1993Int. J. Syst. Bacteriol. 43, 249–255). This differentiation is based on several distinguishing characteristics easily identifiable by the skilled artisan. These differences include the following: DNA-DNA characterization studies; phenotypic presence (Photorhabdus) or absence (Xenorhabdus) of catalase activity; presence (Photorhabdus) or absence (Xenorhabdus) of bioluminescence; the Family of the nematode host in that Xenorhabdus is found in Steinernematidae and Photorhabdus is found in Heterorhabditidae); as well as comparative, cellular fatty-acid analyses (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 (Rainey et al. 1995, Int. J. Syst. Bacteriol., 45, 379–381) and restriction analysis (Brunel et al., 1997, App. Environ. Micro., 63, 574–580) of 16S rRNA genes also support the separation of these two genera.
The expected traits for Xenorhabdus are the following: Gram stain negative rods, white to yellow/brown colony pigmentation, presence of inclusion bodies, absence of catalase, inability to reduce nitrate, absence of bioluminescence, ability to uptake dye from medium, positive gelatin hydrolysis, growth on Enterobacteriaceae selective media, growth temperature below 37° C., survival under anaerobic conditions, and motility.
Currently, the bacterial genus Xenorhabdus is comprised of four recognized species, Xenorhabdus nematophilus, Xenorhabdus poinarii, Xenorhabdus bovienii and Xenorhabdus beddingii (Brunel et al., 1997, App. Environ. Micro., 63, 574–580). A variety of related strains have been described in the literature (e.g., 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).
Photorhabdus and Xenorhabdus spp. are Gram-negative bacteria that entomopathogenically and symbiotically associate with soil nematodes. These bacteria are found in the gut of entomopathogenic nematodes that invade and kill insects. When the nematode invades an insect host, the bacteria are released into the insect haemocoel (the open circulatory system), and both the bacteria and the nematode undergo multiple rounds of replication; the insect host typically dies. These bacteria can be cultured away from their nematode hosts. For a more detailed discussion of these bacteria, see Forst and Nealson, 60 Microbiol. Rev. 1 (1996), pp. 21–43. Unfortunately, as reported in a number of articles, the bacteria only had pesticidal activity when injected into insect larvae and did not exhibit biological activity when delivered orally.
Xenorhabdus and Photorhabus bacteria secrete a wide variety of substances into the culture medium. See R. H. ffrench-Constant et al. 66 AEM No. 8, pp. 3310–3329 (August 2000), for a review of various factors involved in Photorhabdus virulence of insects.
It has been difficult to effectively exploit the insecticidal properties of the nematode or its bacterial symbiont. Thus, proteinaceous agents from Photorhabdus/Xenorhabdus bacteria that have oral activity are desirable so that the products produced therefrom could be formulated as a sprayable insecticide, or the genes encoding said proteinaceous agents could be isolated and used in the production of transgenic plants.
There has been substantial progress in the cloning of genes encoding insecticidal toxins from both Photorhabdus luminescens and Xenorhabdus nematophilus. Toxin-complex encoding genes from P. luminescens were examined first. See WO 98/08932. Parallel genes were more recently cloned from X. nematophilus. Morgan et al., Applied and Environmental Microbiology 2001, 67:20062–69. WO 95/00647 relates to the use of Xenorhabdus protein toxin to control insects, but it does not recognize orally active toxins. WO 98/08388 relates to orally administered pesticidal agents from Xenorhabdus. U.S. Pat. No. 6,048,838 relates to protein toxins/toxin complexes, having oral activity, obtainable from Xenorhabdus species and strains.
Four different toxin complexes (TCs)—Tca, Tcb, Tcc and Tcd—have been identified in Photorhabdus spp. Each of these toxin complexes resolves as either a single or dimeric species on a native agarose gel but resolution on a denaturing gel reveals that each complex consists of a range of species between 25–280 kDa. The ORFs that encode the typical TCs from Photorhabdus, together with protease cleavage sites (vertical arrows), are illustrated in
Genomic libraries of P. luminescens were screened with DNA probes and with monoclonal and/or polyclonal antibodies raised against the toxins. Four tc loci were cloned: tca, tcb, tcc and tcd. The tca locus is a putative operon of three open reading frames (ORFs), tcaA, tcaB, and tcaC, transcribed from the same DNA strand, with a smaller terminal ORF (tcaZ) transcribed in the opposite direction. The tcc locus also is comprised of three ORFs putatively transcribed in the same direction (tccA, tccB, and tccc). The tcb locus is a single large ORF (tcbA), and the tcd locus is composed of two ORFs (tcdA and tcdB); tcbA and tcdA, each about 7.5 kb, encode large insect toxins. TcdB has some level of homology to TcaC. It was determined that many of these gene products were cleaved by proteases. For example, both TcbA and TcdA are cleaved into three fragments termed i, ii and iii (e.g. TcbAi, TcbAii and TcbAiii). Products of the tca and tcc ORFs are also cleaved. See
Bioassays of the Tca toxin complexes revealed them to be highly toxic to first instar tomato hornworms (Manduca sexta) when given orally (LD50 of 875 ng per square centimeter of artificial diet). R. H. ffrench-Constant and Bowen 1999. Feeding was inhibited at Tca doses as low as 40 ng/cm2. Given the high predicted molecular weight of Tca, on a molar basis, P. luminescens toxins are highly active and relatively few molecules appear to be necessary to exert a toxic effect. R. H. ffrench-Constant and Bowen, Current Opinions in Micriobiology, 1999, 12:284–288.
None of the four loci showed overall similarity to any sequences of known function in GenBank. Regions of sequence similarity raised some suggestion that these proteins (TcaC and TccA) may overcome insect immunity by attacking insect hemocytes. R. H. ffrench-Constant and Bowen, Current Opinions in Microbiology, 1999, 12:284–288.
TcaB, TcbA and TcdA all show amino acid conservation (˜50% identity), compared with each other, immediately around their predicted protease cleavage sites. This conservation between three different Tc proteins suggests that they may all be processed by the same or similar proteases. TcbA and TcdA also share ˜50% identity overall, as well as a similar predicted pattern of both carboxy- and amino-terminal cleavage. It was postulated that these proteins might thus be homologs of one another. Furthermore, the similar, large size of TcbA and TcdA, and also the fact that both toxins appear to act on the gut of the insect, may suggest similar modes of action. R. H. ffrench-Constant and Bowen, Current Opinions in Microbiology, 1999, 12:284–288.
Deletion/knock-out studies suggest that products of the tca and tcd loci account for the majority of oral toxicity to lepidopterans. Deletion of either of the tca or tcd genes greatly reduced oral activity against Manduca sexta. That is, products of the tca and tcd loci are oral lepidopteran toxins on their own; their combined effect contributed most of the secreted oral activity. R. H. ffrench-Constant and D. J. Bowen, 57 Cell. Mol. Life. Sci. 831 (2000). Interestingly, deletion of either of the tcb or tcc loci alone also reduces mortality, suggesting that there may be complex interactions among the different gene products. Thus, products of the tca locus may enhance the toxicity of tcd products. Alternatively, tcd products may modulate the toxicity of tca products and possibly other complexes. Noting that the above relates to oral activity against a single insect species, tcb or tcc loci may produce toxins that are more active against other groups of insects (or active via injection directly into the insect haemocoel—the normal route of delivery when secreted by the bacteria in vivo). R. H. ffrench-Constant and Bowen, Current Opinions in Microbiology, 1999, 12:284–288.
The insect midgut epithelium contains both columnar (structural) and goblet (secretory) cells. Ingestion of tca products by M sexta leads to apical swelling and blebbing of large cytoplasmic vesicles by the columnar cells, leading to the eventual extrusion of cell nuclei in vesicles into the gut lumen. Goblet cells are also apparently affected in the same fashion. Products of tca act on the insect midgut following either oral delivery or injection. R. H. ffrench-Constant and D. J. Bowen, Current Opinions in Microbiology, 1999, 12:284–288. Purified tca products have shown oral toxicity against Manduca sexta (LD50 of 875 ng/cm2). R. H. ffrench-Constant and D. J. Bowen, 57 Cell. Mol. Life Sci. 828–833 (2000).
WO 99/42589 and U.S. Pat. No. 6,281,413 disclose TC-like ORFs from Photorhabdus luminescens. WO 00/30453 and WO 00/42855 disclose TC-like proteins from Xenorhabdus. WO 99/03328 and WO 99/54472 (and U.S. Pat. Nos. 6,174,860 and 6,277,823) relate to other toxins from Xenorhabdus and Photorhabdus.
While the exact molecular interactions of the TCs with each other, and their mechanism(s) of action, are not currently understood, it is known, for example, that the Tca toxin complex of Photorhabdus is toxic to Manduca sexta. In addition, some TC proteins are known to have “stand alone” insecticidal activity, while other TC proteins are known to potentiate or enhance the activity of the stand-alone toxins. It is known that the TcdA protein is active, alone, against Manduca sexta, but that TcdB and TccC, together, can be used (in conjunction with TcdA) to greatly enhance the activity of TcdA. TcbA is the other main, stand-alone toxin from Photorhabdus. The activity of this toxin (TcbA) can also be greatly enhanced by TcdB- together with TccC-like proteins.
Some Photorhabdus TC proteins have some level of sequence homology with other Photorhabdus TC proteins. As indicated above, TccA has some level of homology with the N terminus of TcdA, and TccB has some level of homology with the C terminus of TcdA. Furthermore, TcdA is about 280 kDa, and TccA together with TccB are of about the same size, if combined, as that of TcdA. Though TccA and TccB are much less active on SCR than TcdA, TccA and TccB from Photorhabdus strain W14 are called “Toxin D.” “Toxin A” (TcdA), “Toxin B” (Tcb or TcbA), and “Toxin C” (TcaA and TcaB) are also indicated above.
Furthermore, TcaA has some level of homology with TccA and likewise with the N terminus of TcdA. Still further, TcaB has some level of homology with TccB and likewise with the N terminus of TcdA. TcdB has a significant level of similarity to TcaC.
Relatively recent cloning efforts in Xenorhabdus nematophilus also appear to have identified novel insecticidal toxin genes with homology to the P. luminescens tc loci. See, e.g., WO 98/08388 and Morgan et al., Applied and Environmental Microbiology 2001, 67:20062–69. In R. H. ffrench-Constant and D. J. Bowen Current Opinions in Micriobiology, 1999,12:284–288, cosmid clones were screened directly for oral toxicity to another lepidopteran, Pieris brassicae. One orally toxic cosmid clone was sequenced. Analysis of the sequence in that cosmid suggested that there are five different ORF's with similarity to Photorhabdus tc genes; orf2 and orf5 both have some level of sequence relatedness to both tcbA and tcdA, whereas orf1 is similar to tccB, orf3 is similar to tccC and orf4 is similar to tca C. Importantly, a number of these predicted ORFs also share the putative cleavage site documented in P. luminescens, suggesting that active toxins may also be protealytically processed.
There are five typical Xenorhabdus TC proteins: XptA1, XptA2, XptB1, XptC1, and XptD1. XptA1 is a “stand-alone” toxin. XptA2 is the other TC protein from Xenorhabdus that has stand-alone toxin activity. XptB1 and XptC1 are the Xenorhabdus potentiators that can enhance the activity of either (or both) of the XptA toxins. XptD1 has some level of homology with TccB.
XptC1 was known to have some level of similarity to TcaC. The XptA2 protein of Xenorhabdus was known to have some degree of similarity to the TcdA protein. XptB 1 has some level of similarity to TccC.
The finding of somewhat similar, toxin-encoding loci in these two different bacteria is interesting in terms of the possible origins of these virulence genes. The X. nematophilus cosmid also appears to contain transposase-like sequences whose presence may suggest that these loci can be transferred horizontally between different strains or species of bacteria. A range of such transfer events may also explain the apparently different genomic organization of the tc operons in the two different bacteria. Further, only a subset of X. nematophilus and P. luminescens strains appear toxic to M. sexta, suggesting either that different strains lack the tc genes or that they carry a different tc gene compliment. Detailed analysis of both a strain and toxin phylogeny within, and between, these bacterial species should help clarify the likely origin of the toxin genes and how they are maintained in different bacterial populations. R. H. ffrench-Constant and Bowen, Current Opinions in Microbiology, 1999, 12:284–288.
TC proteins and genes have more recently been described from other insect-associated bacteria such as Serratia entomophila, an insect pathogen. Waterfield et al., TRENDS in Microbiology, Vol. 9, No. 4, April 2001.
In summary, toxin complex proteins from P. luminescens and X. nematophilus appear to have little homology to previously identified bacterial toxins and should provide useful alternatives to toxins derived from B. thuringiensis. Although they have similar toxic effects on the insect midgut to other orally active toxins, their precise mode of action remains obscure. Future work could clarify their mechanism of action.
Bacteria of the genus Paenibacillus are distinguishable from other bacteria by distinctive rRNA and phenotypic characteristics (C. Ash et al. (1993), “Molecular identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR probe test: Proposal for the creation of a new genus Paenibacillus,” Antonie Van Leeuwenhoek 64:253–260). Some species in this genusare known to be pathogenic to honeybees (Paenibacillus larvae) and to scarab beetle grubs (P. popilliae and P. lentimorbus.) P. larvae, P. popilliae, and P. lentimorbus are considered obligate insect pathogens involved with milky disease of scarab beetles (D. P. Stahly et al. (1992), “The genus Bacillus: insect pathogens,” p. 1697–1745, In A. Balows et al., ed., The Procaryotes, 2nd Ed., Vol. 2, Springer-Verlag, New York, N.Y.).
A crystal protein, Cry18, has been identified in strains of P. popilliae and P. lentimorbus. Cry18 has scarab and grub toxicity, and has about 40% identity to Cry2 proteins (Zhang et al., 1997; Harrison et al., 2000).
