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WO1998008932A1 - TOXINES PROTEINIQUES INSECTICIDES ISOLEES A PARTIR DE $i(PHOTORHABDUS) - Google Patents

TOXINES PROTEINIQUES INSECTICIDES ISOLEES A PARTIR DE $i(PHOTORHABDUS) Download PDF

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Publication number
WO1998008932A1
WO1998008932A1 PCT/US1997/007657 US9707657W WO9808932A1 WO 1998008932 A1 WO1998008932 A1 WO 1998008932A1 US 9707657 W US9707657 W US 9707657W WO 9808932 A1 WO9808932 A1 WO 9808932A1
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WIPO (PCT)
Prior art keywords
seq
protein
photorhabdus
dna
purified
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1997/007657
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English (en)
Inventor
Jerald C. Ensign
David J. Bowen
James Petell
Raymond Fatig
Sue Schoonover
Richard H. Ffrench-Constant
Thomas A. Rocheleau
Michael B. Blackburn
Timothy D. Hey
Donald J. Merlo
Gregory L. Orr
Jean L. Roberts
James A. Strickland
Lining Guo
Todd A. Ciche
Kitisri Sukhapinda
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wisconsin Alumni Research Foundation
Corteva Agriscience LLC
Original Assignee
Dow AgroSciences LLC
Wisconsin Alumni Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US1996/018003 external-priority patent/WO1997017432A1/fr
Priority to PL97332033A priority Critical patent/PL332033A1/xx
Priority to CA002263819A priority patent/CA2263819A1/fr
Priority to AU28299/97A priority patent/AU2829997A/en
Priority to EP97922696A priority patent/EP0970185A4/fr
Priority to JP10511612A priority patent/JP2000515024A/ja
Application filed by Dow AgroSciences LLC, Wisconsin Alumni Research Foundation filed Critical Dow AgroSciences LLC
Priority to BR9711441-3A priority patent/BR9711441A/pt
Priority to SK246-99A priority patent/SK24699A3/sk
Priority to IL12859097A priority patent/IL128590A0/xx
Publication of WO1998008932A1 publication Critical patent/WO1998008932A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

  • the present invention relates to toxins isolated from cacteria and the use of said toxins as insecticides
  • insects are widely regarded as pests to homeowners, to picnickers, to gardeners, and to farmers and others whose investments in agricultural products are often destroyed or diminished as a result of insect damage to field crops.
  • significant insect damage can mean the loss of all profits to growers and a dramatic decrease crop yield Scarce supply of particular agricultural products invariably results m higher costs to food processors and, then, to the ultimate consumers of food plants and products derived from those plants.
  • SUBSTTTUTE SHEET (RULE 26) produce the insecticides, government agencies, public interest groups, and the public in general.
  • the development of less intrusive pest management strategies has been spurred along both by societal concern for the environment and by the development of biological tools which exploit mechanisms of insect management.
  • Biological control agents present a promising alternative to chemical insecticides.
  • Organisms at every evolutionary development level have devised means to enhance their own success and survival
  • the use of biological molecules as tools of defense and aggression is known throughout the animal and plant kingdoms
  • the relatively new tools of the genetic engineer allow modifications to biological insecticides to accomplish particular solutions to particular problems.
  • One such agent, Bacillus thu ⁇ ngiensis (Bt) is an effective msecticidal agent, and is widely commercially used as such
  • the msecticidal agent of the Bt bacterium is a protein which has such limited toxicity, it can be used on human food crops on the day of harvest.
  • the St toxin is a digestible non-toxic protein.
  • Another known class of biological insect control agents are certain genera of nematodes known to be vectors of transmission for insect-killing bacterial symbionts. Nematodes containing msecticidal bacteria invade insect larvae. The bacteria then kill the larvae. The nematodes reproduce the larval cadaver The nematode progeny then eat the cadaver from withm. The bacteria- containing nematode progeny thus produced can then invade additional larvae.
  • msecticidal nematodes m the Stemernema and Heteror ajbditis genera were used as insect control agents.
  • each genus of nematode hosts a particular species of bacterium.
  • the symbiotic bacterium is Photorhabdus luminescens .
  • the native toxins are protein complexes that are produced and secreted by growing bacteria cells of the genus Photorhabdus, of interest are the proteins produced by the species Photorhabdus luminescens .
  • the protein complexes with a molecular size of approximately 1,000 kDa, can be separated by SDS-PAGE gel analysis into numerous component proteins.
  • the toxins contain no hemolysin, lipase, type C phospholipase, or nuclease activities.
  • the toxins exhibit significant toxicity upon exposure administration to a number of insects.
  • the present invention provides an easily administered msecticidal protein as well as the expression of toxin in a heterologous system.
  • the present invention also provides a method for delivering msecticidal toxins that are functional active and effective against many orders of insects.
  • Fig. 1 is an illustration of a match of cloned DNA isolates used as a part of sequence genes for the toxin of the present invention.
  • Fig. 2 is a map of three plasmids used in the sequencing process .
  • Fig. 3 is a map illustrating the mter-relationship of several partial DNA fragments.
  • Fig. 4 is an illustration of a homology analysis between the protein sequences of TcbAi ! and TcaBu proteins.
  • FIG. 6 is an illustration of the genomic maps of the W-14 Strain.
  • Fig. 6A is an illustration of the tea and tcb loci and primary gene products .
  • Fig. 7 is a phenogram of Photorhabdus strains as defined by rep-PCR.
  • the upper axis of Fig. 7 measures the percentage similarity of strains based on scoring of rep-PCR products (i.e.,
  • the present inventions are directed to the discovery of a unique class of msecticidal protein toxins from the genus
  • Photorhabdus that have oral toxicity against insects A unique feature of Photorhabdus is its biolum escence .
  • Photorhabdus may be isolated from a variety of sources.
  • One such source is nematodes, more particularly nematodes of the genus Heterorhabdi is.
  • Another such source is from human clinical samples from wounds, see Farmer et al . 1989 J Clin. Microbiol. 27
  • SUBST ⁇ TUTE SHEET (RULE 26) pp. 1594-1600. These saprohytic strains are deposited m the American Type Culture Collection (Rockville, MD) ATCC #s 43948, 43949, 43950, 43951, and 43952, and are incorporated herein by reference. It is possible that other sources could harbor Photorhabdus bacteria that produce msecticidal toxins . Such sources in the environment could be either terrestrial or aquatic based.
  • the genus Photorhabdus is taxonom cally defined as a member of the Family Enterobacte ⁇ aceae, although it has certain traits atypical of this family For example, strains of this genus are nitrate reduction negative, yellow and red pigment producing and biolummescent . This latter trait is otherwise unknown with the En t er oba ctena ceae . Photorhabdus has only recently been described as a genus separate from the Xenorhabdus (Boemare et al . , 1993 Int. J. Syst. Bacteriol . 43, 249-255).
  • This differentiation is based on DNA-DNA hybridization studies, phenotypic differences (e.g., presence [ Photorhabdus) or absence ⁇ Xenorhabdus) of catalase and biolummescence) and the Family of the nematode host [Xenorhabdus ; Stemernejnatidae, Photorhabdus ; Heterorhabdi tidae) 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) support the separation of Photorhabdus from Xenorhabdus .
  • the strain collection disclosed herein was comprised of Photorhabdus strains
  • the strains were characterized based on recognized traits which define Photorhabdus and differentiate it from other Enterobacte ⁇ aceae and Xenorhabdus species.
  • the traits studied were the following: gram stain negative rods, organism size, colony pigmentation, inclusion bodies, presence of catalase, ability to reduce nitrate, biolummescence, dye uptake, gelatin hydrolysis, growth on selective media, growth temperature, survival under anerobic conditions and motility Fatty acid analysis was used to confirm that the strains herein all belong to the single genus Photorhabdus .
  • Photorhabdus luminescens ATCC Type strain #29999, Pomar et al . , 1977, Nematologica 23, 97-102.
  • a variety of related strains have been described m the literature (e.g., Akhurst et al . 1988 J. Gen. Microbiol., 134, 1835-1845; Boemare
  • Photorhabdus luminescens are Photorhabdus luminescens .
  • the scope of the invention herein is to any Photorhabdus species or strains which produce proteins that have functional activity as insect control agents, regardless of other traits and characteristics.
  • the bacteria of the genus Photorhabdus produce proteins that have functional activity as defined herein.
  • proteins produced by the species Photorhabdus luminescens are proteins produced by the species Photorhabdus luminescens .
  • the inventions herein should in no way be limited to the strains which are disclosed herein. These strains illustrate for the first time that proteins produced by diverse isolates of Photorhabdus are toxic upon exposure to insects.
  • the inventions described herein are the strains specified herein and any mutants thereof, as well as any strains or species of the genus Photorhabdus that have the functional activity described herein.
  • the protein toxin (s) function as insect control agents in that the proteins are orally active, or have a toxic effect, or are able to disrupt or deter feeding, which may or may not cause death of the insect.
  • formulated protein compositions (s) sprayable protein composition (s) , a bait matrix or other delivery system
  • the results are typically death of the insect, or the insects do not feed upon the source which makes the toxins available to the insects.
  • genetic material By the use of the term “genetic material” herein, it is meant to include all genes, nucleic acid, DNA and RNA.
  • homology it is meant an ammo acid sequence that has a similarity index of at least 33% and/or an identity index of at least 26% to a reference W-14 toxm polypeptide am o acid sequence, as scored by the GAP algorithm using the BlOsum 62 protein scoring matrix (Wisconsin Package Version 9.0, Genetics Computer Group (GCG) , Madison, WI) .
  • identity is meant an ammo acid sequence that contains an identical residue at a given position, following alignment with a reference W-14 tox polypeptide ammo acid sequence by the GAP algorithm.
  • insecticides typically referred to as "insecticides" By insecticides it is meant herein that the protein toxins have a “functional activity” as further defined herein and are used as insect control agents.
  • oligonucleotides By the use of the term “oligonucleotides” it is meant a macromolecule consisting of a short chain of nucleotides of either RNA or DNA. Such length could be at least one nucleotide, but typically are in the range of about 10 to about 12 nucleotides. The determination of the length of the oligonucleotide is well withm the skill of an artisan and should not be a limitation herein therefore, oligonucleotides may be less than 10 or greater than 12.