TC proteins and lepidopteran-toxic Cry proteins have very recently been discovered in Paenibacillus. See U.S. Ser. No. 60/392,633 (Bintrim et al.), filed Jun. 28, 2002.
Although some Xenorhabdus TC proteins were found to “correspond” (have a similar function and some level of sequence homology) to some of the Photorhabdus TC proteins, the “corresponding” proteins share only about 40% (approximately) sequence identity with each other. This is also true for the more recently discovered TC proteins from Paenibacillus (those proteins and that discovery are the subject of co-pending U.S. Ser. No. 60/392,633).
In light of concerns about insects developing resistance to a given pesticidal toxin, and in light of other concerns—some of which are discussed above, there is a continuing need for the discovery of new insecticidal toxins and other proteins that can be used to control insects.
The subject invention relates to novel Xenorhabdus toxin complex (TC) proteins and genes that encode these proteins. More specifically, the subject invention relates to TC proteins and genes obtainable from Xenorhabdus strain Xwi.
The subject invention also provides an exochitinase obtainable from the Xwi strain. This exochitinase can be used to control insects using methods known in the art.
SEQ ID NO:1 is the N-terminus of ToxinXwiA 220 kDa protein.
SEQ ID NO:2 is an internal peptide of ToxinXwiA purified toxin.
SEQ ID NO:3 is an internal peptide of ToxinXwiA purified toxin.
SEQ ID NO:4 is an internal peptide of ToxinXwiA purified toxin.
SEQ ID NO:5 is an internal peptide of ToxinXwiA purified toxin.
SEQ ID NO:6 is the pDAB2097 cosmid insert: 39,005 bp.
SEQ ID NO:7 is the pDAB2097 cosmid ORF1: nucleotides 1–1,533 of SEQ ID NO:6.
SEQ ID NO:8 is the pDAB2097 cosmid ORF1 deduced protein: 511 aa.
SEQ ID NO:9 is the pDAB2097 cosmid ORF2 (xptD1): nucleotides 1,543–5,715 of SEQ ID NO:6.
SEQ ID NO:10 is the pDAB2097 cosmid ORF2 deduced protein: 1,391 aa.
SEQ ID NO:11 is the pDAB2097 cosmid ORF3: nucleotides 5,764–7,707 of SEQ ID NO:6.
SEQ ID NO:12 is the pDAB2097 cosmid ORF3 deduced protein: 648 aa.
SEQ ID NO:13 is the pDAB2097 cosmid ORF4 (xptA1): nucleotides 10,709–18,277 of SEQ ID NO:6.
SEQ ID NO:14 is the pDAB2097 cosmid ORF4 deduced protein: 2,523 aa.
SEQ ID NO:15 is the pDAB2097 cosmid ORF5 (xptB1): nucleotides 18,383–21,430 (C) of SEQ ID NO:6.
SEQ ID NO:16 is the pDAB2097 cosmid ORF5 deduced protein: 1,016 aa.
SEQ ID NO:17 is the pDAB2097 cosmid ORF6 (xptC1): nucleotides 21,487–25,965 (C) of SEQ ID NO:6.
SEQ ID NO:18 is the pDAB2097 cosmid ORF6 deduced protein: 1,493 aa.
SEQ ID NO:19 is the pDAB2097 cosmid ORF7 (xptA2): nucleotides 26,021–33,634 (C) of SEQ ID NO:6.
SEQ ID NO:20 is the pDAB2097 cosmid ORF7 deduced protein: 2,538 aa.
SEQ ID NO:21 is the nucleotide sequence of the pDAB2097 cosmid insert that encodes an exochitinase.
SEQ ID NO:22 is the amino acid sequence of the exochitinase encodes by SEQ ID NO:21.
SEQ ID NO:23 is the deduced amino acid sequence from XptA2, residue numbers 0016–0034.
SEQ ID NO:24 is the deduced amino acid sequence from XptA2, residue numbers 0035–0047.
SEQ ID NO:25 is the deduced amino acid sequence from XptA2, residue numbers 0036–0047.
SEQ ID NO:26 is the deduced amino acid sequence from XptA2, residue numbers 0048–0057.
SEQ ID NO:27 is the deduced amino acid sequence from XptA2, residue numbers 0071–0080.
SEQ ID NO:28 is the deduced amino acid sequence from XptA2, residue numbers 009 1–0099.
SEQ ID NO:29 is the deduced amino acid sequence from XptA2, residue numbers 0100–0124.
SEQ ID NO:30 is the deduced amino acid sequence from XptA2, residue numbers 0128–0141.
SEQ ID NO:31 is the deduced amino acid sequence from XptA2, residue numbers 0 194–0208.
SEQ ID NO:32 is the deduced amino acid sequence from XptA2, residue numbers 0209–0223.
SEQ ID NO:33 is the deduced amino acid sequence from XptA2, residue numbers 0369–0375.
SEQ ID NO:34 is the deduced amino acid sequence from XptA2, residue numbers 0416–0420.
SEQ ID NO:35 is the deduced amino acid sequence from XptA2, residue numbers 0487–0496.
SEQ ID NO:36 is the deduced amino acid sequence from XptA2, residue numbers 0537–0558.
SEQ ID NO:37 is the deduced amino acid sequence from XptA2, residue numbers 0628–0639.
SEQ ID NO:38 is the deduced amino acid sequence from XptA2, residue numbers 0797–0813.
SEQ ID NO:39 is the deduced amino acid sequence from XptA2, residue numbers 0893–0898.
SEQ ID NO:40 is the deduced amino acid sequence from XptA2, residue numbers 0987–1000.
SEQ ID NO:41 is the deduced amino acid sequence from XptA2, residue numbers 1017–1027.
SEQ ID NO:42 is the deduced amino acid sequence from XptA2, residue numbers 1028–1036.
SEQ ID NO:43 is the deduced amino acid sequence from XptA2, residue numbers 1037–1050.
SEQ ID NO:44 is the deduced amino acid sequence from XptA2, residue numbers 1080–1092.
SEQ ID NO:45 is the deduced amino acid sequence from XptA2, residue numbers 1093–1115.
SEQ ID NO:46 is the deduced amino acid sequence from XptA2, residue numbers 1116–1124.
SEQ ID NO:47 is the deduced amino acid sequence from XptA2, residue numbers 1143–1166.
SEQ ID NO:48 is the deduced amino acid sequence from XptA2, residue numbers 1165–1179.
SEQ ID NO:49 is the deduced amino acid sequence from XptA2, residue numbers 1195–1199.
SEQ ID NO:50 is the deduced amino acid sequence from XptA2, residue numbers 1277–1284.
SEQ ID NO:51 is the deduced amino acid sequence from XptA2, residue numbers 1290–1304.
SEQ ID NO:52 is the deduced amino acid sequence from XptA2, residue numbers 1346–1363.
SEQ ID NO:53 is the deduced amino acid sequence from XptA2, residue numbers 1364–1372.
SEQ ID NO:54 is the deduced amino acid sequence from XptA2, residue numbers 1421–1437.
SEQ ID NO:55 is the deduced amino acid sequence from XptA2, residue numbers 1438–1451.
SEQ ID NO:56 is the deduced amino acid sequence from XptA2, residue numbers 1593–1605.
SEQ ID NO:57 is the deduced amino acid sequence from XptA2, residue numbers 1594–1605.
SEQ ID NO:58 is the deduced amino acid sequence from XptA2, residue numbers 1606–1620.
SEQ ID NO:59 is the deduced amino acid sequence from XptA2, residue numbers 1635–1649.
SEQ ID NO:60 is the deduced amino acid sequence from XptA2, residue numbers 1668–1677.
SEQ ID NO:61 is the deduced amino acid sequence from XptA2, residue numbers 1681–1692.
SEQ ID NO:62 is the deduced amino acid sequence from XptA2, residue numbers 1885–1890.
SEQ ID NO:63 is the deduced amino acid sequence from XptA2, residue numbers 1891–1898.
SEQ ID NO:64 is the deduced amino acid sequence from XptA2, residue numbers 1999–2003.
SEQ ID NO:65 is the deduced amino acid sequence from XptA2, residue numbers 2026–2050.
SEQ ID NO:66 is the deduced amino acid sequence from XptA2, residue numbers 2051–2057.
SEQ ID NO:67 is the deduced amino acid sequence from XptA2, residue numbers 2106–2121.
SEQ ID NO:68 is the deduced amino acid sequence from XptA2, residue numbers 2131–2145.
SEQ ID NO:69 is the deduced amino acid sequence from XptA2, residue numbers 2186–2191.
SEQ ID NO:70 is the deduced amino acid sequence from XptA2, residue numbers 2220–2228.
SEQ ID NO:71 is the deduced amino acid sequence from XptA2, residue numbers 2221–2228.
SEQ ID NO:72 is the deduced amino acid sequence from XptA2, residue numbers 2222–2228.
SEQ ID NO:73 is the deduced amino acid sequence from XptA2, residue numbers 2281–2287.
SEQ ID NO:74 is the deduced amino acid sequence from XptA2, residue numbers 2315–2325.
SEQ ID NO:75 is the deduced amino acid sequence from XptA2, residue numbers 2352–2359.
SEQ ID NO:76 is the deduced amino acid sequence from XptA2, residue numbers 2387–2392.
SEQ ID NO:77 is the deduced amino acid sequence from XptA2, residue numbers 2423–2435.
SEQ ID NO:78 is the deduced amino acid sequence from XptA2, residue numbers 2439–2455.
SEQ ID NO:79 is the deduced amino acid sequence from XptA2, residue numbers 2456–2468.
SEQ ID NO:80 is a forward primer sequence used to amplify XptA2.
SEQ ID NO:81 is a reverse primer sequence used to amplify XptA2.
SEQ ID NO:82 is a forward primer sequence used to amplify XptC1.
SEQ ID NO:83 is a reverse primer sequence used to amplify XptC1.
SEQ ID NO:84 is a forward primer sequence used to amplify XptB1.
SEQ ID NO:85 is a reverse primer sequence used to amplify XptB1.
The subject invention relates to novel Xenorhabdus toxin complex (TC) proteins and genes that encode these proteins. More specifically, the subject invention relates to TC genes and proteins obtainable from Xenorhabdus strain Xwi.
The subject invention also provides an exochitinase obtainable from the Xwi strain. This exochitinase can be used to control insects using methods known in the art. See, e.g., U.S. Pat. No. 5,173,419. The polynucleotide of SEQ ID NO:21 can be inserted into the genome of a plant so that the plant produces the protein of SEQ ID NO:22. Insects consuming the plant tissues that produce (and contain) this protein thereby contact the protein and will be controlled in this manner. The TC protein genes can be used in similar manners (i.e., expression in plants) to control insects and other like pests. Preferably, a plant is produced that expresses the XptA1 and/or XptA2 gene of SEQ ID NOs:13 and 19 so that the subject XptA1 and/or XptA2 toxin proteins of the subject invention are produced by and preferably present in the cells of the plant. The plant can be constructed to co-express the XptC1 and XptB1 genes of SEQ ID NOs:17 and 15, respectively, so that the XptC1 and XptB1 proteins potentiate or enhance the XptA1 and/or XptA2 TC protein toxins. The XptD1 gene of the subject invention can also be used, similarly, as would be known in the art.
Other methods of administering the subject proteins to insects and other pests are well known in the art. Furthermore, the subject proteins are not limited to use with each other; they can be used individually (or in combination) with other proteins, as would be known in the art.
Proteins and toxins. The present invention provides easily administered, functional proteins. The present invention also provides a method for delivering insecticidal toxins that are functionally active and effective against many orders of insects, preferably lepidopteran insects. By “functional activity” (or “active against”) it is meant herein that the protein toxins function as orally active insect control agents (alone or in combination with other proteins), that the proteins have a toxic effect (alone or in combination with other proteins), or are able to disrupt or deter insect- growth and/or feeding which may or may not cause death of the insect. When an insect comes into contact with an effective amount of a “toxin” of the subject invention delivered via transgenic plant expression, formulated protein composition(s), sprayable protein composition(s), a bait matrix or other delivery system, the results are typically death of the insect, inhibition of the growth and/or proliferation of the insect, and/or prevention of the insects from feeding upon the source (preferably a transgenic plant) that makes the toxins available to the insects. Functional proteins of the subject invention can also work together or alone to enhance or improve the activity of one or more other toxin proteins. The terms “toxic,” “toxicity,” or “toxin” as used herein are meant to convey that the subject “toxins” have “functional activity” as defined herein.
Complete lethality to feeding insects is preferred but is not required to achieve functional activity. If an insect avoids the toxin or ceases feeding, that avoidance will be useful in some applications, even if the effects are sublethal or lethality is delayed or indirect. For example, if insect resistant transgenic plants are desired, the reluctance of insects to feed on the plants is as useful as lethal toxicity to the insects because the ultimate objective is avoiding insect-induced plant damage.
There are many other ways in which toxins can be incorporated into an insect's diet. For example, it is possible to adulterate the larval food source with the toxic protein by spraying the food with a protein solution, as disclosed herein. Alternatively, the purified protein could be genetically engineered into an otherwise harmless bacterium, which could then be grown in culture, and either applied to the food source or allowed to reside in the soil in an area in which insect eradication was desirable. Also, the protein could be genetically engineered directly into an insect food source. For instance, the major food source for many insect larvae is plant material. Therefore the genes encoding toxins can be transferred to plant material so that said plant material expresses the toxin of interest.
Transfer of the functional activity to plant or bacterial systems typically requires nucleic acid sequences, encoding the amino acid sequences for the toxins, integrated into a protein expression vector appropriate to the host in which the vector will reside. One way to obtain a nucleic acid sequence encoding a protein with functional activity is to isolate the native genetic material from the bacterial species which produce the toxins, using information deduced from the toxin's amino acid sequence, as disclosed herein. The native sequences can be optimized for expression in plants, for example, as discussed in more detail below. Optimized polynucleotide can also be designed based on the protein sequence.