  • Photorhabdus tox any protein produced by a Photorhabdus microorganism strain which has functional activity against insects, where the Photorhabdustox could be formulated as a sprayable composition, expressed by a transgenic plant, formulated as a bait matrix, delivered via baculov rus, or delivered by any other applicable host or delivery system.
  • truncated peptide it is meant herein to include any peptide that is fragment (s) of the peptides observed to have functional activity.
  • substantially sequence homology is meant either, a DNA fragment having a nucleotide sequence sufficiently similar to another DNA fragment to produce a protein having similar biochemical properties; or a polypeptide having an am o acid sequence sufficiently similar to another polypeptide to exhibit similar biochemical properties.
  • Fermentation broths from selected strains reported Table 20 were used to determine the following: breadth of msecticidal tox production by the Photorhabdus genus, the msecticidal spectrum of these toxins, and to provide source material to purify the toxm complexes.
  • the strains characterized herein have been shown to have oral toxicity against a variety of insect orders Such insect orders include but are not limited to Coleoptera , Homoptera, Lepidoptera, Diptera, Acarina, Hymenoptera and Dic yoptera .
  • the rate of mutation of the bacteria in a population causes many related toxins slightly different in sequence to exist.
  • Toxins of interest here are those which produce protein complexes toxic to a variety of insects upon exposure, as described herein
  • the toxins are active against Lepidoptera , Coleoptera , Ho opotera, Diptera, Hymenoptera, Di ctyoptera and Acarina .
  • the inventions herein are intended to capture the protein toxins homologous to protein toxins produced by the strains herein and any derivative strains thereof, as well as any protein toxins produced by Photorhabdus These homologous proteins may differ in sequence, but do not differ m function from those toxins described herein.
  • Homologous toxins are meant to include protein complexes of between 300 kDa to 2,000 kDa and are comprised of at least two (2) subunits, where a subunit is a peptide which may or may not be the same as the other subunit.
  • Various protein subunits have been identified and are taught in the Examples herein.
  • the protein subunits are between about 18 kDa to about 230 kDa; between about 160 kDa to about 230 kDa; 100 kDa to 160 kDa; about 80 kDa to about 100 kDa; and about 50 kDa to about 80 kDa.
  • some Photorhabdus strains can be isolated from nematodes.
  • Some nematodes elongated cylindrical parasitic worms of the p .ylum Nematoda , have evolved an ability to exploit insect larvae as a favored growth environment The insect larvae provide a source of food for growing nematodes and an environment in which to reproduce.
  • One dramatic effect that follows invasion of larvae by certain nematodes is larval death.
  • Larval death results from the presence of, in certain nematodes, bacteria that produce an msecticidal tox which arrests larval growth and inhibits feeding activity.
  • the sequences listed above are grouped by genomic region. More specifically, the Photorhabdus luminesence bacteria (W-14) has at least four distinct genomic regions- tea, tcb, tec and ted. As can be seen in Table 1, peptide products are produced from these distinct genomic regions. Furthermore, as illustrated the Examples, specifically Examples 15 and 21, individual gene products produced from three genomic regions are associated with insect activity. There is also considerable homology between these four genomic regions.
  • the tcbA gene was expressed m E. coli as two possible biological active protein fragments (TcbA and TcbAn/m) .
  • the tcdA gene was also expressed in E. coli .
  • Example 16 when the native unprocessed TcbA tox was treated with the endogeneous metalloproteases or insect gut contents containing proteases, the TcbA protein toxin was processed into smaller subunits that were less than the size of the native peptides and Southern Corn Rootworm activity increased. The smaller tox peptides remained associated as part of a tox complex.
  • truncated peptide in some situations, it may be more desirable to use truncated peptide (s) in some applications, i.e., commercial transgenic plant applications.
  • W-14 strain there are other species withm the Photorhabdus genus that have functional activity which is differential (specifically see Tables 20 and 36) Even though there is differential activity, the amino acid sequences in some cases have substantial sequence homology.
  • the molecular probes indicate that some genes contained in the strains are homologous to the genes contained in the W-14 strain. In fact all of the strains illustrated herein have one or more homologs of W-14 toxm genes.
  • Example 26 The antibody data in Example 26 and the N- terminal sequence data in Example 25 further support the conclusion that there is homology and identity (based on am o acid sequence) between the protein toxm(s) produced by these strains.
  • the W-14 gene probes indicated that the homologs or the W-14 genes themselves (Tables 37, 38, and 39) are dispersed throughout the Photorhabdus genus. Further, it is possible that new toxm genes exist in other strains which are not homologous to W-14, but maintain overall protein attributes (see specifically Examples 14 and 25) .
  • the antibodies may be used to rapidly screen bacteria of the genus Photorhabdus or the family En erbacteracaea for homologous toxm products as illustrated in Example 26.
  • Those skilled in the art are quite familiar with the use of antibodies as an analysis or screening tool (see US Patent No. 5,430,137, which is incorporated herein by reference) .
  • ammo acid residue segments that tend to occupy exposed surface of polypeptides
  • ammo acid consist of contiguous am o acid residues, however, m certain cases they may be formed by non-contiguous ammo acids that are constrained by specific conformation.
  • the ammo acid segments recognized by antibodies are highly specific and commonly referred to epitopes.
  • the ammo acid fragment can be generated by chemical and/or enzymatic cleavage of the native protein, by automated, " solid-phase peptide synthesis, or by production from genetic engineering organisms.
  • Polypeptide fragments- can be isolated by a variety and/or combination of HPLC and FPLC chromatographic methods known in the art. Selection of polypeptide fragment can be aided by the use of algorithms, for example Kyte and Doolittle, 1982, Journal of Molecular Biology 157: 105-132 and Chou and Fasman, 1974, Biochemistry 13: 222-245, that predict those sequences most likely to exposed on the surface of the protein.
  • polypeptides are covalently coupled using chemical reactions to carrier proteins such as keyhole limpet hemocyanm via free amino (lysi ⁇ e) , sulfhydyl (cysteine) , phenolic (tyrosme) or carboxylic (aspartate or glutamate) groups.
  • carrier proteins such as keyhole limpet hemocyanm via free amino (lysi ⁇ e) , sulfhydyl (cysteine) , phenolic (tyrosme) or carboxylic (aspartate or glutamate) groups.
  • Immunogen with an adjuvant is injected n animals, such as mice or rabbits, or
  • Analysis of antibody titer in antisera of inject animals against polypeptide fragment can be determined by a variety of immunological methods such as ELISA and Western blot.
  • monoclonal antibodies can be prepared using spleen cells of the injected animal for fusion with tumor cells to produce immortalized hybndomas cells producing a single antibody species.
  • Hybndomas cells are screened using immunological methods to select lines that produce a specific antibody to the polypeptide fragment of interest. Purification of antibodies from different sources can be performed by a variety of antigen affinity or antibody affinity columns or other chromatographic HPLC or FPLC methods.
  • the toxins described herein are quite unique in that the toxins have functional activity, which is key to developing an insect management strategy
  • it is possible to delay or circumvent the protein degradation process by injecting a protein directly into an organism, avoiding its digestive tract.
  • the protein administered to the organism will retain its function until it is denatured, non-specifically degraded, or eliminated by the immune system in higher organisms.
  • Injection into insects of an msecticidal toxm has potential application only in the laboratory, and then only on large insects which are easily in j ected.
  • the Photorhabdus toxins may be administered to insects a purified form.
  • the toxins may also be delivered in amounts from about 1 to about 100 mg / liter of broth. This may vary upon formulation condition, conditions of the inoculum source, techniques for isolation of the toxm, and the like.
  • the toxins may be administered as an exudate secretion or cellular protein originally expressed in a heterologous prokaryotic or eukaryotic host Bacteria are typically the hosts in which proteins are expressed.
  • Eukaryotic hosts could include but are not limited to plants, insects and yeast.
  • the toxins may be produced in bacteria or transgenic plants in the field or in the insect by a baculovirus vector.
  • the toxins will be introduced to the insect by incorporating one or more of the toxins into the insects' feed
  • SUBST ⁇ UTE SHEET (RULE 26) Complete lethality to feeding insects is useful but is not required to achieve useful toxicity. If the insects avoid the toxm or cease feeding, that avoidance will be useful m some applications, even if the effects are sublethal . For example, if insect resistant transgenic crop plants are desired, a reluctance of insects to feed on the plants is as useful as lethal toxicity to the insects since the ultimate objective is protection of the plants rather than killing the insect.
  • toxins can be incorporated into an insect's diet.
  • 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 m the soil in an area m which insect eradication was desirable.
  • the protein could be genetically engineered directly into an insect food source.
  • the major food source of many insect larvae is plant material.
  • Such techniques include acceleration of genetic material coated onto icroparticles directly into cells (U.S. Patents 4,945,050 to Georgia and 5,141,131 to DowElanco) . Plants may be transformed using Agrrobacterium technology, see U.S. Patent 5,177,010 to University of Toledo, 5,104,310 to Texas A&M,
  • European Patent Application 0131624B1 European Patent Applications 120516, 159418B1 and 176,112 to Schilperoot
  • U.S. Patents 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 MaxPlanck
  • European Patent Applications 604662 and 627752 to Japan Tobacco
  • European Patent Applications 0267159, and 0292435 and U.S. Patent 5,231,019 all to Ciba Ge gy
  • U.S Patents 5,463,174 and 4,762,785 both to Calgene
  • SUBST ⁇ UTE SHEET (RULE 25) 5,159,135 both to Agracetus .
  • Other transformation technology includes whiskers technology, see U.S. Patents 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, 5,472,869 and 5,384,253 both to Dekalb, WO9209696 and W09321335 both to PGS . All of these transformation patents and publications are incorporated by reference. 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 type I and II, hypocotyl , meristem, and the like. Almost all plant tissues may be transformed during dedifferentiation using appropriate techniques withm the skill of an artisan. Another variable is the choice of a selectable marker. The 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.
  • selectable markers include but are not limited to ammoglycoside phosphotransferase gene of transposon Tn5 (Aph II) which encodes resistance to the antibiotics kana ycm, neomycm and G418, as well as those genes which code for resistance or tolerance to glyphosate; hygromycm; methotrexate; phosphmothric (bialophos) ; lmidazolmones, sulfonylureas and triazolopyrimidme herbicides, such as chlorosulfuron,- bromoxynil, dalapon and the like.
  • reporter gene In addition to a selectable marker, it may be desirous to use a reporter gene . In some instances a reporter gene may be used without a selectable marker. Reporter genes are genes which are typically not present or expressed in the recipient organism or tissue. The reporter gene typically encodes for a protein which provides for some phenotypic change or enzymatic property Examples of such genes are provided in K. Weismg et al . Ann. Rev. Genetics, 22, 421 (1988), which is incorporated herein by reference. A preferred reporter gene is the glucuronidase (GUS) gene .
  • GUS glucuronidase
  • the gene is preferably incorporated into a gene transfer vector adapted to express the Photorhabdus toxins in the plant cell by including m the vector a plant promoter.
  • promoters from a variety of sources can be used efficiently in plant cells to express foreign genes.
  • promoters of bacterial origin such as the octop e synthase promoter, the nopalme synthase
  • SUBST ⁇ TUTE SHEET (RULE 26) promoter the marmop e synthase promoter; promoters of viral origin, such as the cauliflower mosaic virus (35S and 19S) , reengineered 35S, known as 35T (see PCT/US96/16582 , WO 97/13402 published April 17, 1997, which is incorporated herein by reference) and the like may be used.
  • Plant promoters include, but are not limited to r ⁇ bulose-1 , 6-b ⁇ sphosphate (RUBP) carboxylase small subunit (ssu) , beta-conglyc in promoter, phaseolm promoter, ADH promoter, heat-shock promoters and tissue specific promoters.
  • Promoters may also contain certain enhancer sequence elements that may improve the transcription efficiency. Typical enhancers include but are not limited to Adh-mtron 1 and Adh- intron 6 Constitutive promoters may be used. Constitutive promoters direct continuous gene expression in all cells types and at all times (e.g., act , ubiquitin, CaMV 35S) Tissue specific promoters are responsible for gene expression specific cell or tissue types, such as the leaves or seeds (e.g., zein, oleosin, napin, ACP) and these promoters may also be used. Promoters may also be are active during a certain stage of the plants' development as well as active in plant tissues and organs. Examples of such promoters include but are not limited to pollen-specific, embryo specific, corn silk specific, cotton fiber specific, root specific, seed endosperm specific promoters and the like.
  • an mducible promoter 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, and stress.
  • a specific signal such as. physical stimulus (heat shock genes) , light (RUBP carboxylase) ; hormone (Em); metabolites, and stress.
  • Other desirable transcription and translation elements that function plants may be used.
  • Numerous plant-specific gene transfer vectors are known to the art.
  • SUBST ⁇ UTE SHEET (RULE 26) transcription of the gene(s) .
  • 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
  • AAUAAA polyadenylation signal sequences
  • plant optimized gene(s) one goal in the design of reengmeered bacterial gene(s), 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 plant genes coding for metabolic enzymes.
  • Another goal m the design of the plant optimized gene(s) is to generate a DNA sequence that not only has a higher G+C content, but by modifying the sequence changes, should be made so as to not hinder translation.
  • G+C content An example of a plant that has a high G+C content is maize.
  • the table below illustrates how high the G+C content is in maize. As in maize, it is thought that G+C content n other plants is also high.
  • SUBST ⁇ JTE SHEET (RULE 26) 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 MacVectorTM program (IBI, New Haven, CT) . Intron sequences were ignored in the calculations. Group I and II storage protein gene sequences were distinguished by their marked difference m base composition.
  • the codon bias is the statistical codon distribution that the plant uses for coding its proteins 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 .
  • the amino acid sequence of the protein of interest is reverse translated so that the resulting nucleic acid sequence codes for the same protein as the native bacterial gene, but the resulting nucleic acid sequence corresponds to the first preferred codons of the desired plant
  • the new sequence is 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.
  • exon -.intron 5' or 3 ' junctions Other sites in the sequence which could affect the transcription or translation of the gene of interest are the exon -.intron 5' or 3 ' junctions, 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.
  • SUBST ⁇ UTE SHEET (RULE 26) 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.
  • the plant optimized genets contains about 63% of first choice codons, between about 22% to about 37% second choice codons, and between 15% and 0% third choice codons, wherein the total percentage is 100%.
  • Most preferred the plant optimized gene(s) contain 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 ammo acid sequence of the toxins are reverse translated into a DNA sequence, utilizing a nonredundant genetic code established from a codon bias table compiled for the gene DNA sequence for the particular plant being transformed
  • 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.
  • bacterial genes may be more easily expressed n plants if the bacterial genes are expressed in the plastids. Thus, it may be possible to express bacterial genes in plants, without optimizing the genes for plant expression, and obtain high express of the protein. See U.S. Patent Nos. 4,762,785; 5,451,513 and 5,545,817, which are incorporated herein by reference.
  • SUBST ⁇ TUTE SHEET for a sprayable application or could be molecular combinations.
  • Plants could be transformed with Photorhabdus genes that produce insect toxins and other insect toxm genes such as Bt as with other insect tox genes such as Bt.
  • European Patent Application 0400246A1 describes transformation of 2 Bt m a plant, which could be any 2 genes.
  • Another way to produce a transgenic plant that contains more than one insect resistant gene would be to produce two plants, with each plant containing an insect resistant gene. These plants would be backcrossed using traditional plant breeding techniques to produce a plant containing more than one insect resistant gene.
  • a genetically engineered, easily isolated protein tox fusing together both a molecule attractive to insects as a food source and the msecticidal activity of the toxin may be engineered and expressed in bacteria or m eukaryotic cells using standard, well-known techniques. After purification in the laboratory such a toxic agent with "built-in" bait could be packaged inside standard insect trap housings.
  • 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 Photorhabdus toxins. Infectious baculovirus harboring an expression construct for the Photorhabdus toxins could be introduced into areas of insect infestation to thereby intoxicate or poison infected insects.
  • Transfer of the msecticidal properties requires nucleic acid sequences encoding the coding the ammo acid sequences for the Photorhabdus 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 msecticidal properties is to isolate the native genetic material which produces the toxins from Photorhabdus, using information deduced from the toxin's ammo acid sequence, large portions of which are set forth below. As described below, methods of purifying the proteins responsible for tox activity are also disclosed.
  • oligonucleotides complementary to all, or a section of, the DNA bases that encode the first am o acids of the tox can be constructed. These oligonucleotides can be radiolabeled and used as
  • SUBST ⁇ UTE SHEET molecular probes to isolate the genetic material from a genomic genetic library built from genetic material isolated from strains of Photorhabdus.
  • the genetic library can be cloned in plasmid, cosmid, phage or phagemid vectors.
  • the library could be transformed into Escherichia coli and screened for toxm production by the transformed cells using antibodies raised against the tox or direct assays for insect toxicity.
  • the degenerate genetic code allows an ammo acid to be encoded the DNA by any of several three-nucleotide combinations.
  • the ammo acid argmme can be encoded by nucleic acid triplets CGA, CGC, CGG, CGT, AGA, and AGG. Since one cannot predict which triplet is used at those positions the tox gene, one must prepare oligonucleotides with each potential triplet represented. More than one DNA molecule corresponding to a protein subunit may be necessary to construct a sufficient number of oligonucleotide probes to recover all of the protein subunits necessary to achieve oral toxicity.
  • a typical expression vector is a DNA plasmid, though other transfer means including, but not limited to, cosmids, phagemids and phage are also envisioned.
  • protein expression vectors normar-ly additionally require an expression cassette which incorporates the cis-actmg sequences necessary fcrr-tr-anscription and translation of the gene of interest
  • the cis-actmg sequences required for expression in prokaryotes differ from those required in eukaryotes and plants.
  • a eukaryotic expression cassette requires a transcriptional promoter upstream (5') to the gene of interest, a transcriptional termination region such as a poly-A addition site, and a ribosome binding site upstream of the gene of interest's first codon.
  • a useful transcriptional promoter that could be mcluded in the vector is the T7 RNA Polymerase-binding promoter.
  • Promoters are known to efficiently promote transcription of mRNA Also upstream from the gene of interest the vector may include a nucleotide sequence encoding a
  • SUBST ⁇ UTE SHEET (RULE 26) signal sequence known to direct a covalently linked protein to a particular compartment of the host cells such as the cell surface.
  • Insect viruses or baculoviruses
  • Insect viruses are known to infect and adversely affect certain insects.
  • the affect of the viruses on insects is slow, and viruses do not stop the feeding of insects.
  • viruses are not viewed as being useful as insect pest control agents .
  • Combining the Photorhabdus toxins genes into a baculovirus vector could provide an efficient way of transmitting the toxins while increasing the lethality of the virus.
  • 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.
  • the proteins may be encapsulated using Bacillus thuringiensis encapsulation technology such as but not limited to U.S. Patent Nos. 4,695,455; 4,695,462; 4,861,595 which are all incorporated herein by reference.
  • Bacillus thuringiensis encapsulation technology such as but not limited to U.S. Patent 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.
  • SUBST ⁇ UTE SHEET (RULE 26) resistance development, altered stability, or modified target species specificity.
  • the scope of the present invention is intended to include related nucleic acid sequences which encode ammo acid biopolymers homologous to the toxm proteins and which retain the toxic effect of the Photorhabdus proteins in insect species after oral mgestion.
  • the toxms used in the present invention seem to first inhibit larval feeding before death ensues.
  • genetic engineers placing the tox gene into plants could modulate its potency or its mode of action to, for example, keep the eating- inhibitory activity while eliminating the absolute toxicity to the larvae. This change could permit the transformed plant to survive until harvest without having the unnecessarily dramatic effect on the ecosystem of wiping out all target insects. All such modifications of the gene encoding the toxm, or of the protein encoded by the gene, are envisioned to fall withm the scope of the present invention.
  • nucleic acid examples include the addition of targeting sequences to direct the toxm to particular parts of the insect larvae for improving its efficiency.
  • T ⁇ s t ⁇ s (hydroxymethyl) amino methane
  • SDS sodium dodecyl sulfate
  • EDTA ethylenediammetetraacetic acid
  • IPTG lsopropylthio-B- galactoside
  • X-gal 5-bromo-4-chloro-3-mdoyl-B-D-galactos ⁇ de
  • CTAB cetyltrimethylammonium bromide
  • kbp kilobase pairs
  • - dATP dCTP
  • dGTP dGTP
  • dTTP 2 ' -deoxynucleoside 5 ' -t ⁇ phosphates of adenme, cytosine, guanine, thymme, and mosme, respectively
  • - ATP adenosine 5' t ⁇ phosphate .