The subject invention provides new classes of toxins having advantageous pesticidal activities. One way to characterize these classes of toxins and the polynucleotides that encode them is by defining a polynucleotide by its ability to hybridize, under a range of specified conditions, with an exemplified nucleotide sequence (the complement thereof and/or a probe or probes derived from either strand) and/or by their ability to be amplified by PCR using primers derived from the exemplified sequences.
There are a number of methods for obtaining the pesticidal toxins of the instant invention.
For example, antibodies to the pesticidal toxins disclosed and claimed herein can be used to identify and isolate other toxins from a mixture of proteins. Specifically, antibodies may be raised to the portions of the toxins which are most constant and most distinct from other toxins. These antibodies can then be used to specifically identify equivalent toxins with the characteristic activity by immunoprecipitation, enzyme linked immunosorbent assay (ELISA), or western blotting. Antibodies to the toxins disclosed herein, or to equivalent toxins, or to fragments of these toxins, can be readily prepared using standard procedures. Toxins of the subject invention can be obtained from a variety of sources/source microorganisms.
One skilled in the art would readily recognize that toxins (and genes) of the subject invention can be obtained from a variety of sources. A toxin “from” or “obtainable from” the subject Xwi isolate means that the toxin (or a similar toxin) can be obtained from Xwi or some other source, such as another bacterial strain or a plant. For example, one skilled in the art will readily recognize that, given the disclosure of a bacterial gene and toxin, a plant can be engineered to produce the toxin. Antibody preparations, nucleic acid probes (DNA and RNA), and the like may be prepared using the polynucleotide and/or amino acid sequences disclosed herein and used to screen and recover other toxin genes from other (natural) sources.
Polynucleotides and probes. The subject invention further provides nucleotide sequences that encode the toxins of the subject invention. The subject invention further provides methods of identifying and characterizing genes that encode pesticidal toxins. In one embodiment, the subject invention provides unique nucleotide sequences that are useful as hybridization probes and/or primers for PCR techniques. The primers produce characteristic gene fragments that can be used in the identification, characterization, and/or isolation of specific toxin genes. The nucleotide sequences of the subject invention encode toxins that are distinct from previously described toxins.
The polynucleotides of the subject invention can be used to form complete “genes” to encode proteins or peptides in a desired host cell. For example, as the skilled artisan would readily recognize, the subject polynucleotides can be appropriately placed under the control of a promoter in a host of interest, as is readily known in the art.
As the skilled artisan knows, DNA typically exists in a double-stranded form. In this arrangement, one strand is complementary to the other strand and vice versa. As DNA is replicated in a plant (for example), additional complementary strands of DNA are produced. The “coding strand” is often used in the art to refer to the strand that binds with the anti-sense strand. The mRNA is transcribed from the “anti-sense” strand of DNA. The “sense” or “coding” strand has a series of codons (a codon is three nucleotides that can be read as a three-residue unit to specify a particular amino acid) that can be read as an open reading frame (ORF) to form a protein or peptide of interest. In order to produce a protein in vivo, a strand of DNA is typically transcribed into a complementary strand of mRNA which is used as the template for the protein. Thus, the subject invention includes the use of the exemplified polynucleotides shown in the attached sequence listing and/or equivalents including the complementary strands. RNA and PNA (peptide nucleic acids) that are functionally equivalent to the exemplified DNA are included in the subject invention.
In one embodiment of the subject invention, bacterial isolates can be cultivated under conditions resulting in high multiplication of the microbe. After treating the microbe to provide single-stranded genomic nucleic acid, the DNA can be contacted with the primers of the invention and subjected to PCR amplification. Characteristic fragments of toxin-encoding genes will be amplified by the procedure, thus identifying the presence of the toxin-encoding gene(s).
Further aspects of the subject invention include genes and isolates identified using the methods and nucleotide sequences disclosed herein. The genes thus identified encode toxins active against pests.
Toxins and genes of the subject invention can be identified and obtained by using oligonucleotide probes, for example. These probes are detectable nucleotide sequences which may be detectable by virtue of an appropriate label or may be made inherently fluorescent as described in International Application No. WO 93/16094. The probes (and the polynucleotides of the subject invention) may be DNA, RNA, or PNA. In addition to adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U; for RNA molecules), synthetic probes (and polynucleotides) of the subject invention can also have inosine (a neutral base capable of pairing with all four bases; sometimes used in place of a mixture of all four bases in synthetic probes). Thus, where a synthetic, degenerate oligonucleotide is referred to herein, and “n” is used generically, “n” can be G, A, T, C, or inosine. Ambiguity codes as used herein are in accordance with standard IUPAC naming conventions as of the filing of the subject application (for example, R means A or G, Y means C or T, etc.).
As is well known in the art, if a probe molecule hybridizes with a nucleic acid sample,it can be reasonably assumed that the probe and sample have substantial homology/similarity/identity. Preferably, hybridization of the polynucleotide is first conducted followed by washes under conditions of low, moderate, or high stringency by techniques well-known in the art, as described in, for example, Keller, G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169–170. For example, as stated therein, low stringency conditions can be achieved by first washing with 2×SSC (Standard Saline Citrate)/0.1% SDS (Sodium Dodecyl Sulfate) for 15 minutes at room temperature. Two washes are typically performed. Higher stringency can then be achieved by lowering the salt concentration and/or by raising the temperature. For example, the wash described above can be followed by two washings with 0.1×SSC/0. 1% SDS for 15 minutes each at room temperature followed by subsequent washes with 0.1×SSC/0.1% SDS for 30 minutes each at 55° C. These temperatures can be used with other hybridization and wash protocols set forth herein and as would be known to one skilled in the art (SSPE can be used as the salt instead of SSC, for example). The 2×SSC/0.1% SDS can be prepared by adding 50 ml of 20×SSC and 5 ml of 10% SDS to 445 ml of water. 20×SSC can be prepared by combining NaCl (175.3 g/0.150 M), sodium citrate (88.2 g/0.015 M), and water to 1 liter, followed by adjusting pH to 7.0 with 10 N NaOH. 10% SDS can be prepared by dissolving 10 g of SDS in 50 ml of autoclaved water, diluting to 100 ml, and aliquotting.
Detection of the probe provides a means for determining in a known manner whether hybridization has been maintained. Such a probe analysis provides a rapid method for identifying toxin-encoding genes of the subject invention. The nucleotide segments which are used as probes according to the invention can be synthesized using a DNA synthesizer and standard procedures. These nucleotide sequences can also be used as PCR primers to amplify genes of the subject invention.
Hybridization characteristics of a molecule can be used to define polynucleotides of the subject invention. Thus the subject invention includes polynucleotides (and/or their complements, preferably their full complements) that hybridize with a polynucleotide exemplified herein.
As used herein “stringent” conditions for hybridization refers to conditions which achieve the same, or about the same, degree of specificity of hybridization as the conditions employed by the current applicants. Specifically, hybridization of immobilized DNA on Southern blots with 32P-labeled gene-specific probes was performed by standard methods (see, e.g., Maniatis, T., E. F. Fritsch, J. Sambrook [1982] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). In general, hybridization and subsequent washes were carried out under conditions that allowed for detection of target sequences. For double-stranded DNA gene probes, hybridization was carried out overnight at 20–25° C. below the melting temperature (Tm) of the DNA hybrid in 6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is described by the following formula (Beltz, G. A., K. A. Jacobs, T. H. Eickbush, P. T. Cherbas, and F. C. Kafatos [1983] Methods of Enzymology, R. Wu, L. Grossman and K. Moldave [eds.] Academic Press, New York 100:266–285):
Tm=81.5° C.+16.6 Log[Na+]+0.41(% G+C)−0.61(% formamide)−600/length of duplex in base pairs.
Washes are typically carried out as follows:
(1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (low stringency wash).
(2) Once at Tm-20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS (moderate stringency wash).
For oligonucleotide probes, hybridization was carried out overnight at 10–20° C. below the melting temperature (Tm) of the hybrid in 6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm for oligonucleotide probes was determined by the following formula:
Tm(° C.)=2(number T/A base pairs)+4(number G/C base pairs)
(Suggs, S. V., T. Miyake, E. H. Kawashime, M. J. Johnson, K. Itakura, and R. B. Wallace [1981] ICA-UCLA Symp. Dev. Biol. Using Purified Genes, D. D. Brown [ed.], Academic Press, New York, 23:683–693).
Washes were typically carried out as follows:
(1) Twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS (low stringency wash).
(2) Once at the hybridization temperature for 15 minutes in 1×SSPE, 0.1% SDS (moderate stringency wash).
In general, salt and/or temperature can be altered to change stringency. With a labeled DNA fragment >70 or so bases in length, the following conditions can be used:
Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid, and, as noted above, a certain degree of mismatch can be tolerated. Therefore, the probe sequences of the subject invention include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence in many ways, and these methods are known to an ordinarily skilled artisan. Other methods may become known in the future.
PCR technology. Polymerase Chain Reaction (PCR) is a repetitive, enzymatic, primed synthesis of a nucleic acid sequence. This procedure is well known and commonly used by those skilled in this art (see Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki, Randall K., Stephen Scharf, Fred Faloona, Kary B. Mullis, Glenn T. Horn, Henry A. Erlich, Norman Arnheim [1985] “Enzymatic Amplification of β-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia,” Science 230:1350–1354). PCR is based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize to opposite strands of the target sequence. The primers are oriented with the 3′ ends pointing towards each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences, and extension of the annealed primers with a DNA polymerase result in the amplification of the segment defined by the 5′ ends of the PCR primers. The extension product of each primer can serve as a template for the other primer, so each cycle essentially doubles the amount of DNA fragment produced in the previous cycle. This results in the exponential accumulation of the specific target fragment, up to several million-fold in a few hours. By using a thermostable DNA polymerase such as Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus, the amplification process can be completely automated. Other enzymes which can be used are known to those skilled in the art.
The DNA sequences of the subject invention can be used as primers for PCR amplification. In performing PCR amplification, a certain degree of mismatch can be tolerated between primer and template. Therefore, mutations, deletions, and insertions (especially additions of nucleotides to the 5′ end) of the exemplified primers fall within the scope of the subject invention. Mutations, insertions, and deletions can be produced in a given primer by methods known to an ordinarily skilled artisan.
Modification of genes and toxins. The genes and toxins useful according to the subject invention include not only the specifically exemplified full-length sequences, but also portions, segments and/or fragments (including internal and/or terminal deletions compared to the full-length molecules) of these sequences, variants, mutants, chimerics, and fusions thereof. Proteins of the subject invention can have substituted amino acids so long as they retain the characteristic pesticidal/functional activity of the proteins specifically exemplified herein. “Variant” genes have nucleotide sequences that encode the same toxins or equivalent toxins having pesticidal activity equivalent to an exemplified protein. The terms “variant proteins” and “equivalent toxins” refer to toxins having the same or essentially the same biological/functional activity against the target pests and equivalent sequences as the exemplified toxins. As used herein, reference to an “equivalent” sequence refers to sequences having amino acid substitutions, deletions, additions, or insertions which improve or do not adversely affect pesticidal activity. Fragments retaining pesticidal activity are also included in this definition. Fragments and other equivalents that retain the same or similar function, or “toxin activity,” as a corresponding fragment of an exemplified toxin are within the scope of the subject invention. Changes, such as amino acid substitutions or additions, can be made for a variety of purposes, such as increasing (or decreasing) protease stability of the protein (without materially/substantially decreasing the functional activity of the toxin).
Equivalent toxins and/or genes encoding these equivalent toxins can be obtained/derived from wild-type or recombinant bacteria and/or from other wild-type or recombinant organisms using the teachings provided herein. Other Bacillus, Paenibacillus, Photorhabdus, and Xenorhabdus species, for example, can be used as source isolates.
Variations of genes may be readily constructed using standard techniques for making point mutations, for example. In addition, U.S. Pat. No.5,605,793, for example, describes methods for generating additional molecular diversity by using DNA reassembly after random fragmentation. Variant genes can be used to produce variant proteins; recombinant hosts can be used to produce the variant proteins. Using these “gene shuffling” techniques, equivalent genes and proteins can be constructed that comprise any 5, 10, or 20 contiguous residues (amino acid or nucleotide) of any sequence exemplified herein. As one skilled in the art knows, the gene shuffling techniques can be adjusted to obtain equivalents having, for example, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409,410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445; 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 contiguous residues (amino acid or nucleotide), corresponding to a segment (of the same size) in any of the exemplified sequences (or the complements (full complements) thereof). Similarly sized segments, especially those for conserved regions, can also be used as probes and/or primers.
Fragments of full-length genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Also, genes which encode active fragments may be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of these toxins.
It is within the scope of the invention as disclosed herein that toxins may be truncated and still retain functional activity. By “truncated toxin” is meant that a portion of a toxin protein may be cleaved and yet still exhibit activity after cleavage. Cleavage can be achieved by proteases inside or outside of the insect gut. Furthermore, effectively cleaved proteins can be produced using molecular biology techniques wherein the DNA bases encoding said toxin are removed either through digestion with restriction endonucleases or other techniques available to the skilled artisan. After truncation, said proteins can be expressed in heterologous systems such as E. coli, baculoviruses, plant-based viral systems, yeast and the like and then placed in insect assays as disclosed herein to determine activity. It is well-known in the art that truncated toxins can be successfully produced so that they retain functional activity while having less than the entire, full-length sequence. It is well known in the art that B.t. toxins can be used in a truncated (core toxin) form. See, e.g., Adang et al., Gene 36:289–300 (1985), “Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp kurstaki HD-73 and their toxicity to Manduca sexta.” There are other examples of truncated proteins that retain insecticidal activity, including the insect juvenile hormone esterase (U.S. Pat. No.5,674,485 to the Regents of the University of California). As used herein, the term “toxin” is also meant to include functionally active truncations.
Certain toxins of the subject invention have been specifically exemplified herein. As these toxins are merely exemplary of the toxins of the subject invention, it should be readily apparent that the subject invention comprises variant or equivalent toxins (and nucleotide sequences coding for equivalent toxins) having the same or similar pesticidal activity of the exemplified toxin. Equivalent toxins will have amino acid similarity (and/or homology) with an exemplified toxin. The amino acid identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and can be greater than 95%. Preferred polynucleotides and proteins of the subject invention can also be defined in terms of more particular identity and/or similarity ranges. For example, the identity and/or similarity can be 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequence exemplified herein.
Unless otherwise specified, as used herein percent sequence identity and/or similarity of two nucleic acids is determined using the algorithm of Karlin and Altschul (1990), Proc. Natl. Acad. Sci. USA 87:2264–2268, modified as in Karlin and Altschul (1993), Proc. Natl. Acad. Sci. USA 90:5873–5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990), J. Mol. Biol. 215:402–410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12. To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997), Nucl. Acids Res. 25:3389–3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) are used. See NCBI/NIH website. The scores can also be calculated using the methods and algorithms of Crickmore et al. as described in the Background section, above.
The amino acid homology/similarity/identity will be highest in critical regions of the toxin which account for biological activity or are involved in the determination of three-dimensional configuration which is ultimately responsible for the biological activity. In this regard, certain amino acid substitutions are acceptable and can be expected to be tolerated. For example, these substitutions can be in regions of the protein that are not critical to activity. Analyzing the crystal structure of a protein, and software-based protein structure modeling, can be used to identify regions of a protein that can be modified (using site-directed mutagenesis, shuffling, etc.) to actually change the properties and/or increase the functionality of the protein.
Various properties and three-dimensional features of the protein can also be changed without adversely affecting the toxin activity/functionality of the protein. Conservative amino acid substitutions can be expected to be tolerated/to not adversely affect the three-dimensional configuration of the molecule. Amino acids can be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution is not adverse to the biological activity of the compound. Table 1 provides a listing of examples of amino acids belonging to each class.
In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutions must not significantly detract from the functional/biological activity of the toxin.
As used herein, reference to “isolated” polynucleotides and/or “purified” toxins refers to these molecules when they are not associated with the other molecules with which they would be found in nature. Thus, reference to “isolated” and/or “purified” signifies the involvement of the “hand of man” as described herein. For example, a bacterial toxin “gene” of the subject invention put into a plant for expression is an “isolated polynucleotide.” Likewise, a Xenorhabdus protein, exemplified herein, produced by a plant is an “isolated protein.”
Because of the degeneracy/redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequences disclosed herein. It is well within the skill of a person trained in the art to create alternative DNA sequences that encode the same, or essentially the same, toxins. These variant DNA sequences are within the scope of the subject invention.
Optimization of sequence for expression in plants. To obtain high expression of heterologous genes in plants it may be preferred to reengineer said genes so that they are more efficiently expressed in (the cytoplasm of) plant cells. Maize is one such plant where it may be preferred to re-design the heterologous gene(s) prior to transformation to increase the expression level thereof in said plant. Therefore, an additional step in the design of genes encoding a bacterial toxin is reengineering of a heterologous gene for optimal expression.
One reason for the reengineering of a bacterial toxin for expression in maize is due to the non-optimal G+C content of the native gene. For example, the very low G+C content of many native bacterial gene(s) (and consequent skewing towards high A+T content) results in the generation of sequences mimicking or duplicating plant gene control sequences that are known to be highly A+T rich. The presence of some A+T-rich sequences within the DNA of gene(s) introduced into plants (e.g., TATA box regions normally found in gene promoters) may result in aberrant transcription of the gene(s). On the other hand, the presence of other regulatory sequences residing in the transcribed mRNA (e.g., polyadenylation signal sequences (AAUAAA), or sequences complementary to small nuclear RNAs involved in pre-mRNA splicing) may lead to RNA instability. Therefore, one goal in the design of genes encoding a bacterial toxin for maize expression, more preferably referred to as plant optimized gene(s), is to generate a DNA sequence having a higher G+C content, and preferably one close to that of maize genes coding for metabolic enzymes. Another goal in the design of the plant optimized gene(s) encoding a bacterial toxin is to generate a DNA sequence in which the sequence modifications do not hinder translation.
The table below (Table 2) illustrates how high the G+C content is in maize. For the data in Table 2, coding regions of the genes were extracted from GenBank (Release 71) entries, and base compositions were calculated using the MacVector.™ program (IBI, New Haven, Conn.). Intron sequences were ignored in the calculations.
Due to the plasticity afforded by the redundancy/degeneracy of the genetic code (i.e., some amino acids are specified by more than one codon), evolution of the genomes in different organisms or classes of organisms has resulted in differential usage of redundant codons. This “codon bias” is reflected in the mean base composition of protein coding regions. For example, organisms with relatively low G+C contents utilize codons having A or T in the third position of redundant codons, whereas those having higher G+C contents utilize codons having G or C in the third position. It is thought that the presence of “minor” codons within a mRNA may reduce the absolute translation rate of that mRNA, especially when the relative abundance of the charged tRNA corresponding to the minor codon is low. An extension of this is that the diminution of translation rate by individual minor codons would be at least additive for multiple minor codons. Therefore, mRNAs having high relative contents of minor codons would have correspondingly low translation rates. This rate would be reflected by subsequent low levels of the encoded protein.
In engineering genes encoding a bacterial toxin for maize (or other plant, such as cotton or soybean) expression, the codon bias of the plant has been determined. The codon bias for maize is the statistical codon distribution that the plant uses for coding its proteins and the preferred codon usage is shown in Table 3. After determining the bias, the percent frequency of the codons in the gene(s) of interest is determined. The primary codons preferred by the plant should be determined as well as the second and third choice of preferred codons. Afterwards, the amino acid sequence of the bacterial toxin of interest is reverse translated so that the resulting nucleic acid sequence codes for exactly the same protein as the native gene wanting to be heterologously expressed. The new DNA sequence is designed using codon bias information so that it corresponds to the most preferred codons of the desired plant. The new sequence is then analyzed for restriction enzyme sites that might have been created by the modification. The identified sites are further modified by replacing the codons with second or third choice preferred codons. Other sites in the sequence which could affect transcription or translation of the gene of interest are the exon intronjunctions (5′ or 3′), poly A addition signals, or RNA polymerase termination signals. The sequence is further analyzed and modified to reduce the frequency of TA or GC doublets. In addition to the doublets, G or C sequence blocks that have more than about four residues that are the same can affect transcription of the sequence. Therefore, these blocks are also modified by replacing the codons of first or second choice, etc. with the next preferred codon of choice.
It is preferred that the plant optimized gene(s) encoding a bacterial toxin contain about 63% of first choice codons, between about 22% to about 37% second choice codons, and between about 15% to about 0% third choice codons, wherein the total percentage is 100%. Most preferred the plant optimized gene(s) contains about 63% of first choice codons, at least about 22% second choice codons, about 7.5% third choice codons, and about 7.5% fourth choice codons, wherein the total percentage is 100%. The preferred codon usage for engineering genes for maize expression are shown in Table 3. The method described above enables one skilled in the art to modify gene(s) that are foreign to a particular plant so that the genes are optimally expressed in plants. The method is further illustrated in PCT application WO 97/13402.
In order to design plant optimized genes encoding a bacterial toxin, the amino acid sequence of said protein is reverse translated into a DNA sequence utilizing a non-redundant genetic code established from a codon bias table compiled for the gene sequences for the particular plant, as shown in Table 2. The resulting DNA sequence, which is completely homogeneous in codon usage, is further modified to establish a DNA sequence that, besides having a higher degree of codon diversity, also contains strategically placed restriction enzyme recognition sites, desirable base composition, and a lack of sequences that might interfere with transcription of the gene, or translation of the product mRNA.
Thus, synthetic genes that are functionally equivalent to the toxins/genes of the subject invention can be used to transform hosts, including plants. Additional guidance regarding the production of synthetic genes can be found in, for example, U.S. Pat. No. 5,380,831.
In some cases, especially for expression in plants, it can be advantageous to use truncated genes that express truncated proteins. Höfte et al. 1989, for example, discussed in the Background Section above, discussed protoxin and core toxin segments of B.t. toxins. Preferred truncated genes will typically encode 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the full-length toxin.
Transgenic hosts. The toxin-encoding genes of the subject invention can be introduced into a wide variety of microbial or plant hosts. In preferred embodiments, transgenic plant cells and plants are used. Preferred plants (and plant cells) are corn, maize, and cotton.
In preferred embodiments, expression of the toxin gene results, directly or indirectly, in the intracellular production (and maintenance) of the pesticide proteins. Plants can be rendered insect-resistant in this manner. When transgenic/recombinant/transformed/transfected host cells (or contents thereof) are ingested by the pests, the pests will ingest the toxin. This is the preferred manner in which to cause contact of the pest with the toxin. The result is control (killing or making sick) of the pest. Sucking pests can also be controlled in a similar manner. Alternatively, suitable microbial hosts, e.g., Pseudomonas such as P. fluorescens, can be applied where target pests are present; the microbes can proliferate there, and are ingested by the target pests. The microbe hosting the toxin gene can be treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell, which retains the toxic activity, can then be applied to the environment of the target pest.
Where the toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, certain host microbes should be used. Microorganism hosts are selected which are known to occupy the “phytosphere” (phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.
A large number of microorganisms are known to inhabit the phylloplane (the surface of the plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., genera Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobirum melioti, Alcaligenes entrophus, and Azotobacter vinlandii; 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. Also of interest are pigmented microorganisms.
Insertion of genes to form transgenic hosts. One aspect of the subject invention is the transformation/transfection of plants, plant cells, and other host cells with polynucleotides of the subject invention that express proteins of the subject invention. Plants transformed in this manner can be rendered resistant to attack by the target pest(s).
A wide variety of methods are available for introducing a gene encoding a pesticidal protein into the target host under conditions that allow for stable maintenance and expression of the gene. These methods are well known to those skilled in the art and are described, for example, in U.S. Pat. No. 5,135,867.
For example, a large number of cloning vectors comprising a replication system in E. coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, the sequence encoding the toxin can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli. The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted. The use of T-DNA for the transformation of plant cells has been intensively researched and described in EP 120 516; Hoekema (1985) In: The Binary Plant Vector System, Offset-durkkerij Kanters B. V., Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci. 4:1–46; and An et al. (1985) EMBO J. 4:277–287.
A large number of techniques are available for inserting DNA into a plant host cell. Those techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistics (microparticle bombardment), or electroporation as well as other possible methods. If Agrobacteria are used for the transformation, the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the T-DNA. The Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria. The intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid (conjugation). Binary vectors can replicate themselves both in E. coli and in Agrobacteria. They comprise a selection marker gene and a linker or polylinker which are framed by the right and left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et al. [1978] Mol. Gen. Genet. 163:181–187). The Agrobacterium used as host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained. The bacterium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension-cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection. The plants so obtained can then be tested for the presence of the inserted DNA. No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives
The transformed cells grow inside the plants in the usual manner. They can form germ cells and transmit the transformed trait(s) to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.
In some preferred embodiments of the invention, genes encoding the bacterial toxin are expressed from transcriptional units inserted into the plant genome. Preferably, said transcriptional units are recombinant vectors capable of stable integration into the plant genome and enable selection of transformed plant lines expressing mRNA encoding the proteins.
Once the inserted DNA has been integrated in the genome, it is relatively stable there (and does not come out again). It normally contains a selection marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as kanamycin, G418, bleomycin, hygromycin, or chloramphenicol, inter alia. The individually employed marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted DNA. The gene(s) of interest are preferably expressed either by constitutive or inducible promoters in the plant cell. Once expressed, the mRNA is translated into proteins, thereby incorporating amino acids of interest into protein. The genes encoding a toxin expressed in the plant cells can be under the control of a constitutive promoter, a tissue-specific promoter, or an inducible promoter.
Several techniques exist for introducing foreign recombinant vectors into plant cells, and for obtaining plants that stably maintain and express the introduced gene. Such techniques include the introduction of genetic material coated onto microparticles directly into cells (U.S. Pat. Nos. 4,945,050 to Cornell and U.S. Pat. No. 5,141,131 to DowElanco, now Dow AgroSciences, LLC). In addition, plants may be transformed using Agrobacterium technology, see U.S. Pat. No. 5,177,010 to University of Toledo; U.S. Pat. No. 5,104,310 to Texas A&M; European Patent Application 0131624B1; European Patent Applications 120516, 159418B1 and 176,112 to Schilperoot; U.S. Pat. Nos. 5,149,645, 5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot; European Patent Applications 116718, 290799, 320500 all to Max Planck; European Patent Applications 604662 and 627752, and U.S. Pat. No. 5,591,616, to Japan Tobacco; European Patent Applications 0267159 and 0292435, and U.S. Pat. No. 5,231,019, all to Ciba Geigy, now Novartis; U.S. Pat. Nos. 5,463,174 and 4,762,785, both to Calgene; and U.S. Pat. Nos. 5,004,863 and 5,159,135, both to Agracetus. Other transformation technology includes whiskers technology. See U.S. Pat. Nos. 5,302,523 and 5,464,765, both to Zeneca. Electroporation technology has also been used to transform plants. See WO 87/06614 to Boyce Thompson Institute; U.S. Pat. Nos. 5,472,869 and 5,384,253, both to Dekalb; and WO 92/09696 and WO 93/21335, both to Plant Genetic Systems. Furthermore, viral vectors can also be used to produce transgenic plants expressing the protein of interest. For example, monocotyledonous plant can be transformed with a viral vector using the methods described in U.S. Pat. Nos. 5,569,597 to Mycogen Plant Science and Ciba-Giegy, now Novartis, as well as U.S. Pat. Nos. 5,589,367 and 5,316,931, both to Biosource.