  • the msecticidal protein toxm of the present invention was purified from Photorhabdus luminescens strain W-14, ATCC Accession Number 55397 Stock cultures of Photorhabdus luminescens were maintained on petri dishes containing 2% Proteose Peptone No. 3 (i.e., PP3, Difco Laboratories, Detroit MI) in 1.5% agar, incubated at 25°C and transferred weekly. Colonies of the primary form of the bacteria were inoculated into 200 ml of PP3 broth supplemented with 0.5% polyoxyethylene sorbitan mono-stearate (Tween 60, Sigma Chemical Company, St. Louis, MO) in a one liter flask.
  • the broth cultures were grown for 72 hours at 30°C on a rotary shaker.
  • the toxm proteins can be recovered from cultures grown in the presence or absence of Tween,- however, the absence of Tween can affect the form of the bacteria grown and the profile of proteins produced by the bacteria.
  • a variant shift occurs insofar as the molecular weight of at least one identified toxm subunit shifts from about 200 kDa to about 185 kDa.
  • the 72 hour cultures were centrifuged at 10,000 x g for 30 minutes to remove cells and debris
  • the supernatant fraction that contained the msecticidal activity was decanted and brought to 50 mM K 2 HPOoir by adding an appropriate volume of 1.0 M K ⁇ HPO, .
  • the pH was acfjuste ⁇ 3 to 8.6 by adding potassium hydroxide.
  • This supernatant fraction was then mixed with DEAE-Sephace-z (Pharmacia LKB Biotechnology) which had been equilibrated with 50 mM K 2 HP0 4 .
  • the toxic activity was adsorbed to the DEAE resin.
  • This mixture was then poured into a 2.6 x 40 cm column and washed with 50 mM K 2 HP0 4 at room temperature at a flow rate of 30 l/hr until the effluent reached a steady baseline UV absorbance at 280 nm.
  • the column was then washed with 150 mM KC1 until the effluent again reached a steady 280 nm baseline. Finally the column was washed with 300 mM KC1 and fractions were collected.
  • Fractions containing the toxin were pooled and filter sterilized using a 0.2 micron pore membrane filter The toxm was then concentrated and equilibrated to 100 mM KP0 4 , pH 6.9, using an ultraflltration membrane with a molecular weight cutoff of 100 kDa
  • the toxic fractions were pooled and concentrated using the Centr ⁇ prep-100 and were then analyzed by HPLC using a 7.5 mm x 60 cm TSK-GEL G-4000 SW gel permeation column with 100 mM potassium phosphate, pH 6.9 eluent buffer running at 0 4 ml/min.
  • This analysis revealed the toxm protein to be contained with a single sharp peak that eluted from the column with a retention time of approximately 33 6 minutes. This retention time corresponded to an estimated molecular weight of 1,000 kDa. Peak fractions were collected for further purification while fractions not containing this protein were discarded.
  • the peak eluted from the HPLC absorbs UV light at 218 and 280 nm but did not absorb at 405 nm. Absorbance at 405 nm was shown to be an attribute of xenorhabdm antibiotic compounds.
  • Electrophoresis of the pooled peak fractions m a non- denaturing agarose gel showed that two protein complexes are present in the peak.
  • the peak material buffered in 50 mM Tris-HCl, pH 7.0, was separated on a 1.5% agarose stacking gel buffered with 100 mM Tris-HCl at pH 7.0 and 1.9% agarose resolving gel buffered with 200 mM Tris-borate at pH 8.3 under standard buffer conditions (anode buffer 1M Tris-HCl, pH 8.3; cathode buffer 0.025 M Tris, 0.192 M glycme) .
  • the gels were run at 13 mA constant current at 15°C until the phenol red tracking dye reached the end of the gel. Two protein bands were visualized in the agarose gels using Coomassie brilliant blue staining
  • the slower migrating band was referred to as "protein band 1" and faster migrating band was referred to as "protein band 2.”
  • the two protein bands were present m approximately equal amounts .
  • the Coomassie stained agarose gels were used as a guide to precisely excise the two protein bands from unstained portions of the gels.
  • the excised pieces containing the protein bands were macerated and a small amount of sterile water was added.
  • As a control a portion of the gel that contained no protein was also excised and treated the same manner as the gel pieces containing the protein. Protein was recovered from the gel pieces by electroelution into
  • HPLC-purifled toxm When HPLC-purifled toxm was applied to larval diet at a concentration of 7.5 ⁇ g/larva, it caused a halt m larval weight gam (24 larvae tested) . The larvae begin to feed, but after consuming only a very small portion of the toxm treated diet they began to show pathological symptoms induced by the toxm and the larvae cease feeding. The insect frass became discolored and most larva showed signs of diarrhea. Significant insect mortality resulted when several 5 ⁇ g toxm doses were applied to the diet over a 7-10 day period.
  • Agarose-separated protein band 1 significantly inhibited larval weight gam at a dose of 200 ng/larva.
  • Larvae fed similar concentrations of protein band 2 were not inhibited and gained weight at the same rate as the control larvae .
  • SUBST ⁇ JTE SHEET (RULE 26) Further analysis of protein bands 1 and 2 by SDS-PAGE under denaturing conditions showed that each band was composed of several smaller protein subunits. Proteins were visualized by Coomassie brilliant blue staining followed by silver staining to achieve maximum sensitivity.
  • Protein band 1 contains 8 protein subunits of 25.1, 56.2, 60.8, 65.6, 166, 171, 184 and 208 kDa
  • Protein band 2 had an identical profile except that the 25.1, 60.8, and 65.6 kDa proteins were not present.
  • the 56.2, 60.8, 65.6, and 184 kDa proteins were present in the complex of protein band 1 at approximately equal concentrations and represent 80% or more of the total protein content of that complex.
  • the native HPLC-purifled toxm was further characterized as follows.
  • the toxm was heat labile in that after being heated to 60°C for 15 minutes it lost its ability to kill or to inhibit weight gam when injected or fed to Manduca sexta larvae
  • Assays were designed to detect lipase, type C phospholipase, nuclease or red blood cell hemolysis activities and were performed with purified toxin. None of these activities were present.
  • Antibiotic zone inhibition assays were also done and the purified toxin failed to inhibit growth of Gram-negative or -positive bacteria, yeast or filamentous fungi, indicating that the toxic is not a xenorhabd antibiotic.
  • the native HPLC-purifled tox was tested for ability to kill insects other than Manduca sexta Table 3 lists insects killed by the HPLC-purifled Photorhabdus luminescens toxin in this study.
  • Mealworm Coleoptera Tenebrio mol i tor Oral Pharaoh ant Hymenoptera Mono orium pharoanis Oral
  • the tox protein complex was subjected to further characterization from W-14 growth medium
  • the culture conditions and initial purification steps through the S-400 HR column were identical to those described above
  • the toxic fractions were equilibrated with 10 mM Tris- HCl, pH 8.6, and concentrated in the centriplus 100 (Amicon) concentrators
  • the protein toxin complex was then applied to a weak anion exchange (WAX) column, Vydac 301VPH575 (Hesparia, CA) , at a flow rate of 0.5 ml/mm.
  • WAX weak anion exchange
  • the proteins were eluted with a linear potassium chloride gradient, 0-250 mM KC1 , in 10 mM Tris-HCl pH 8.6 for 50 mm. Eight protein peaks were detected by absorbance at 280 nm. Bioassays using neonate southern corn rootworm (JD ⁇ abrot ⁇ ca undeci punctata howardi , SCR) larvae and tobacco horn worm [Manduca sexta , THW) were performed on all fractions eluted from the HPLC column. THW were grown on Gypsy Moth wheat germ diet (ICN) at 25 ⁇ C with a 16 hr light 8 hr dark cycle. SCR were grown on Southern Corn Rootworm Larval Insecta-Diet (BioServ) at 25°C with a 16 hr light / 8 hr dark cycle.
  • ICN Gypsy Moth wheat germ diet
  • BioServ Southern Corn Rootworm Larval Insecta-Diet
  • Peak 6 which was further analyzed by native agarose gel electrophoresis, as described herein, migrated as a single band with similar mobility to that of band 1.
  • the protein concentration of the purified peak 6 tox protein was determined using the BCA reagents (Pierce) Dilutions of the protein were made in 10 mM Tris, pH 8.6 and applied to the diet bioassays. After 240 hours all neonate larvae on diet bioassays that received ng or greater of the peak 6 protein fraction were dead. The grou of larvae that received 90 ng of the same fraction
  • SUBST ⁇ UTE SHEET (RULE 26) had 40% mortality. After 240 hrs the survivors that received 90 ng and 20 ng of peak 6 protein fraction were ca. 10% and 70%, respectively, of the control weight.
  • Photorhabdus luminescens utility and toxicity were further characterized.
  • Photorhabdus luminescens (strain W-14) culture broth was produced as follows.
  • the production medium was 2% Bacto Proteose Peptone ® Number 3 (PP3, Difco Laboratories, Detroit, Michigan) in Milli-Q * deionized water.
  • Photorhabdus culture broth and protein toxm(s) purified from this broth showed activity (mortality and/or growth inhibition, reduced adult emergence) against a number of insects More specifically, the activity is seen against corn rootworm (larvae and adult) , Colorado potato beetle, and turf grubs, which are members of the insect order Coleoptera .
  • Other members of the Coleoptera include wireworms, pollen beetles, flea beetles, seed beetles and weevils.
  • Activity has also been observed against aster leafhopper, which is a member of the order, Homoptera
  • Other members of the Homoptera include planthoppers, pear pyslla, apple
  • SUBST ⁇ UTE SHEET sucker, scale insects, whiteflies, and spittle bugs, as well as numerous host specific aphid species.
  • the broth and purified fractions are also active against beet armyworm, cabbage looper, black cutworm, tobacco budworm, European corn borer, corn earworm, and codling moth, which are members of the order Lepidoptera .
  • Other typical members of this order are clothes moth, Indian mealmoth, leaf rollers, cabbage worm, cotton bollworm, bagworm, Eastern tent caterpillar, sod webworm, and fall armyworm Activity is also seen against fruitfly and mosquito larvae, which are members of the order Diptera.