As mentioned previously, the manner in which the DNA construct is introduced into the plant host is not critical to this invention. Any method which provides for efficient transformation may be employed. For example, various methods for plant cell transformation are described herein and include the use of Ti or Ri-plasmids and the like to perform Agrobacterium mediated transformation. In many instances, it will be desirable to have the construct used for transformation bordered on one or both sides by T-DNA borders, more specifically the right border. This is particularly useful when the construct uses Agrobacterium tumefaciens or Agrobacterium rhizogenes as a mode for transformation, although T-DNA borders may find use with other modes of transformation. Where Agrobacterium is used for plant cell transformation, a vector may be used which may be introduced into the host for homologous recombination with T-DNA or the Ti or Ri plasmid present in the host. Introduction of the vector may be performed via electroporation, tri-parental mating and other techniques for transforming gram-negative bacteria which are known to those skilled in the art. The manner of vector transformation into the Agrobacterium host is not critical to this invention. The Ti or Ri plasmid containing the T-DNA for recombination may be capable or incapable of causing gall formation, and is not critical to said invention so long as the vir genes are present in said host.
In some cases where Agrobacterium is used for transformation, the expression construct being within the T-DNA borders will be inserted into a broad spectrum vector such as pRK2 or derivatives thereof as described in Ditta et al., (PNAS USA (1980) 77:7347–7351 and EPO 0 120 515, which are incorporated herein by reference. Included within the expression construct and the T-DNA will be one or more markers as described herein which allow for selection of transformed Agrobacterium and transformed plant cells. The particular marker employed is not essential to this invention, with the preferred marker depending on the host and construction used.
For transformation of plant cells using Agrobacterium, explants may be combined and incubated with the transformed Agrobacterium for sufficient time to allow transformation thereof. After transformation, the Agrobacteria are killed by selection with the appropriate antibiotic and plant cells are cultured with the appropriate selective medium. Once calli are formed, shoot formation can be encouraged by employing the appropriate plant hormones according to methods well known in the art of plant tissue culturing and plant regeneration. However, a callus intermediate stage is not always necessary. After shoot formation, said plant cells can be transferred to medium which encourages root formation thereby completing plant regeneration. The plants may then be grown to seed and said seed can be used to establish future generations. Regardless of transformation technique, the gene encoding a bacterial toxin is preferably incorporated into a gene transfer vector adapted to express said gene in a plant cell by including in the vector a plant promoter regulatory element, as well as 3′ non-translated transcriptional termination regions such as Nos and the like.
In addition to numerous technologies for transforming plants, the type of tissue which is contacted with the foreign genes may vary as well. Such tissue would include but would not be limited to embryogenic tissue, callus tissue types I, II, and III, hypocotyl, meristem, root tissue, tissues for expression in phloem, and the like. Almost all plant tissues may be transformed during dedifferentiation using appropriate techniques described herein.
As mentioned above, a variety of selectable markers can be used, if desired. Preference for a particular marker is at the discretion of the artisan, but any of the following selectable markers may be used along with any other gene not listed herein which could function as a selectable marker. Such selectable markers include but are not limited to aminoglycoside phosphotransferase gene of transposon Tn5 (Aph II) which encodes resistance to the antibiotics kanamycin, neomycin and G418, as well as those genes which encode for resistance or tolerance to glyphosate; hygromycin; methotrexate; phosphinothricin (bialaphos); imidazolinones, sulfonylureas and triazolopyrimidine herbicides, such as chlorsulfuron; bromoxynil, dalapon and the like.
In addition to a selectable marker, it may be desirous to use a reporter gene. In some instances a reporter gene may be used with or without a selectable marker. Reporter genes are genes which are typically not present in the recipient organism or tissue and typically encode for proteins resulting in some phenotypic change or enzymatic property. Examples of such genes are provided in K. Wising et al. Ann. Rev. Genetics, 22, 421 (1988). Preferred reporter genes include the beta-glucuronidase (GUS) of the uidA locus of E. coli, the chloramphenicol acetyl transferase gene from Tn9 of E. coli, the green fluorescent protein from the bioluminescent jellyfish Aequorea victoria, and the luciferase genes from firefly Photinus pyralis. An assay for detecting reporter gene expression may then be performed at a suitable time after said gene has been introduced into recipient cells. A preferred such assay entails the use of the gene encoding beta-glucuronidase (GUS) of the uidA locus of E. coli as described by Jefferson et al., (1987 Biochem. Soc. Trans. 15, 17–19) to identify transformed cells.
In addition to plant promoter regulatory elements, promoter regulatory elements from a variety of sources can be used efficiently in plant cells to express foreign genes. For example, promoter regulatory elements of bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, the mannopine synthase promoter; promoters of viral origin, such as the cauliflower mosaic virus (35S and 19S), 35T (which is a re-engineered 35S promoter, see U.S. Pat. No. 6,166,302, especially Example 7E) and the like may be used. Plant promoter regulatory elements include but are not limited to ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu), beta-conglycinin promoter, beta-phaseolin promoter, ADH promoter, heat-shock promoters, and tissue specific promoters. Other elements such as matrix attachment regions, scaffold attachment regions, introns, enhancers, polyadenylation sequences and the like may be present and thus may improve the transcription efficiency or DNA integration. Such elements may or may not be necessary for DNA function, although they can provide better expression or functioning of the DNA by affecting transcription, mRNA stability, and the like. Such elements may be included in the DNA as desired to obtain optimal performance of the transformed DNA in the plant. Typical elements include but are not limited to Adh-intron 1, Adh-intron 6, the alfalfa mosaic virus coat protein leader sequence, the maize streak virus coat protein leader sequence, as well as others available to a skilled artisan. Constitutive promoter regulatory elements may also be used thereby directing continuous gene expression in all cells types and at all times (e.g., actin, ubiquitin, CaMV 35S, and the like). Tissue specific promoter regulatory elements are responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (e.g., zein, oleosin, napin, ACP, globulin and the like) and these may also be used.
Promoter regulatory elements may also be active during a certain stage of the plant's development as well as active in plant tissues and organs. Examples of such include but are not limited to pollen-specific, embryo-specific, corn-silk-specific, cotton-fiber-specific, root-specific, seed-endosperm-specific promoter regulatory elements and the like. Under certain circumstances it may be desirable to use an inducible promoter regulatory element, which is responsible for expression of genes in response to a specific signal, such as: physical stimulus (heat shock genes), light (RUBP carboxylase), hormone (Em), metabolites, chemical, and stress. Other desirable transcription and translation elements that function in plants may be used. Numerous plant-specific gene transfer vectors are known in the art.
Standard molecular biology techniques may be used to clone and sequence the toxins described herein. Additional information may 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.
Resistance Management. With increasing commercial use of insecticidal proteins in transgenic plants, one consideration is resistance management. That is, there are numerous companies using Bacillus thuringiensis toxins in their products, and there is concern about insects developing resistance to B.t. toxins. One strategy for insect resistance management would be to combine the TC toxins produced by Xenorhabdus, Photorhabdus, and the like with toxins such as B.t. crystal toxins, soluble insecticidal proteins from Bacillus stains (see, e.g., WO 98/18932 and WO 99/57282), or other insect toxins. The combinations could be formulated for a sprayable application or could be molecular combinations. Plants could be transformed with bacterial genes that produce two or more different insect toxins (see, e.g., Gould, 38 Bioscience 26–33 (1988) and U.S. Pat. No. 5,500,365; likewise, European Patent Application 0 400 246 A1 and U.S. Pat. Nos. 5,866,784; 5,908,970; and 6,172,281 also describe transformation of a plant with two B.t. crystal toxins). Another method of producing a transgenic plant that contains more than one insect resistant gene would be to first produce two plants, with each plant containing an insect resistance gene. These plants could then be crossed using traditional plant breeding techniques to produce a plant containing more than one insect resistance gene. Thus, it should be apparent that the phrase “comprising a polynucleotide” as used herein means at least one polynucleotide (and possibly more, contiguous or not) unless specifically indicated otherwise.
Formulations and Other Delivery Systems. Formulated bait granules containing spores and/or crystals of the subject Paenibacillus isolate, or recombinant microbes comprising the genes obtainable from the isolate disclosed herein, can be applied to the soil. Formulated product can also be applied as a seed-coating or root treatment or total plant treatment at later stages of the crop cycle. Plant and soil treatments of cells may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersants, or polymers.
As would be appreciated by a person skilled in the art, the pesticidal concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used directly. The pesticide will be present in at least 1% by weight and may be 100% by weight. The dry formulations will have from about 1–95% by weight of the pesticide while the liquid formulations will generally be from about 1–60% by weight of the solids in the liquid phase. The formulations will generally have from about 102 to about 104 cells/mg. These formulations will be administered at about 50 mg (liquid or dry) to 1 kg or more per hectare.
The formulations can be applied to the environment of the pest, e.g., soil and foliage, by spraying, dusting, sprinkling, or the like.
Another delivery scheme is the incorporation of the genetic material of toxins into a baculovirus vector. Baculoviruses infect particular insect hosts, including those desirably targeted with the toxins. Infectious baculovirus harboring an expression construct for the toxins could be introduced into areas of insect infestation to thereby intoxicate or poison infected insects.
Insect viruses, or baculoviruses, are known to infect and adversely affect certain insects. The affect of the viruses on insects is slow, and viruses do not immediately stop the feeding of insects. Thus, viruses are not viewed as being optimal as insect pest control agents. However, combining the toxin genes into a baculovirus vector could provide an efficient way of transmitting the toxins. In addition, since different baculoviruses are specific to different insects, it may be possible to use a particular toxin to selectively target particularly damaging insect pests. A particularly useful vector for the toxins 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 virus-toxin gene recombinant may be constructed in an orally transmissible form. Baculoviruses normally infect insect victims through the mid-gut intestinal mucosa. The toxin gene inserted behind a strong viral coat protein promoter would be expressed and should rapidly kill the infected insect.
In addition to an insect virus or baculovirus or transgenic plant delivery system for the protein toxins of the present invention, the proteins may be encapsulated using Bacillus thuringiensis encapsulation technology such as but not limited to U.S. Pat. Nos. 4,695,455; 4,695,462; 4,861,595 which are all incorporated herein by reference. Another delivery system for the protein toxins of the present invention is formulation of the protein into a bait matrix, which could then be used in above and below ground insect bait stations. Examples of such technology include but are not limited to PCT Patent Application WO 93/23998, which is incorporated herein by reference.
Plant RNA viral based systems can also be used to express bacterial toxin. In so doing, the gene encoding a toxin can be inserted into the coat promoter region of a suitable plant virus which will infect the host plant of interest. The toxin can then be expressed thus providing protection of the plant from insect damage. Plant RNA viral based systems are described in U.S. Pat. Nos. 5,500,360 to Mycogen Plant Sciences, Inc. and U.S. Pat. Nos. 5,316,931 and 5,589,367 to Biosource Genetics Corp.
In addition to producing a transformed plant, there are other delivery systems where it maybe desirable to reengineer the bacterial gene(s). For example, a protein toxin can be constructed by fusing together a molecule attractive to insects as a food source with a toxin. After purification in the laboratory such a toxic agent with “built-in” bait could be packaged inside standard insect trap housings.
Mutants. Mutants of the Xenorhabdus Xwi isolate of the invention can be made by procedures that are well known in the art. For example, asporogenous mutants can be obtained through ethylmethane sulfonate (EMS) mutagenesis of an isolate. The mutants can be made using ultraviolet light and nitrosoguanidine by procedures well known in the art.
All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety to the extent they are not inconsistent with the explicit teachings of this specification.
Following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
It was shown previously (U.S. Pat. No. 6,048,838) that Xenorhabdus strain Xwi (NRRL B-21733, deposited Apr. 29, 1997) produced extracellular proteins with oral insecticidal activity against members of the insect orders Coleoptera, Lepidoptera, Diptera, and Acarina. Full-length gene and TC protein sequences obtainable from strain Xwi are disclosed herein.
Production and processing of Xenorhabdus fermentation broths. Xenorhabdus strain Xwi was grown on 2% proteose peptone #3 (hereafter designated as PP3) agar containing 0.0025% bromthymol blue (20 g/L proteose peptone #3, 0.025 g/L bromthymol blue, 15 g/L Bacto agar; Difco Laboratories, Detroit, Mich.) for 72 hours at 28° C. Seed flasks were produced by inoculating single, bromthymol blue-adsorbing colony into a 500 mL tri-baffled flask containing 175 mL of sterile PP3 plus 1.25% NaCl. Following 16 hr incubation at 28° C. on a rotary shaker at 150 rpm, seed cultures were transferred into production flasks. Two mL of the seed culture was inoculated into each production flask, which was a 500 mL tri-baffled flask containing 175 mL of sterile PP3 plus 1.25% NaCl. Production flasks were incubated at 28° C. and shaken on a rotary shaker at 150 rpm. After incubation for 48–72 hrs, the production fermentation broths were pooled, dispensed into sterile 1.0 L polyethylene bottles, centrifuged at 2,400×g for 1 hr at 10° C., and decanted from the cell and debris pellet. The fermentation broth was then either filter sterilized through a 0.22 μM filter, or further clarified using a tangential flow microfiltration device (Pall Filtron, Northborough, Mass.) using a 0.5 μM open channel poly-ether sulfone membrane filter. The filter-sterilized fermentation broths were then used as the starting material for the biochemical fractionation and purification of proteins responsible for the insecticidal activities observed in these broths.
Insect bioassay of biochemically fractionated and purified protein samples. To aid in the purification and specific activity determination of Xenorhabdus proteins possessing insecticidal activity, biochemically fractionated protein samples and serially diluted purified protein preparations were tested in insect feeding bioassays. The insect species used in these assays included Diabrotica undecimpunctata howardi (Barber) (southern corn rootworm, SCR), Helicoverpa zea (Boddie) (corn earworm, CEW), Heliothis virescens (Fabricius) (tobacco budworm, TBW), Spodoptera exigua (Hübner) (beet armyworm, BAW), Manduca sexta (Linnaeus) (tobacco hornworm, THW), and Ostrinia nubilalis (Hübner) (European corn borer, ECB). The artificial diet used to bioassay SCR was as described in Rose, R. I. & J. M. McCabe (1973), “Laboratory rearing techniques for the southern corn rootworm,” J. Econ. Entomol. 66(2):398–400. The Multiple Species Diet (Southland Products, Inc., Lake Village, Ark.) was used in bioassays with ECB, CEW, TBW, and THW.
Samples were bioassayed by applying 40 μL aliquots of each sample directly to the surface of the artificial diet (˜1.5 cm2) in 8 or 16 wells of a 128-well bioassay tray (BIO-BA-128, CD International, Pitman, N.J.). Treated diet wells were allowed to dry under a constant air flow in a biological safety cabinet, then each well was infested with a single, neonate insect hatched from surface sterilized eggs. Assay trays were sealed with a vented lid (BIO-CV, CD International), then placed in an environmentally controlled chamber [28° C., relative humidity of 40%, photoperiod of 16:8 (L:D)] for the duration of the assay. Mortality and growth inhibiton were assessed after 3–5 days.
Insect Bioassay of Expressed Toxin
Complex Genes. The biological activity of expressed toxin complex genes was tested in insect feeding assays. These assays were performed as described previously except that the artificial diets used were modified from those described by Marrone, P. G., F. D. Ferri, T. R. Mosely, & L. J. Meinke (1985), “Improvements in laboratory rearing of the southern corn rootworm, Diabrotica undecimpunctata howardi Barber (Coleoptera: Chrysomelidae), on artificial diets and corn,” J. Econ. Entomol. 78(1):290–293, and King, E. G. & G. G. Hartley (1985), page 323 in P. Singh & R. F. Moore [eds.], Handbook of Insect Rearing, vol. 2, Elsevier, New York, and that mortality and growth inhibition were assessed after 5–7 days.
In summary, proteinaceous insecticidal actives with oral activity against Lepidoptera were biochemically-purified from Xenorhabdus strain Xwi and was designated as ToxinXwiA. The purified active had an apparent native molecular weight of about 860 kDa as determined by gel filtration column chromatography. When examined by SDS-PAGE analysis, a Coomassie-staining band>220 kDa was observed for the purified toxin. These data indicate that the native toxin may exist as a tetramer of>220 kDa monomers. When tested for oral insecticidal activity in insect bioassay, this purified toxin exhibited mortality and/or growth inhibition against THW, TBW, CEW, and BAW.
More specifically, five liters of filter-sterilized of Xenorhabdus strain Xwi fermentation broth were concentrated using an Amicon (Beverly, Mass.) spiral ultrafiltration cartridge Type S1Y100 (100 kDa molecular weight cut off) attached to an Amicon M-12 filtration device according to the manufacturer's recommendations. The retentate material was diafiltered with 10 mM sodium phosphate, pH 7.0 (hereafter referred to as Buffer A) and applied at 5 mL/min to a Q Sepharose XL anion exchange column (1.6×10 cm, Amersham Biosciences Corp., Piscataway, N.J.). [For this and subsequent protein purification steps, all operations were performed at room temperature unless otherwise noted.] The column was washed with 5 bed volumes of Buffer A to remove unbound proteins. Protein fractions containing the THW activity were eluted by 0.4 M NaCl in Buffer A and loaded onto a gel filtration column (2.6×100 cm) of Sepharose CL-4B previously equilibrated with Buffer A. Protein was eluted in Buffer A at a flow rate of 0.75 mL/min. An activity peak against THW eluted between retention times 320 min to 450 min. Protein fractions with THW activitywere pooled and further purified.
The pooled protein fractions were applied at a flow rate of 1 mL/min to a Mono Q column (1.0×10 cm, Amersham Biosciences Corp.) previously equilibrated with 20 mM Tris-HCl, pH 7.0 (hereafter referred to as Buffer B). Bound proteins were eluted by a linear gradient of 0 to 1 M NaCl in Buffer B at 2 mL/min for 60 min. Two mL fractions were collected and THW activity was determined by testing a dilution series of each fraction in insect bioassay.
Solid (NH4)2SO4 was added to those protein fractions containing THW activity to a final concentration of 1.7 M. The fractions were then applied at 1 mL/min to a phenyl-Superose column (1.0×10 cm, Amersham Biosciences Corp.) previously equilibrated with 1.7 M (NH4)2SO4 in 50 mM potassium phosphate buffer, pH 7.0 (hereafter referred to as Buffer C). After washing the column with 10 mL of Buffer C, bound proteins were eluted with a linear gradient Buffer C to 5 mM potassium phosphate, pH 7.0 at 1 mL/min for 120 min. Protein fractions were then dialyzed overnight against Buffer A.
The protein fractions were assayed for THW activity and the most active fractions were pooled and applied at 1 mL/min to a Mono Q column (0.5×5 cm) that was previously equilibrated with Buffer B. Bound proteins were eluted at 1 mL/min by a linear gradient of 0 to 1 M NaCl in Buffer B.
The molecular weight of the purified insecticidal protein was examined by a gel-filtration column containing Superdex S-200, and it appeared to have a native molecular weight of approximately 860 kDa. SDS-PAGE analyses of this insecticidal protein showed a predominant Coomassie blue staining band of estimated size >220 kDa. The purified toxin was designated as ToxinXwiA.
The LD50s of ToxinXwiA were determined to be as follows: 50 ng/cm2 against THW, 100 ng/cm2 against ECB, 250 ng/cm2 against TBW, and>1,000 ng/cm2 against CEW.
The amino acid sequences of the N-terminal and some internal peptides of ToxinXwiA are given below. These sequences were obtained as described below.
N-terminal and internal amino acid sequence analysis of Xenorhabdus toxins. To facilitate the cloning and characterization of nucleotide sequences encoding insecticidal toxins, N-terminal and internal amino acid sequences were obtained for some of the toxin peptides identified. Two methods for the determination of amino acid sequences of the highly purified Xenorhabdus protein toxins are described.
N-terminal Sequence Analysis. Proteins described herein were electrophoresed by SDS PAGE and transblotted to Immuno Blot™ PVDF Membrane (Bio-Rad Laboratories, Hercules, Calif.). Proteins of interest were localized on the membrane by staining with 1×Amido Black Staining Solution (0.1% (w/v) amido black, 25% (v/v) isopropanol, and 10% (v/v) acetic acid, Sigma Chemical Co., St. Louis, Mo.) for approximately 3 min at room temperature followed by partial destaining in several changes of distilled water. The bands of interest were excised from the membrane and subjected to Edman degradation for amino acid sequence analysis at the Harvard University Microchemistry Facility (Cambridge, Mass.). The N-terminal sequences obtained for insecticidal protein toxins purified from Xenorhabdus Xwi are listed below.
Internal Peptide Sequence Analysis. Purified insecticidal protein toxins were resolved by SDS-PAGE, excised from gels, digested ‘in-situ’ with trypsin, and analyzed by MALDI-TOF . Approximately one picomole of the proteolytic digest was mixed with the matrix solution (α-cyano-4-hydroxycinnamic acid), and then air-dried. Positive-ion post source decay (PSD) MALDI-TOF MS was performed using a Voyager DE™-STR equipped with a delayed-extraction system (PerSeptive Biosystems, Framingham, Mass.) with a 3 meter flight tube in the reflectron mode. A specific peptide mass was analyzed from a mixed population of peptide masses by utilizing a timed ion selector. Fragment ions were generated as a result of metastable decay. The segments of the product ion spectra, measured successively at each potential on the reflectron, are stitched together to create a complete product ion spectrum. Internal amino acid sequences of insect active proteins from strain Xwi was determined by MALDI-PSD and are listed below.
As a prerequisite for the production of Xenorhabdus insect toxin proteins in heterologous hosts, and for other uses, it is necessary to isolate and characterize the genes that encode those peptides. One cloning approach is based on the use of N-terminal and internal amino acid sequence data to design degenerate oligonucleotides for use as hybridization probes, or in amplification reactions by polymerase chain reaction (PCR). Another approach, described in this example, involves the construction of a cosmid library and screening for heterologous expression of insect toxin proteins in an insect bioassay.
Isolation of total cellular DNA from Xenorhabdus. Xenorhabdus strain Xwi was grown on PP3 agar containing 0.0025% bromthymol blue for 72 hours at 28° C. A single bromthymol blue-adsorbing colony was selected and used to inoculate 500 mL tri-baffled flasks containing 175 mL of PP3. Shake flasks were shaken at 150 rpm and incubated at 28° C. for approximately 24 hrs. Fifty mL of this culture was centrifuged at 2,400×g to pellet the cells. The supernatant fluid was removed and the cell pellet was frozen at −20° C. until it was thawed for total cellular DNA isolation.
Total cellular DNA was isolated from the :strain using a Genomic DNA purification kit (Qiagen Inc., Valencia, Calif.). Frozen bacterial cell pellets were resuspended in 1 1 mL of Buffer B1 (50.mM Tris/HCl, pH 8.0; 50 mM EDTA, pH 8.0; 0.5% Tween 20,0.5% Triton X-100) containing 11 μL of Qiagen RNase A solution (100 mg/mL) by vortexing. To this suspension, 300 μL of a lysozyme (100 mg/mL; Sigma Chemical Co.) stock solution and 500 μL of a proteinase K (50 mg/mL; Sigma Chemical Co.) stock solution were added. The suspension was mixed by vortexing and incubated at 37° C. for 30 min. Four mL of Buffer B2 (3 M guanidine HCl; 20% Tween 20) was added to the bacterial lysates and mixed into solution by gentle inversion of the tubes. The bacterial lysates were incubated at 50° C. for 30 min. Total cellular DNA was isolated from the bacterial lysates using Qiagen Genomic-tip 500/G tips as per manufacturer's instructions (Qiagen Genomic DNA Handbook). The resulting purified DNA was dissolved in 500 L TE buffer (10 mM Tris/HCl pH 8.0; 1 mM EDTA pH 8.0) and stored at 4° C.
Construction of cosmid libraries. Partial Sau3A I digests were made of the total cellular DNA isolated from the Xenorhabdus strain based on section 3.1.3 of Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., New York, N.Y.). 400 μg of Xenorhabdus total cellular DNA was incubated with 9 units of Sau3A I (Invitrogen, Carlsbad, Calif.) for 15 min at 37° C. in 800 μL total volume of 1×React 4 Buffer (supplied as 10× by the manufacturer). The reaction was heated at 65° C. for 20 min to inactivate the enzyme. The partially digested Xenorhabdus total cellular DNA was dephosphorylated by incubating with 20 units of shrimp alkaline phosphatase (Roche Molecular Biochemicals, Indianapolis, Ind.) for 2 hrs at 37° C. in 1.2 mL total volume of 1×SAP buffer (supplied as 10× by the manufacturer). The dephosphorylated insert DNA was mixed with an equal volume of an equilibrated phenol-chloroform (50:50; v/v) solution, mixed by gentle inversion, centrifuged at 14,000×g for 15 min, and the aqueous phase was removed and mixed with an equal volume of a chloroform-isoamyl alcohol (24:1; v/v) solution. After mixing the two phases by gentle inversion, the solution was centrifuged at 14,000×g for 15 min, the aqueous phase was removed to a fresh tube, and 0.1 volume of 3 M sodium acetate (pH 5.2) was added. Two volumes of ice-cold 100% ethanol were added and the solution was mixed by inversion. and placed at −70° C. overnight. DNA was pelleted by centrifugation at 14,000×g for 20 min, and the DNA pellet was resuspended in 50 μL double-distilled water and stored at −20° C.
Cosmid vector SuperCos 1 (Stratagene, La Jolla, Calif.) was prepared as recommended by the manufacturer. Insert DNA was ligated [20 units of T4 DNA Ligase (New England BioLabs Inc., Beverly, MA) overnight at 16° C. in 1×T4 DNA Ligase Buffer (supplied as 10× by manufacturer)] into the BamHI site of SuperCos I using a 3:1 ratio of partially-digested insert to vector DNA. Ligation mixtures were packaged using Gigapack III Gold Packaging Extract (Stratagene) and recombinant phage were titered using Escherichia coli strain XL1-Blue MR cells as described in the supplier's instructions. Library source plates were prepared from aliquots (20–40 μL) of the recombinant phage plus host cell culture spread onto LB agar (10 g/L Bacto-tryptone, 10 g/L NaCl, 5 g/L Bacto-yeast extract, 15g/L Bacto agar; Difco Laboratories) containing ampicillin (100 mg/L; Sigma Chemical Co.) and incubated overnight at 37° C. Master plates of the cosmid libraries for freezer storage were prepared from single colonies inoculated into individual wells of sterile 96-well microwell plates containing 100–1000 μL of Terrific Broth (TB media: 12 g/L Bacto-tryptone, 24 g/L Bacto-yeast extract, 0.4% v/v glycerol, 17 mM KH2PO4, 72 mM K2HP2O4) plus either 100 ampicillin or 50 mg/L kanamycin (Sigma Chemical Co.), incubated without shaking overnight at 37° C. Copy plates from the master plates were made using a 96-well microplate replicator (V & P Scientific, Inc., San Diego, Calif.) to inoculate wells of a sterile 96-well microwell plate containing 100–1000 μL of LB broth containing 100 mg/L ampicillin. Copy plates were incubated without shaking at 37° C. overnight. For both master and copy plates, an equal volume (100–1000 μL) of filter-sterilized TB:glycerol or LB:glycerol (1:4; v:v) was added to the plates and the cultures and glycerol solutions were mixed using a multichannel pipetter. Plates were sealed with Biomek Seal and Sample aluminum foil lids (Beckman Instruments, Inc., Fullerton, Calif.) and placed at −70° C. for storage.