  • Diptera Other members of the order Diptera are pea midge, carrot fly, cabbage root fly, turnip root fly, onion fly, crane fly, house fly, and various mosquito species. Activity is seen against carpenter ant and Argentine ant, which are members of the order that also includes fire ants, oderous house ants, and little black ants.
  • the broth/fraction is useful for reducing populations of insects and were used in a method of inhibiting an insect population.
  • the method may comprise applying to a locus of the insect an effective insect inactivating amount of the active described. Results are reported in Table 4.
  • the diet plates were allowed to air-dry in a sterile flow-hood and the wells were infested with single, neonate Diabrotica undecimpunctata howardi (Southern corn rootworm, SCR) hatched from sterilized eggs, with second nstar SCR grown on artificial diet or with second instar Diabrotica virgif ra virgifera (Western corn rootworm, WCR) reared on corn seedlings grown in Metromix . Second instar larvae were weighed prior to addition to the diet. The plates were sealed, placed in a humidified growth chamber and maintained at 27°C for the appropriate period (4 days for neonate and adult SCR, 2-5 days for WCR larvae, 7-14 days for second instar SCR) .
  • SUBST ⁇ UTE SHEET (RULE 26) 50 ⁇ l of treatment and was allowed to air dry. Individual second instar Colorado potato beetle [Leptmotarsa decemlinea ta , CPB) larvae were then placed onto the diet and mortality was scored after 4 days Ten larvae per treatment were used in all studies. Control mortality was 3.3%.
  • Turf grubs [ Popillia japonica , 2-3rd instar) were collected from infested lawns and maintained in the laboratory in soil/peat mixture with carrot slices added as additional diet. Turf beetles were pheromone-trapped locally and maintained m the laboratory in plastic containers with maple leaves as food Following application of undiluted Photorhabdus culture broth or control medium to corn rootworm artificial diet (30 ⁇ l/1.54 cm 2 , beetles) or carrot slices (larvae), both stages were placed singly m a diet well and observed for any mortality and feeding In both cases there was a clear reduction in the amount of feeding (and feces production) observed.
  • Activity against mosquito larvae was tested as follows. The assay was conducted in a 96-well microtiter plate Each well contained 200 ⁇ l of aqueous solution [ Photorhabdus culture broth, control medium or H 2 0) and approximately 20, 1-day old larvae [Aedes aegypti ) There were 6 wells per treatment. The results were read at 2 hours after infestation and did not change over the three day observation period. No control mortality was seen.
  • Activity against fruitflies was tested as follows. Purchased Drosophila elanogaster medium was prepared using 50% dry medium and a 50% liquid of either water, control medium or Photorhabdus culture broth.
  • the mgestion assay for aster leafhopper [Macrosteles severini ) is designed to allow mgestion of the active without other external contact
  • the reservoir for the active/ "food” solution is made by making 2 holes in the center of the bottom portion of a 35 x 10 mm Petri dish. A 2 inch Parafilm " square is placed across the top of
  • SUBST ⁇ JTE SHEET (RULE 26) the dish and secured with an "O" ring.
  • a 1 oz. plastic cup is then infested with approximately 7 leafhoppers and the reservoir is placed on top of the cup, Parafilm down.
  • the test solution is then added to the reservoir through the holes.
  • the broth and control medium were dialyzed against water to reduce control mortality Mortality is reported at day 2 where 26.5% control mortality was seen.
  • purified fractions 200 mg protem/ml
  • a final concentration of 5% sucrose was used in all treatments to improve survivability of the aster leafhoppers.
  • the assay was held in an incubator at 28 °C, 70% RH with a 16/8 photoperiod.
  • the assay was graded for mortality at 72 hours. Control mortality was 5.5%.
  • Photorhabdus culture broth were performed as follows. Each plastic bioassay container (7 1/8" x 3") held fifteen workers, a paper harborage and 10 ml of broth or control media m a plastic shot glass. A cotton wick delivered the treatment to the ants through a hole m the shot glass lid. All treatments contained 5% sucrose Bioassays were held in the dark at room temperature and graded at 19 days. Control mortality was 9%. Assays delivering purified fractions utilized artificial ant diet mixed with the treatment (purified fraction or control solution) at a rate of 0.2 ml treatment/2.0 g diet in a plastic test tube.
  • the final protein concentration of the purified fraction was less than 10 ⁇ g/g diet
  • Ten ants per treatment, a water source, harborage and the treated diet were placed in sealed plastic containers and maintained m the dark at 27 °C in a humidified incubator Mortality was scored at day 10. No control mortality was seen.
  • SUBST ⁇ UTE SHEET (RULE 26) applied directly to the surface (about 1.5 cm ) of 0.25 ml of standard artificial diet in 30 ⁇ l aliquots following dilution in control medium or 10 mM sodium phosphate buffer, pH 7.0, respectively.
  • the diet plates were allowed to air-dry in a sterile flow-hood and the wells were infested with single, neonate larva.
  • European corn borer Ostrinia nubilalis
  • corn earworm corn earworm [Helicoverpa zea) eggs were supplied from commercial sources and hatched m-house, whereas beet armyworm [ ⁇ podoptera exigua) , cabbage looper [ Trichoplusia ni ) , tobacco budworm [Heliothis virescens) , codling moth (Laspeyresia pomonella) and black cutworm [Agrotis ipsilon) larvae were supplied internally. Following infestation with larvae, the diet plates were sealed, placed a humidified growth chamber and maintained in the dark at 27 °C for the appropriate period.
  • SUBST ⁇ UTE SHEET (RULE 26) Metromix ® was tested as follows (Table 5) . The test was run using corn seedlings (United Agriseeds brand CL614) that were germinated m the light on moist filter paper for 6 days. After roots were approximately 3-6 cm long, a single kernel/seedlmg was planted in a 591 ml clear plastic cup with 50 gm of dry MetromixX Twenty neonate SCR or WCR were then placed directly on the roots of the seedling and covered with Metromix". Upon infestation, the seedlings were then drenched with 50 ml total volume of a diluted broth solution. After drenching, the cups were sealed and left at room temperature in the light for 7 days.
  • the unsealed cups were incubated in a high relative humidity chamber (80%) at 78°F Afterwards, the seedlings were washed to remove all soil and the roots " //ere excised and weighed. Activity was rated as the percentage of corn root remaining relative to the ⁇ c ⁇ ⁇ rtrrol plants and as leaf damage induced by feeding. Leaf damage was scored visually and rated as either -, +, ++, or +++, with - representing no damage and +++ representing severe damage.
  • SUBST ⁇ UTE SHEET (RULE 26) removed and replaced with fresh larvae.
  • the cups were sealed and placed in a 28 °C incubator, in the dark. After seven days, larvae were removed from the Metromix" and scored for mortality. Activity was rated the percentage of mortality relative to control .
  • Control medium (1.0% w/v) 12/19 63
  • Photorhabdus broth against European corn borer was seen when the broth was applied directly to the surface of maize leaves (Table 8)
  • Photorhabdus broth was diluted 100 -fold with culture medium and applied manually to the surface of excised maize leaves at a rate of about 6.0 ⁇ l/cm 2 of leaf surface.
  • the leaves were air dried and cut into equal sized strips approximately 2 x 2 inches.
  • the leaves were rolled, secured with paper clips and placed in 1 oz plastic shot glasses with 0.25 inch of 2% agar on the bottom surface to provide moisture. Twelve neonate European corn borers were then placed onto the rolled leaf and the cup was sealed. After incubation for 5 days at 27°C in the dark, the samples were scored for feeding damage and recovered larvae .
  • SUBSTTTUTE SHEET (RULE 26) polyacrylamide electrophoresis gel with a ratio of 30:0.8 (acrylamide : BIS-acrylamide) . This gel matrix facilitates better resolution of the larger proteins.
  • the gel system used to estimate the Band 1 and Band 2 subunit molecular weights Example 1 was an 18% gel with a ratio of 38:0.18 (acrylamide :BIS-acrylam ⁇ de) , which allowed for a broader range of size separation, but less resolution of higher molecular weight components.
  • Am o acid sequences were determined for the N- terminal portions of five of the 10 resolved peptides Table 10 + correlates the molecular weight of the proteins and the identified sequences.
  • certain analyses suggest that the prol e at residue 5 may be an asparag e (asn) .
  • SEQ ID NO: 3 certain analyses suggest that the am o acid residues at positions 13 and 14 are both arg ine (arg)
  • SEQ ID NO: 4 certain analyses suggest that the ammo acid residue at position 6 may be either alanme (ala) or ser e (ser) .
  • SEQ ID NO: 5 certain analyses suggest that the ammo acid residue at position 3 may be aspartic acid (asp) .
  • New N-terminal sequence SEQ ID NO: 15, Ala Gin Asp Gly Asn Gin Asp Thr Phe Phe Ser Gly Asn Thr, was obtained by further N- terminal sequencing of peptides isolated from Native HPLC-purifled tox as described m Example 5, Part A, above.
  • This peptide comes from the tcaA gene.
  • the peptide labeled TcaA n starts at position 254 and goes to position 491, where the TcaA xll peptide starts, SEQ ID NO: 4
  • the toxm protein complex was re- lsolated from the Photorhabdus luminescens growth medium (after culture without Tween) by performing a 10% - 80% ammonium sulfate precipitation followed by an ion exchange chromatography step (Mono Q) and two molecular sizing chromatography steps. These conditions were like those used in Example 1 During the first molecular sizing step, a second biologically active peak was found at about 100 ⁇ 10 kDa. Based upon protein measurements, this fraction was 20 - 50 fold less active than the larger, or primary, active peak of about 860 ⁇ 100 kDa (native) .
  • a second, prominent 185 kDa protein was consistently present in amounts comparable to that of protein 3 from Table 10, and may be the same protein or protein fragment.
  • the N-terminal sequence of this 185 kDa protein is shown at SEQ ID NO: 7. Additional N-terminal amino acid sequence data were also obtained from isolated proteins. None of the determined N-terminal sequences appear identical to a protein identified in Table 10. Other proteins were present in isolated preparation.
  • One such protein has an estimated molecular weight of 108 kDa and an N- terminal sequence as shown in SEQ ID NO: 8.
  • a second such protein has an estimated molecular weight of 80 kDa and an N-terminal sequence as shown in SEQ ID NO: 9.
  • the toxic activity was also retained by a dialysis membrane, again confirming the large size of the native tox complex
  • ammonium sulfate precipitation of Photorhabdus proteins was performed by adjusting Photorhabdus broth, typically 2-3 liters, to a final concentration of either 10% or 20% by the slow addition of ammonium sulfate crystals. After stirring for 1 hour at 4°C, the material was centrifuged at 12,000 x g for 30 minutes.
  • the supernatant was ad ⁇ usted to 80% ammonium sulfate, stirred at 4°C for 1 hour, and centrifuged at 12,000 x g for 60 minutes The pellet was resuspended in one-tenth the volume of 10 mM Na 2 P0 4 , pH
  • proteins could be eluted by a single 0.4 M NaCl wash without prior elution with 0.1 M NaCl.
  • SUBST ⁇ TUTE SHEET (RULE 26) collected and concentrated using a Centriprep 100. Two milliliter aliquots of concentrated Superose 12 samples were loaded at 0.5 ml/min onto a HR 16/50 Sepharose 4B-CL (Pharmacia) gel filtration column equilibrated with 10 mM Na 2 ' P0 4 , pH 7.0. The column was washed with the same buffer for 240 min at 0.5 ml/min and 2 min samples were collected.
  • the excluded protein peak was subjected to a second fractionation by application to a gel filtration column that used a Sepharose CL-4B resin, which separates proteins ranging from about 30 kDa to 1000 kDa. This fraction was resolved into two peaks,- a minor peak at the void volume (>1000 kDa) and a major peak which eluted at an apparent molecular weight of about 860 kDa. Over a one week period subsequent samples subjected to gel filtration showed the gradual appearance of a third peak (approximately 325 kDa) that seemed to arise from the major peak, perhaps by limited proteolysis.
  • Milligram quantities of peak toxin complex fractions determined to be "P" or "S” peptide patterns were subjected to preparative SDS PAGE, and transblotted with TRIS-glycine (SeprabuffTM to PVDF membranes (ProBlottTM, Applied Biosystems) for 3-4 hours. Blots were sent for amino acid analysis and N-terminal amino acid sequencing at Harvard MicroChem and Cambridge ProChem, respectively. Three peptides in the "S" pattern had unique N- terminal amino acid sequences compared to the sequences identified in the previous example.
  • TcdA ⁇ A 201 kDa (TcdA ⁇ ) peptide set forth as SEQ ID NO: 13 below shared between 33% amino acid identity and 50% similarity (similarity and identity were calculated by hand) with SEQ ID NO:l (TcbA ⁇ ) (in Table 10 vertical lines denote ammo acid
  • a limited N- terminal ammo acid sequence, SEQ ID NO:16 (TcbA), of a peptide of at least 235 kDa was identical with the ammo acid sequence, SEQ ID NO:12, deduced from a cloned gene [ tcbA) , SEQ ID NO:ll, containing a deduced ammo acid sequence corresponding to SEQ ID NO:l (TcbA i) .
  • TcaBx (68 kDa peptide)
  • TcaBi-PTl ⁇ (SEQ ID NO: 19)
  • TcaBi-PTlO ⁇ (SEQ ID NO: 20)
  • TcbA 11 -PT103 (SEQ ID N0:21)
  • TcbA ⁇ :L -PT56 (SEQ ID NO:22)
  • TcbA ⁇ ;L -PT81 (a) (SEQ ID NO: 23)
  • TcbAn-PT81 (b) (SEQ ID NO: 24).
  • I I I I F I Q G Y S D L F G N - A SEQ ID NO : 1
  • Photorhabdus luminescens strain W-14 (ATCC accession number 55397) was grown on 2% proteose peptone #3 agar (Difco Laboratories, Detroit, MI) and msecticidal tox competence was maintained by repeated bioassay after passage, using the method described in Example 1 above.
  • a 50 ml shake culture was produced in a 175 ml baffled flask in 2% proteose peptone #3 medium, grown at 28°C and 150 rpm for approximately 24 hours. 15 ml of this culture was pelleted and frozen in its medium at -20°C until it was thawed for DNA isolation.
  • Genomic DNA was isolated by an adaptation of the CTAB method described in section 2.4.1 of Current Protocols m Molecular Biology (Ausubel et al .
  • the pelleted bacterial cells were resuspended m TE buffer (10 mM T ⁇ s- HCl, 1 mM EDTA, pH 8.0) to a final volume of 10 ml, then 12 ml of 5 M NaCl was added; this mixture was centrifuged 20 mm at 15,000 x g. The pellet was resuspended in 5.7 ml TE and 300 ml of 10% SDS and 60 ml of 20 mg/ l protemase K (Gibco BRL Products, Grand Island, NY; sterile distilled water) were added to the suspension.
  • m TE buffer 10 mM T ⁇ s- HCl, 1 mM EDTA, pH 8.0
  • 12 ml of 5 M NaCl was added; this mixture was centrifuged 20 mm at 15,000 x g.
  • the pellet was resuspended in 5.7 ml TE and 300 ml of 10% SDS and 60 ml of 20 mg/
  • the DNA was precipitated with 0.6 volume of isopropanol.
  • the DNA precipitate was gently removed with a glass rod, washed twice with 70% ethanol, dried, and dissolved in 2 ml STE (10 mM Tris-HCl pH 8.0 , 10 mM NaCl, 1 mM EDTA) .
  • This preparation contained 2 5 mg/ml DNA, as determined by optical density at 260 nm (i.e., OD 260 ) .
  • the molecular size range of the isolated genomic DNA was evaluated for suitability for library construction.
  • CHEF gel analysis was performed in 1.5% agarose (Seakem ⁇ LE, FMC BioProducts, Rockland, ME) gels with 0.5 X TBE buffer (44.5 mM Tris-HCl pH 8.0, 44.5 mM H 3 B0 3 , 1 mM EDTA) on a BioRad CHEF-DR II apparatus with a Pulsewave 760 Switcher (Bio-Rad Laboratories, Inc., Richmond, CA) .
  • the running parameters were: initial A time, 3 sec,- final A time, 12 sec; 200 volts; running temperature, 4-18°C; run time, 16.5 hr . Ethidium bromide staining and examination of the gel under ultraviolet light indicated the DNA ranged from 30-250 kbp in size.
  • the supernatant was removed and the precipitate was dried in a vacuum oven at 40°C, then resuspended m 400 ⁇ l STE .
  • Spectrophotomet ⁇ c assay indicated about 40% recovery of the input DNA.
  • the digested DNA was size fractionated on a_ sucrose gradient according to section 5.3.2 of CPMB [ op . ci t . ) .
  • a 10% to 40% (w/v) linear sucrose gradient was prepared with a gradient maker m Ultra-ClearTM tubes (Beckman Instruments, Inc., Palo Alto, CA) and the DNA sample was layered on top.
  • the DNA was collected by centrifugation (17,000 x g, 15 mm), dried, redissolved in TE, pooled into a final volume of 80 ⁇ l , and reprecipitated with the addition of 8 ⁇ l 3 M sodium acetate and 220 ⁇ l ethanol. The pellet collected by centrifugation as above was resuspended in 12 ⁇ l TE. Concentration of the DNA was determined by Hoechst 33258 dye (Polysciences, Inc., Warrmgton, PA) fluorometry a Hoefer TKO100 fluo ⁇ meter (Hoefer Scientific Instruments, San Francisco, CA) . Approximately 2.5 ⁇ g of the size-fractionated DNA was recovered.
  • Ligation of the size-fractionated Sau3A 1 fragments to the BamH 1 -digested and phosphatased pWE15 vector was accomplished using T4 ligase (NEB) by a modification (i.e., use of premixed 10X ligation buffer supplied by the manufacturer) of the protocol section 3.33 of Ausubel. Ligation was carried out overnight m a total volume of 20 ⁇ l at 15°C, followed by storage at - 20°C.
  • SUBST ⁇ UTE SHEET (RULE 26) (Stratagene) according to the G ⁇ gapa,ck" III Gold protocols ("Titermg the Cosmid Library"), as follows.
  • XLI Blue MR cells were grown in LB medium (g/L: Bacto-tryptone , 10; Bacto-yeast extract, 5; Bacto-agar, 15; NaCl, 5; [Difco Laboratories, Detroit, MI]) containing 0.2% (w/v) maltose plus 10 mM MgS0 4 , at 37°C. After 5 hr growth, cells were pelleted at 700 x g (15 min) and resuspended m 6 ml of 10 mM MgSO, .
  • the packaged cosmid library was diluted 1:10 or 1.20 with sterile SM medium (0 1 M NaCl, 10 mM MgSO, 50 mM Tris-HCl pH 7.5, 0.01% w/v gelatin), and 25 ⁇ l of the diluted preparation was mixed with 25 ⁇ l of the diluted XLI Blue MR cells. The mixture was incubated at 25°C for 30 mm (without shaking) , then 200 ⁇ l of LB broth was added, and incubation was continued for approximately 1 hr with occasional gentle shaking.
  • a positive control colony (a bacterial clone containing a GZ4 sequence insert, see below) was grown on a separate Magna NT membrane (Nunc, 0.45 micron, 82 mm circle) on LB medium supplemented with 35 mg/1 chloramphenicol (i.e., LB-Cam 35 ) , and processed alongside the library colony membranes.
  • a pool of degenerate oligonucleotides (pool S4Psh) was synthesized by standard ⁇ -cyanoethyl chemistry on an Applied BioSystem ABI394 DNA7RNA Synthesizer (Perkm Elmer, Foster City, CA) .
  • the oligonucleotides were deprotected 8 hours at 55°C ⁇ "" dissolved in water, quantitated by spectrophotometric measurement, and diluted for use.
  • This pool corresponds to the determined N-termmal ammo acid sequence of the TcaC peptide.
  • the determined am o acid sequence and the corresponding degenerate DNA sequence are given below, where A, C, G, and T are the standard DNA bases, and I represents mosme:
  • oligonucleotides were used as primers in Polymerase Cham Reactions (PCR S , Roche Molecular Systems, Branchburg, NJ) to amplify a specific DNA fragment from genomic DNA prepared from Photorhabdus strain W-14 (see above) .
  • a typical reaction contained 125 pmol of each primer pool P2Psh and P2.3.5R, 253 ng of genomic template DNA, 10 nmol each of dATP, dCTP, dGTP, and dTTP, IX GeneAmp"' PCR buffer, and 2.5 units of AmpliTaq " DNA polymerase (both from Roche Molecular Systems,- 10X GeneAmp ® buffer is 100 mM Tris-HCl pH 8.3, 500 mM KC1 , 0.01% w/v gelatin).