The average insert size of selected recombinant cosmids was assessed by isolating cosmid DNA using the NucleoSpin Nucleic Acid Purification Kit (CLONTECH Laboratories, Inc., Palo Alto, Calif.). The recovered DNA was digested with 20 units of Eco RI (New England BioLabs) for 1 hr at 37° C. and fragments were separated through a 1.0% agarose gel. DNA fragments were visualized with UV light following 0.5% ethidium bromide (Sigma Chemical Co.) staining and the relative sizes of fragments were estimated by comparison with 1 Kb DNA ladder (Invitrogen). Average insert size of individual cosmids ranged from 30–45 Kb.
Screening of cosmid libraries and identification of cosmids expressing insecticidal activity. Fresh cultures of the cosmid libraries were screened in insect bioassay to identify clones that expressed insecticidal activity. Copy plates of the libraries were removed from storage at −70° C. and thawed at 25° C. A 96-well microplate replicator was used to inoculate wells of a sterile 96-well microwell plate containing 2 mL of LB broth containing 100 mg/L ampicillin. The newly-inoculated plates were incubated without shaking at 28° C. for 2 days. Cell pellets of the cultures were obtained by centrifugation of the plates at 2,200×g for 1 hr. After centrifugation, 1.8 mL of the supernatant fluid was removed and the cell pellet was resuspended in the remaining supernatant fluid (approximately 200 μL). This process concentrated the cell pellet about 10×relative to the original culture.
As shown previously, culture broths from Xenorhabdus strain Xwi showed differential insecticidal activity (mortality and/or growth inhibition) against a number of insects from the orders Coleoptera, Diptera, Arcina, and Lepidoptera. Recombinant cosmids that expressed insecticidal activity against THW larvae (Lepidoptera) were identified by testing aliquots of the concentrated cell pellets in an insect bioassay. Concentrated cell pellets of the recombinant cosmid clones were applied directly to the surface (approximately 1.5 cm2) of Multiple Species Diet in 40–100 μL aliquots. Experimental controls included in the assays and treated analogously were: LB media plus 100 mg/L ampicillin; and concentrated cell pellets of the E. coli host strain XL1-Blue MR containing the SuperCos I vector without insert. The diet plates were allowed to air-dry in a sterile flow-hood and each well was infested with two neonate THW larvae. The plates were sealed, placed in a humidified growth chamber and maintained in the dark at 27° C. Mortality and visible growth inhibition relative to control treatments were scored after 5–7 days of incubation. Generally, 8 larva (4 wells containing two insects each) per treatment were assayed. Approximately 600–1200 recombinant clones were screened from each of the cosmid libraries tested.
Spectrum of activity of recombinant cosmid clones expressing insecticidal activity. The spectrum of insecticidal activity encoded by the clones identified in the cosmid screening was assayed against THW, TBW, CEW, ECB, and BAW using concentrated cell pellets of the clones, prepared and tested as described for the library screening. These assays showed that the recombinant cosmid clones obtained from the Xwi cosmid libraries had insecticidal activity (mortality and/or growth inhibition) against all species of insects tested (Table 4).
Xenorhabdus cosmid
To determine the open reading frame(s) (ORFs) responsible for the insecticidal activity observed from the recombinant cosmid pDAB2097 isolated in Example 3, the nucleotide sequence of the insert DNA in this cosmid was determined and analyzed.
Nucleotide Sequencing of pDAB2097 Insert DNA. Cosmid DNA was purified according to manufacturer's instructions using a NucleoSpin Nucleic Acid Purification Kit (CLONTECH). The DNA was partially digested in a series of enzyme dilutions as described in section 3.1.3 of Ausubel et al. (ibid.) to fragments ranging in size from 800–1,800 bp. Digestion reactions consisted of 20–40 μg cosmid DNA with 10 units/μL of diluted restriction enzyme HinPI (New England BioLabs) in 1×NEBuffer 2 (supplied as a 10×stock by the manufacturer) at 37° C. for approximately 12 minutes. Following incubation, reactions were heat inactivated by incubation at 65° C. for 30 minutes. Partial digests were gel purified using an 0.8% agarose gel (Invitrogen) and fragments were excised from the gel and purified using a QIAEX II Gel Extraction Kit, as described by the manufacturer (Qiagen).
Bacteriophage M13mp19RF vector (Roche Molecular Biochemicals) was prepared by completely digesting 5 μg of DNA with restriction enzyme AccI (10 units/μL) (New England BioLabs) in 1×NEBuffer 4 (supplied as a 10×stock by the manufacturer) at 37° C. The reaction was heat inactivated at 65° C. for 30 minutes, then the DNA was dephosphorylated using 1 unit of shrimp alkaline phosphatase (SAP) (Roche Molecular Biochemicals) in 1×SAP buffer (supplied as a 10×stock by the manufacturer) and incubation for 1 hr at 37° C. The vector DNA was then extracted once with 1 volume of phenol:chloroform:isoamyl (25:24:1; v/v/v) and once with 1 volume of chloroform:isoamyl (24:1; v/v) before precipitation by adding 0.1 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of 100% ethanol, and incubating in a dry ice/ethanol bath for 30 minutes. The precipitated vector was spun at 14,000×g and the pellet washed with 1 volume of 70% ethanol before resuspending in 10 μL of distilled sterile water.
Partially digested HinPI cosmid fragments (0.2 μg) were ligated to AccI digested, dephosphorylated M13mp19RF fragments (0.2 μg) using 20 units of T4 DNA Ligase (New England BioLabs) in 1×T4 DNA Ligase Buffer with overnight incubation at room temperature. The ligation reaction was ethanol precipitated with 0.1 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of 100% ethanol, then resuspended in a final volume of 20 μl TE buffer.
Transformation of host E. coli cells (electrocompetent XL1-Blue MRF′, Stratagene) by electroporation was performed using a Bio-Rad Gene Pulser (200 ohms, 25 μF, 1.25 V) and 0.1 cm cuvette (Bio-Rad). Prior to transformation, 5 μL of ligation reaction mixture was added to 50 μL cells and incubated on ice. Immediately following electroporation, 1 mL of YT Broth [8 g/L Bacto tryptone, 5 g/L Bacto yeast extract, 5 g/L NaCl; pH 7.0] was added directly to the cuvette and then transferred to a 1.7 mL Eppendorf tube. Cells were pelleted by centrifuging for 30 sec at 10,000×g and the supernatant fluid was removed. Cells were resuspended in 1 mL YT Broth and repelleted by centrifuging for 30 sec at 10,000×g. The supernatant fluid was removed and the pelleted cells were resuspended in 200 μL YT Broth. Following a 1 hr recovery period at 37° C., the transformed cells were diluted and mixed with 50 μL XL1-Blue MRF′ E. coli. This mixture was plated onto YT agar supplemented with X-gal (40 mg/L), IPTG (12 mg/L) and tetracycline (25 mg/L), and incubated overnight at 37° C. Clear phage plaques were then picked and used to infect XL1-Blue MRF′ E. coli. Phage DNA was isolated using 20% PEG 8000 and 2.5 M NaCl precipitation. M13mp19RF vector containing cosmid DNA fragments were recovered by normal miniprep plasmid isolation from the remaining E. coli pellet (Sambrook, J., et al., 1989). The recovered phage and plasmid were used as templates in dye terminator cycle sequencing reactions using the DNA Sequencing Kit with AmpliTaq® DNA Polymerase, FS and protocols supplied with the PRISM™ sequencing kit (ABI/Perkin Elmer, Great Britain). Reaction primers were pUC/M13 reverse (17-mer) and pUC/M13 forward (17-mer) (Promega, Madison, Wis.). All sequencing reactions were incubated in a Perkin-Elmer 9600 Thermal Cycler. With phage DNA as template, the thermocycler parameters were: 5 cycles of 95° C. for 4 sec; 55° C. for 10 sec; and 70° C. for 60 sec, followed by 10 cycles of 95° C. for 4 sec and 70° C. for 60 sec. For plasmid DNA as template, the thermocycler parameters were: 25 cycles of 96° C. for 30 sec; 50° C. for 15 sec, and 60° C. for 4 min. The DNA sequence was obtained analysis of the DNA samples on an ABI Model 377 DNA Sequencer (ABI/Perkin Elmer).
The resulting sequence data were sorted and aligned using the Sequencher software package (Version 3.1.1; Gene Codes Corporation, Ann Arbor, Mich.). Gaps in the alignment of sequence contigs or second strand sequence reactions were solved through direct primer design and walking using cosmid DNA or a subclone derivative as template. All oligonucleotides were synthesized using a 394 DNA/RNA Synthesizer (ABI/Perkin Elmer). Double stranded nucleotide sequence was obtained for the entire insert contained in the pDAB2097 recombinant cosmid. PHRED-PHRAP analysis software (University of Washington, Seattle, Wash., USA) was used to assess the quality of the double-stranded sequence determined for the entire 39 kb insert contained in cosmid pDAB2097. Nucleotide positions that had quality scores <15 were resolved by repeated sequencing with the standard M13/pUC primers or with specifically designed primers, until high quality nucleotide sequence was obtained.
Nucleotide sequence analysis of the pDAB2097 insert DNA. The 39,005 bp sequence obtained from the pDAB2097 cosmid (SEQ ID NO. 6) was analyzed using the Vector NTI™ Suite (Informax, Inc. North Bethesda, Md., USA) to identify encoded ORFs (Open Reading Frames). Six full length ORFs and one partial ORF were identified (
The nucleotide sequences of the identified ORFs and the deduced amino acid sequences encoded by these ORFs were used to search the databases at the National Center for Biotechnology Information by using BLASTn, BLASTp, and BLASTx, via the “.gov” (government) website of ncbi/nih for BLAST. These analyses showed that the ORFs identified in the pDAB2097 insert had significant amino acid sequence identity to genes previously identified in Photorhabdus luminescens and Xenorhabdus nematophilus (Table 6). It is noteworthy that the xpt gene sequences presented in GenBank accession number AJ308438 were obtained from a recombinant cosmid that expressed oral insecticidal activity.
Since ORF2, ORF4, ORF5, ORF6, and ORF7 were shown to have at least 95% amino acid sequence identity to previously identified genes, the same gene nomenclature was adopted for further studies on the ORFs identified in the pDAB2097 insert sequence (Table 7).
From comparison of the deduced amino sequences of the xpt genes found in pDAB2097 with the biochemical data obtained from the characterization of ToxinXwiA, it was concluded that xptA2 encodes the ToxinXwiA protein. The data supporting this conclusion are as follows (Table 8). First, the N-terminal sequence obtained for ToxinXwiA (SEQ ID NO. 1) exactly matches the first 20 amino acids encoded by xptA2. Second, the four internal amino acid sequences obtained from ToxinXwiA are found in the xptA2 deduced amino acid sequence.
As described in Example 3, the recombinant cosmid clone pDAB2097 demonstrated insecticidal activity against THW, TBW, CEW, ECB, and BAW (Table 4). The nature of the insecticidal activity encoded by this cosmid was investigated by biochemical purification and characterization. Insect bioassay using THW, as described in Example 1, was used during the purification process to monitor the biochemical purification of insecticidal activities.
Concentrated cell pellets of E. coli cells harboring pDAB2097 were produced by processing 5 liters of fermentation broths prepared as follows. A single colony of the recombinant clone was inoculated into 1 L LB plus 100 μg/mL ampicillin in 2.8 L Fernbach flasks. Inoculated flasks were shaken on a rotary shaker at 150 rpm at 28° C. for 2 days, the cultures were dispensed into sterile 1.0 L polyethylene bottles, and then centrifuged at 12,400×g for 30 min at 4° C. Supernatant fluid was removed and discarded. Cell pellets were resuspended in 50 mM potassium phosphate buffer, pH 7.0 and lysed by mechanical disruption in a Bead Beater® Blender with 0.1 mm beads according to the manufacture's protocol. The cell debris was removed by filtering through cheesecloth and centrifugation at 27,000×g for 15 minutes at 4° C. The supernatant liquid was applied to a Q Sepharose XL anion exchange column (1.6×10 cm) at 5 mL/min, and bound proteins were then eluted with 30 mL of 20 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl.
The protein fraction was loaded onto a gel filtration column (2.6×100 cm) of Sepharose CL-4B which was equilibrated with Buffer A. Proteins were eluted in Buffer A at a flow rate of 0.75 mL/min. Bioassays were performed on each fraction against THW. Active fractions were pooled and applied at a flow rate of 1 mL/min to a Mono Q column (1.0×10 cm) equilibrated with Buffer A.
The proteins bound to the column were eluted with a linear gradient of 0 to 1 M NaCl in Buffer A at 2 mL/min for 60 min. Two mL fractions were collected and activity was determined in a dilution series of each fraction in insect bioassay.
Solid ammonium sulfate was added to the above protein fractions to a final concentration of 1.7 M, and the solution was applied at 1 mL/min to a phenyl-Superose column (0.5×5 cm) equilibrated with 1.7 M (NH4)2SO4 in 50 mM potassium phosphate buffer, pH 7.0 (Buffer B). After washing the column with 10 mL of Buffer C, proteins bound to the column were eluted with a linear gradient Buffer B to 5 mM potassium phosphate, pH 7.0 at 1 mL/min for 120 min. Fractions were dialyzed overnight against Buffer A. The most active fractions, as determined by bioassay on THW, were pooled and applied at 1 mL/min to a Mono Q column (0.5×5 cm) equilibrated with Buffer B. The proteins bound to the column were eluted at 1 mL/min by a linear gradient of 0 to 1 M NaCl in Buffer A.