  • Amplifications were performed in a Perkm Elmer Cetus DNA Thermal Cycler (Perkin Elmer, Foster City, CA) using 35 cycles of 94°C (1.0 mm), 55°C (2.0 mm), 72°C (3.0 mm), followed by an extension period of 7.0 mm at 72 °C Amplification products were analyzed by electrophoresis through 2% w/v NuSieve" 3:1 agarose (FMC
  • m TEA buffer 40 mM Tris-acetate, 2 mM EDTA, pH 8.0
  • a specific product of estimated size 250 bp was observed amongst numerous other amplification products by ethidium bromide (0.5 ⁇ g/ l) staining of the gel and examination under ultraviolet light.
  • the region of the gel containing an approximately 250 bp product was excised, and a small plug (0.5 mm dia.) was removed and used to supply template for PCR amplification (40 cycles)
  • the reaction 50 ⁇ l) contained the same components as above, minus genomic template DNA.
  • DNA fragments were separated from residual primers by electrophoresis through 1% w/v GTG" agarose (FMC) in TEA.
  • FMC 1% w/v GTG" agarose
  • a gel slice containing fragments of apparent size 250 bp was excised, and the DNA was extracted using a Qiaex kit (Qiagen Inc., Chatsworth, CA) .
  • the extracted DNA fragments were ligated to plasmid vector pBC KS(+) (Stratagene) that had been digested to completion with restriction enzyme Sma 1 and extracted in a manner similar to that desc ⁇ ned for pWE15 DNA above.
  • a typical ligation reaction (16.3 ⁇ l) contained 100 ng of digested pBC KS(+) DNA, 70 ng of 250 bp fragment DNA, 1 nmol [Co (NH 3 ) J Cl 3 , and 3.9 Weiss units of T4 DNA ligase (Collaborative Biomedical Products, Bedford, MA), IX
  • SUBSTITUTE SHEET (RULE 25) ligation buffer (50 mM Tris-HCl, pH.7.4; 10 mM MgCl : ; 10 mM dithiothreitol; 1 mM spermidine, 1 mM ATP, 100 mg/ml bovine serum albumin) . Following overnight incubation at 14 °C, the ligated products were transformed into frozen, competent Escherichia coli DH5 ⁇ cells (Gibco BRL) according to the suppliers' recommendations, and plated on LB-Cam 35 plates , containing IPTG (119 ⁇ g/ l) and X-gal (50 ⁇ g/ml) .
  • ligation buffer 50 mM Tris-HCl, pH.7.4; 10 mM MgCl : ; 10 mM dithiothreitol; 1 mM spermidine, 1 mM ATP, 100 mg/ml bovine serum albumin
  • plasmid DNA was prepared by a modified alkaline-lysis/PEG precipitation method (PRISMTM Ready Reaction DyeDeoxyTM Terminator Cycle Sequencing Kit Protocols; ABI/Perkin Elmer).
  • the nucleotide sequence of both strands of the insert DNA was determined, using T7 primers [pBC KS(+) bases 601-623: TAAAACGACGGCCAGTGAGCGCG) and LacZ primers [pBC KS(+) bases 792-816: ATGACCATGATTACGCCAAGCGCGC) and protocols supplied with the PRISMTM sequencing kit (ABI/Perkin Elmer) .
  • Nonincorporated dye-terminator dideoxyribonucleotides were removed by passage through Centri-Sep 100 columns (Princeton Separations, Inc., Adelphia, NJ) according to the manufacturer's instructions.
  • the DNA sequence was obtained by analysis of the samples on an ABI Model 373A DNA Sequencer (ABI/Perkin Elmer) .
  • the DNA sequences of two isolates, GZ4 and HB14, were found to be as illustrated in Fig. 1.
  • bases 1- 20 represent one of the 64 possible sequences of the S4Psh degenerate oligonucleotides, ii) the sequence of amino acids 1-3 and 6-12 correspond exactly to that determined for the N-terminus of TcaC (disclosed as SEQ ID NO: 2), iii) the fourth amino acid encoded is a cysteine residue rather than serine.
  • the fifth amino acid encoded is proline, corresponding to the TcaC N-terminal sequence given as SEQ ID NO:2, v) bases 257-276 encode one of the 192 possible sequences designed into the degenerate pool, vi ) the TGA termination codon introduced at bases 268-270 is the result of complementarity to the degeneracy built into the oligonucleotide pool at the corresponding position, and does not indicate a shortened reading frame for the corresponding gene .
  • DNA fragments corresponding to the above 276 bases were amplified (35 cycles) by PCR S in a 100 ⁇ l reaction volume, using 100 pmol each of P2Psh and P2.3.5R primers, 10 ng of plasmids GZ4 or HB14 as templates, 20 nmol each of dATP, dCTP, dGTP, and dTTP, 5
  • SUBST ⁇ UTE SHEET (RULE 26) units of AmpliTAq * DNA polymerase, and IX concentration of GeneAmp" buffer, under the same temperature regimes as described above.
  • the amplification products were extracted from a 1% GTG " agarose gel by Qiaex kit and quantitated by fluorometry
  • the extracted amplification products from plasmid HB14 template (approximately 400 ng) were split into five aliquots and labeled with "P-dCTP using the High Prime Labeling Mix (Boehringer Mannheim) according to the manufacturer's instructions.
  • Nonmcorporated radioisotope was removed by passage through NucTrap * Probe Purification Columns (Stratagene) , according to the supplier's instructions.
  • the specific activity of the labeled DNA product was determined by scintillation counting to be 3.11 x 10 ⁇ dpm/ ⁇ g This labeled DNA was used to probe membranes prepared from 800 members of the genomic library.
  • the radiolabeled HB14 probe was boiled approximately 10 mm, then added to "minimal hyb” solution.
  • the "minimal hyb” method is taken from a CERES protocol, "Restriction Fragment Length Polymorphism Laboratory Manual version 4.0", sections 4-40 and 4- 47; CERES/NPI, Salt Lake City, UT. NPI is now defunct, with its successors operating as Linkage Genetics] .
  • "Minimal hyb” solution contains 10% w/v PEG (polyethylene glycol, M.W. approx.
  • the filters were washed three times for approximately 10 mm each at 25°C m "minimal hyb wash solution" (0.25X SSC, 0.2% SDS), followed by two 30-mm washes with slow shaking at 60°C in the same solution.
  • the filters were placed on paper covered with Saran Wrap ' (Dow Brands, Indianapolis, IN) in a light-tight autoradiographic cassette and exposed to X-Omat X-ray film (Kodak, Rochester, NY) with two DuPont Cronex Lightnmg-Plus Cl enhancers (Sigma Chemical Co., St. Louis, MO), for 4 hr at -70°C.
  • the twelve putative hybridization-positive colonies were retrieved from the frozen 96-well library plates and grown overnight at 37°C on solid LB-Amp 100 medium They were then patched (3/plate, plus three negative controls: XLI Blue MR cells containing the pWE15 vector) onto solid LB-Amp 100
  • Two sets of membranes (Magna NT nylon, 0.45 micron) were prepared for hybridization. The first set was prepared by placing a filter directly onto the colonies on a patch plate, then removing it with adherent bacterial cells, and processing as below Filters of the second set were placed on plates containing LB-Amp 100 medium, then inoculated by transferring cells from the patch plates onto the filters. After overnight growth at 37°C, the filters were removed from the plates and processed.
  • Bacterial cells on the filters were lysed and DNA denatured by placing each filter colony-side-up on a pool (1.0 ml) of 0 5 N NaOH in a plastic plate for 3 mm.
  • the filters were blotted dry on a paper towel, then the process was repeated with fresh 0 5 N NaOH.
  • the filters were neutralized by placing each on a 1.0 ml pool of 1 M Tris-HCl, pH 7.5 for 3 min, blotted dry, and reneutralised with fresh buffer. This was followed by two similar soak gs (5 mm each) on pools of 0 5 M Tris-HCl pH 7 5 plus 1.5 M NaCl.
  • the DNA was UV crosslinked to the filter (as above) , and the filters were washed (25°C, 100 rpm) in about 100 ml of 3X SSC plus 0.1% (w/v) SDS (4 times, 30 mm each with fresh solution for each wash) They were then placed in a minimal volume of prehybridization solution [6X SSC plus 1% w/v each of Ficoll 400 (Pharmacia), polyvmylpyrrolidone (av. M.W.
  • the membranes were washed 3 times at 25°C (50 rpm, 15 mm) in 3X SSC (about 150 ml each wash) They were then washed for 3 hr at 68 °C (50 rpm)
  • SUBSTTTUTE SHEET (RULE 26) 0.25X SSC plus 0.2% SDS (minimal hyb wash solution), and exposed to X-ray film as described above for 1.5 hr at 25°C (no enhancer screens) .
  • This exposure revealed very strong hybridization signals to cosmid isolates 22G12, 25A10, 26A5, and 26B10, and a very weak signal with cosmid isolate 8B10.
  • No signal was seen with the negative control (pWE15) colonies, and a very strong signal was seen with positive control membranes (DH5 ⁇ cells containing the GZ4 isolate of the PCR product) that had been processed concurrently with the experimental samples.
  • oligonucleotides were used as primers for PCR * using HotStart 50 TubesTM (Molecular Bio-Products, Inc , San Diego, CA) to amplify a specific DNA fragment from genomic DNA prepared from Photorhabdus strain W-14 (see above).
  • HotStart 50 TubesTM Molecular Bio-Products, Inc , San Diego, CA
  • a typical reaction contained (bottom layer) 25 pmol of each primer pool P8F and P8.108.3R, with 2 nmol each of dATP, dCTP, dGTP, and dTTP, m IX GeneAmp * PCR buffer, and (top layer) 230 ng of genomic template DNA, 8 nmol each of dATP, dCTP, dGTP, and dTTP, and 2 5 units of AmpliTaq * DNA polymerase, in IX GeneAmp " PCR buffer Amplifications were performed by 35 cycles as described for the TcaC peptide Amplification products were analyzed by electrophoresis through 0.7% w/v SeaKem" LE agarose (FMC) in TEA buffer. A specific product of estimated size 1600 bp was observed.