The last step of the purification was accomplished by gel filtration through a Superdex 200 column (1.0×30 cm) which was pre-equilibrated with Buffer A. The active fractions were applied to the column at 0.5 mL aliquots and eluted with Buffer A at 0.5 mL/min.
SDS-PAGE analysis of the purified toxin from E. coli harboring cosmid pDAB2097 indicated a predominant peptide of about 220 kDa or more. The native molecular weight of the toxin complex, as determined by gel filtration, was approximately 860 kDa (which would be consistent with a tetramer of the predominant peptides). The purified protein having insecticidal activity, and encoded by the recombinant cosmid pDAB2097 (i.e. Xwi-8C3), was designated as ToxinXwi-8C3. The LD50 for ToxinXwi-8C3 was determined to be approximately 300 ng/cm2 against THW.
MALDI-TOF analysis was used to obtain information regarding the relationship between ToxinXwiA and ToxinXwi-8C3. For this analysis, peptide mass fingerprints were obtained for both ToxinXwiA and Toxin Xwi-8C3, and these data were compared to a theoretical peptide mass fingerprint of the deduced amino acid sequence from ORF xptA2. To generate these peptide mass fingerprints, ToxinXwiA and ToxinXwi-8C3 were digested with trypsin and the mass of the resulting peptides was determined using mass spectroscopy. Such digestion with trypsin generates a specific peptide “fingerprint” for each purified toxin based upon the specific cleavage site of trypsin. Since the alteration of only a single amino acid residue can detectably alter the mass of a given tryptic peptide, the identification of common peptide masses between two fingerprints indicates a degree of amino acid sequence identity.
MALDI-TOF analysis of ToxinXwiA and ToxinXwi-8C3 ToxinXwiA and ToxinXwi-8C3 proteins were subjected to preparative 1-D separation in order to produce well-resolved, purified toxin proteins in quantities sufficient for peptide mass fingerprinting. A standard procedure for protein separation was followed (Laemmli, 1970), and purified protein was loaded in each well of 4–20% gradient sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE; Owl Scientific Co., Mass.) for electrophoresis. Electrophoresis was conducted at constant 35 mA for 2 h. The proteins were visualized by staining in a solution of Coomassie Brilliant Blue R-250 (Bio-Rad).
Following separation of proteins by SDS PAGE, protein bands were excised from gels using a stainless steel scalpel and placed into a 1.5-mL polypropylene Eppendorf tube. After adding 0.7 mL of de-stain solution (50% acetonitrile in 25 mM NH4HCO3), gel pieces were crushed to <1 mm2 using a Kontes Pellet Pestle™, followed by addition of another 0.7 mL of destain solution. Samples were shaken vigorously for 30 minutes and then centrifuged to pellet the gel pieces. The supernatant was discarded and subsequent de-stain steps were performed until gel pieces were translucent in color, at which time the gel pieces were dried under vacuum centrifugation for 15 minutes. Dried gel pieces were covered with a volume (15–20 μL per protein band) of trypsin (50 μg/mL in 25 mM NH4HCO3, pH 8.0) which allowed complete rehydration of the gel pieces. Proteolysis occurred for 16 hours at 37° C. Peptides were extracted with the addition of 0.3 mL of 50% acetonitrile in 0.5% trifluoroacetic acid (TFA), immediately followed by vigorous shaking for 1 hour. After brief centrifugation to pellet the gel pieces, the supernatant was saved in a siliconized 0.5-mL Eppendorf tube. Gel pieces were dried under vacuum centrifugation for 15 minutes. After rehydration with 0.1 mL of 0.5% TFA, the sample was placed in a sonication bath for 10 minutes. Then, 0.1 mL of acetonitrile was added, followed by vigorous shaking for 1 hour. After centrifugation, the supernatant was combined with the first extract and dried using vacuum centrifugation.
To determine peptide mass fingerprints of ToxinXwiA and ToxinXwi-8C3, peptides were solubilized with 10 μl of 0.1% TFA. Soluble peptides (0.6 μl) were mixed by pipetting with 0.6 μl of matrix solution (α-cyano-4-hydroxycinnamic acid, at 10 mg/mL in 50% acetonitrile in 0.5% TFA), placed onto the MALDI plate, and allowed to dry. Internal calibration was performed using autolyic trypsin peptide masses (m/z 805.41 and/or m/z 2163.05). Mass analyses were recorded on a PerSeptive Biosystems (Framingham, Mass.) Voyager DE™-STR delayed extraction time-of-flight reflectron mass spectrometer equipped with a nitrogen laser (337 nm). Mass spectra were collected in positive ion mode with the reflectron flight tube using the following instrument settings: 20 kV ion acceleration, grid voltage of 75%, guide wire voltage of 0.02–0.03%, and a low mass gate setting of 600.
Peptide mass fingerprint analysis of ToxinXwiA and ToxinXwi-8C3. MALDI-TOF MS analysis was used to compare the peptide mass fingerprints obtained for tryptic digests of purified ToxinXwi-8C3 protein prepared from E. coli cells harboring pDAB2097, the in silico tryptic digests predicted from the deduced amino acid sequence encoded by ORF xptA2, and the tryptic digests generated from the native protein ToxinXwiA (Table 9). Fifty-seven tryptic peptide masses of ToxinXwiA matched the in silico digest of the deduced amino acid sequence of XptA2. The relatively high number of matching peptide masses from the observed ToxinXwiA peptides and the theoretical deduced XptA2 peptides indicates that ORF xptA2 encodes the ToxinXwiA protein. Similarly, eleven peptide masses from ToxinXwi-8C3 matched both XptA2 theoretical tryptic masses and native ToxinXwiA tryptic masses (in bold type). These data indicate that the recombinant insecticidal activity purified from E coli harboring cosmid pDAB2097 (i.e. ToxinXwi8C3) is derived from expression of ORF xptA2, and that this cosmid encodes at least one of the proteins responsible for the insecticidal activity of the native Xwi strain.
1564.74
1564.81
1564.78
1831.87
1831.88
1831.90
1973.97
1973.98
1973.99
1544.87
1544.82
1544.88
1012.47
1012.49
1012.49
1770.86
1770.86
1770.87
1269.58
1269.62
1269.59
1492.76
1492.77
1492.78
1702.77
1702.83
1702.85
1264.63
1264.61
1264.66
777.46
777.45
777.46
Xenorhabdus Xwi genes were expressed in E. coli. Several plasmids were constructed in which polycistronic arrangements of up to three genes were constructed. Each gene contained a separate ribosome binding site and start codon, a coding sequence and a stop codon. The expression system was mediated by the strong T7 phage promoter and T7 RNA polymerase (
Construction of pET280-XptA2, pET280-XptC1, and pET280-XptB1. The coding sequences for the XptA2, XptC1, and XptB1 proteins were each PCR amplified from pDAB2097, a recombinant cosmid containing the three genes that encode these proteins (see Example 6). The PCR primer sets used to amplify these coding sequences are listed in Table 10. In all of these primer sets, the forward primer did not change the coding sequence of the gene but provided 5′ non coding SalI and XbaI sites as well as a ribosome binding site. The reverse primers also did not alter the corresponding coding sequences, but provided a 3′ XhoI cloning site. Following amplification with components of the EPICENTRE Fail Safe PCR kit, the engineered XptA2, XptC1, and XptB1 coding sequences were each cloned into pCR2.1. The cloned amplified products were sequence confirmed to ensure that PCR-induced mutations did not alter the coding sequences. Recombinant plasmids that contained unaltered coding sequences for XptA2, XptC1, and XptB1 were identified and designated as pDAB3056, pDAB3064, and pDAB3055, respectively. The coding sequences were each cut from the pCR2.1 derivatives and transferred to a modified pET vector via the 5′ XbaI and 3′ XhoI sites to create plasmids pET280-XptA2, pET280-XptC1, and pET280-XptB1. The plasmid pET280-SS is a modified pET28 (Novagen, Madison, Wis.) plasmid with the multiple cloning site replaced and a streptomycin/spectinomycin gene inserted into the backbone.
GAACGAATGGTATAGC
GGATATGCAGAATGAT
TGTATTACTCAATAAA
ATCGCTCAGGCTCTCC
ATCAGTCCCACTCGCG
ACGG* (SEQ
TTATGCTGTCATTTCA
CCGGCAGTGTCATTTT
ACCTTTGAAACTTGAA
CATCTTCATTCACCAC
ATACCGTCATTGCCCT
C (SEQ ID NO:82)
TATGCTTCGGATTCAT
TATGACGTGCAGAGGC
TCACAGCAATACGCCA
GTTAAAGAAGAAGTTA
TCCGTCACCGTACTGG
TT (SEQ ID
ACAACC (SEQ
Construction of pET280-XptA280-XptC1. Plasmid pET280-XptA2 DNA was cut with XhoI and ligated into the unique SalI site in pDAB3064. The resulting ligated product contained both pCR2.1 and pET280-SS vector backbones and could be recovered by antibiotic selection using a combination of streptomycin (25 μg/mL), spectinomycin (25 μg/mL), and ampicillin (100 μg/mL). DNA of the recovered plasmids was digested with XhoI to check fragment orientation. A plasmid with the XptC1 coding region immediately downstream of the XptA2 coding region was obtained and the DNA was digested with XhoI to remove the pCR2.1 vector backbone. The resulting construct, which contains the pET280-SS vector backbone and the coding sequences for XptA2 and XptC1, was self-ligated to produce pET280-XptA2-XptC1.
Construction of pET280-XptC1-XptB1. Plasmid pET280-XptC1 DNA was cut with XhoI and ligated into the unique SalI site in pDAB3055. The resulting ligated product contained both pCR2.1 and pET280-SS vector backbones and could be recovered by antibiotic selection using a combination of streptomycin (25 μg/mL), spectinomycin (25 μg/mL), and ampicillin (100 μg/mL). DNA of the recovered plasmids was digested with XhoI to check fragment orientation. A plasmid with the XptB1 coding region immediately downstream of the XptC1 coding region was obtained and the DNA was digested with XhoI to remove the pCR2.1 vector backbone. The resulting construct, which contains the pET280-SS vector backbone and the coding sequences for XptC1 and XptB1, was self-ligated to produce pET280-XptC1-XptB1.
Construction of pET280-XptA2-XptB1. Plasmid pET280-XptA2 DNA was cut with XhoI and ligated into the unique SalI site in pDAB3055. The resulting ligated product contained both pCR2.1 and pET280-SS vector backbones and could be recovered by antibiotic selection using a combination of streptomycin (25 μg/mL), spectinomycin (25 μg/mL), and ampicillin (100 μg/mL). DNA of the recovered plasmids was digested with XhoI to check fragment orientation. A plasmid with the XptB1 coding region immediately downstream of the XptA2 coding region was obtained and the DNA was digested with XhoI to remove the pCR2.1 vector backbone. The resulting construct, which contains the pET280-SS vector backbone and the coding sequences for XptA2 and XptB1, was self-ligated to produce pET280-XptA2-XptB1.
Construction of pET280-XptA2-XptC1-XptB1. Plasmid pET280-XptA2-XptC1 DNA was cut with XhoI and ligated into the unique SalI site in pDAB3055. The resulting ligated product contained both pCR2.1 and pET280-SS vector backbones and could be recovered by antibiotic selection using a combination of streptomycin (25 μg/mL), spectinomycin (25 μg/mL), and ampicillin (100 μg/mL). The recovered plasmids were digested with XhoI to check fragment orientation. A plasmid with the XptB1 coding region immediately downstream of the XptC1 coding region was obtained and the DNA was digested with XhoI to remove the pCR2.1 vector backbone. The resulting construct, which contains the pET280-SS vector backbone and the XptA2, XptC1, and XptB1 coding sequences, was self-ligated to produce pET280-XptA2-XptC1-XptB1.
Expression of T7-based constructions. The expression plasmids were transformed into E. coli T7 expression strain BL21(DE3) (Novagen, Madison, Wis.) cells and plated on LB agar containing a combination of streptomycin (25 μg/mL) and spectinomycin (25 μg/mL) and 50 mM glucose, and transformants were grown at 37° C. overnight. Approximately 10–100 well isolated colonies were used to inoculate 200 mL of sterile LB containing a combination of streptomycin (25 μg/mL) and spectinomycin (25 μg/mL) plus 75 μM isopropyl-β-D-thiogalatopyranoside (IPTG) in 500 mL baffled flasks. The cultures were shaken at 200 rpm at 28° C. for 24 hours: Cells were collected by centrifugation (approximately 3000×g) and resuspended in phosphate buffer (30 mM, pH 7.4; NutraMax; Gloucester, Mass.) to a cell density of 30–120 OD600 units/mL. Diluted cells were then used for insect bioassay.
A series of expression experiments was performed using the pET expression system as described above. E. coli cells were transformed, induced and grown overnight at 28° C. The cells were collected, washed, normalized to equal concentrations, and tested for insecticidal activity against Ostrinia nubilalis European corn borer (ECB), corn earworm (CEW), and tobacco budworm (TBW). As shown in Table 11, the highest levels of insecticidal activity were observed when xptA2, xptC1, and xptB1 were present in the same construct.
Further Bioassay Results. E. coli cells were co-transformed with the pET280 and pCoT constructs listed in Table 12. Transformants were induced, processed and bioassayed as described above. In these assays, co-transformants that contained pCOT/pET280-XptA2-XptC1-XptB1 plasmid combinations exhibited the highest levels of insecticidal activity.
This application claims priority to provisional application Ser. No. 60/441,717, filed Jan. 21, 2003.
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