  • the membranes containing the 12 colonies identified m the TcaC-probe library screen were stripped of radioactive TcaC-specific label by boiling twice for approximately 30 mm each time in 1 liter of 0. IX SSC plus 0.1 % SDS. Removal of ra ⁇ iolabel was checked with a 6 hr film exposure The stripped membranes were then incubated with the TcaB peptide-specific probe prepared above The labeled DNA was denatured by boiling for 10 mm, and then added to the filters that had been incubated for L hr in 100 ml of "minimal hyb" solution at 60°C.
  • a 0.15 ⁇ g sample of 26A5 cosmid DNA was used to transform 50 ml of E. coli DH5 ⁇ cells (Gibco BRL) , by the supplier's protocols.
  • a single colony isolate of that strain was inoculated into 4 ml of TB-Amp 100 , and grown for 8 hr at 37°C.
  • Chloramphenicol was added to a final concentration of 225 ⁇ g/ml, incubation was continued for another 24 hr, then cells were harvested by centrifugation and frozen at -20°C.
  • Isolation of the 26A5 cosmid DNA was by a standard alkaline lysis mmiprep (Maniatis et al . , op .
  • cosmid 25A10 from XLI Blue MR cells
  • cosmid 26A5 from chloramphenicol -amplified [ 5 ⁇ cells) were each digested with about 15 units of EcoR I (NEB) for 85 mm, frozen overnight, then heated at 65 °C for five mm, and electrophoresed n a 0.7% agarose gel (Seake * LE, IX TEA, 80 volts, 90 m ) .
  • the DNA was stained with ethidium bromide as described above, and photographed under ultraviolet light.
  • the EcoR I digest of cosmid 25A10 was a complete digestion, but the sample of cosmid 26A5 was only partially digested under these conditions.
  • the agarose gel containing the DNA fragments was subjected to depurmation, denaturation and neutralization, followed by Southern blotting onto a Magna NT nylon membrane, using a high salt (20X SSC) protocol, all as described in section 2.9 of Ausubel et al . (CPMB, op . ci t . )
  • the transferred DNA was then UV-crosslmked to the nylon membrane as before .
  • TcaC-peptide specific DNA fragment corresponding to the insert of plasmid isolate GZ4 was amplified by PCR° in a 100 ml reaction volume as described previously above.
  • the amplification products from three such reactions were pooled and were extracted from a 1% GTG" agarose gel by Qiaex kit, as described above, and quantitated by fluorometry.
  • the gel-purified DNA 100 ng was labeled with 32 P-dCTP using the High Prime Labeling Mix (Boehrmger Mannheim) as described above, to a specific activity of 6 34 x 10 s dpm/ ⁇ g.
  • the 32 P-labeled GZ4 probe was boiled 10 mm, then added to "minimal hyb" buffer (at 1 ng/ml) , and the Southern blot membrane containing the digested cosmid DNA fragments was added, and incubated for 4 hr at 60°C with gentle shaking at 50 rpm. The membrane was then washed 3 times at 25°C for about 5 mm each (minimal hyb wash solution) , followed by two washes for 30 mm each at 60°C.
  • the blot was exposed to film (with enhancer screens) for about 30 mm at -70°C
  • the GZ4 probe hybridized strongly to the 5.0 kbp (apparent size) EcoR I fragment of both these two cosmids, 26A5 and 25A10.
  • the membrane was stripped of radioactivity by boiling for about 30 mm in 0. IX SSC plus 0.1 % SDS, and absence of radiolabel was checked by exposure to film It was then hybridized at 60°C for 3.5 hours with the (denatured) TcaB probe "minimal hyb" buffer previously used for screening the colony membranes (above) , washed as described previously, and exposed to film for 40 mm at -
  • SUBST ⁇ JTE SHEET (RULE 26) 70°C with two enhancer screens. With both cosmids, the TcaBx probe hybridized lightly with the about 5 0 kbp EcoR 1 fragment, and strongly with a fragment of approximately 2.9 kbp .
  • This DNA (2.5 ⁇ g) was digested with about 3 units of EcoR I (NEB) a total volume of 30 ⁇ l for 1.5 hr, to give a partial digest, as confirmed by gel electrophoresis
  • Ten ⁇ g of pBC KS (+) DNA (Stratagene) were digested for 1.5 hr with 20 units of EcoR I in a total volume of 20 ⁇ l , leading to total digestion as confirmed by electrophoresis
  • Both EcoR I -cut DNA preparations were diluted to 50 ⁇ l with water, to each an equal volume of PCI was added, the suspension was gently mixed, spun in a microcentrifuge and the aqueous supernatant was collected.
  • DNA was precipitated by 150 ⁇ l ethanol, and the mixture was placed at -20°C overnight. Following centrifugation and drying, the EcoR I - digested pBC KS (+) was dissolved in 100 ⁇ l TE, the partially digested 26A5 was dissolved in 20 ⁇ l TE DNA recovery was checked by fluorometry. In separate reactions, approximately 60 ng of EcoR I -digested pBC KS(+) DNA was ligated with approximately 180 ng or 270 ng of partially digested cosmid 26A5 DNA.
  • Ligations were carried out m a volume of 20 ⁇ l at 15°C for 5 hr, using T4 ligase and buffer from New England BioLabs The ligation mixture, diluted to 100 ⁇ l with sterile TE, was used to transform frozen, competent DH5 ⁇ cells
  • Varying amounts (25-200 ⁇ l) of the transformed cells were plated on freshly prepared solid LB-Cam 35 medium with 1 mM IPTG and 50 mg/1 X-gal. Plate ' s ' were " incubated at 37 °C about 20 hr, then chilled the dark for approximately 3 hr to intensify color for ⁇ nsertr ⁇ select ⁇ on. White colonies were picked onto patch plates of the same composition and incubated overnight at 37°C.
  • Two colony lifts of each of the selected patch plates were prepared as follows. After picking white colonies to fresh plates, round Magna NT nylon membranes were pressed onto the patch plates, the membrane was lifted off, and subjected to denaturation, neutralization and UV crosslmk g as described above for the library colony membranes. The crosslmked colony lifts were vigorously washed, including gently wiping off the excess cell debris with a tissue. One set was hybridized with the GZ4 (TcaC) probe solution described earlier, and the other set was hybridized with the TcaBx probe solution described earlier, according to the
  • Isolates D38.3 and C44 1 each contain only the 2.9 kbp, TcaBi -hybridizing EcoR I fragment inserted into pBC KS(+)
  • plasmids named pDAB2000 and pDAB2001, respectively, are illustrated in Fig. 2.
  • Isolate A35.3 contains only the approximately 5 kbp, GZ4)- hyb ⁇ dizmg EcoR 1 fragment, inserted into pBC KS(+ )
  • This plasmid was named pDAB2002 (also Fig. 2) These isolates provided templates for DNA sequencing.
  • Plasmids pDAB2000 and pDAB2001 were prepared using the BIGprepTM kit as before Cultures (30 ml) were grown overnight in
  • plasmid was isolated according to the manufacturer's directions. DNA pellets were redissolved in 100 ⁇ l TE each, and sample integrity was checked by EcoR I digestion and gel electrophoretic analysis. Sequencing reactions were run in duplicate, with one replicate using as template pDAB2000 DNA, and the other replicate using as template pDAB2001 DNA.
  • the reactions were carried out using the dideoxy dye terminator cycle sequencing method, as described above for the sequencing of the GZ4/HB14 DNAs
  • SUBST ⁇ UTE SHEET (RULE 26) sequencing "steps" were made by selecting appropriate sequence for new primers. With a few exceptions, primers (synthesized as described above) were 24 bases in length with a 50% G+C composition. Sequencing by this method was carried out on both strands of the approximately 2.9 kbp EcoR I fragment .
  • plasmid DNA from isolate pDAB2002 was prepared by BIGprepTM kit. Sequencing reactions were performed and analyzed as described above. Initially, a T3 primer (pBS SK (+) bases 774-796: CGCGCAATTAACCCTCACTAAAG) and a T7 primer (pBS KS (+) bases 621-643: GCGCGTAATACGACTCACTATAG) were used to prime the sequencing reactions from the flanking vector sequences, reading into the insert DNA.
  • GZ4F GTATCGATTACAACGCTGTCACTTCCC
  • THl3 GGGAAGTGACAGCGTTGTAATCGATAC
  • TH14 ATGTTGGGTGCGTCGGCTAATGGACATAAC
  • LW1-204 Another set of primers, (GZ4F: GTATCGATTACAACGCTGTCACTTCCC; THl3 : GGGAAGTGACAGCGTTGTAATCGATAC; TH14: ATGTTGGGTGCGTCGGCTAATGGACATAAC ; and LW1-204 :
  • GGGAAGTGACAGCGTTGTAATCGATAC was made to prime from internal sequences, which were determined previously by degenerate oligonucleotide-mediated sequencing of subcloned TcaC-peptide PCR products. From the data generated during the initial rounds of sequencing, new sets of primers were designed and used to walk the entire length of the about 5 kbp fragment. A total of 55 oligo primers was used, enabling the identification of 4832 total bp of contiguous sequence.
  • SUBST ⁇ UTE SHEET (RULE 26) region and the lack of a discernible ⁇ bosome binding site immediately upstream of the TcaB n coding region, indicate that peptides TcaB lx and TcaB x are encoded by a single open reading frame of 3567 bp beginning at base pair 65 in SEQ ID NO:25), and are most likely derived from a single primary gene product TcaB of 1189 ammo acids (131,586 Daltons; disclosed herein as SEQ ID NO: 26) by post-translational cleavage.
  • TcaB xl N-termmal peptide represents the C-termmal ammo acid of peptide
  • the predicted mass of TcaB n (627 ammo acids) is 70,814 Daltons (disclosed herein as SEQ ID NO:28) , somewhat higher than the size observed by SDS-PAGE (68 kDa)
  • SEQ ID NO:28 the predicted mass of TcaB n (627 ammo acids) is 70,814 Daltons
  • SEQ ID NO:28 the predicted mass of TcaB n (627 ammo acids) is 70,814 Daltons (disclosed herein as SEQ ID NO:28) , somewhat higher than the size observed by SDS-PAGE (68 kDa)
  • SEQ ID NO:27 the native C-termmus of TcaB x lies somewhat closer to the C- terminus of TcaBi-PTlO ⁇ .
  • the molecular mass of PT108 [3.438 kDa, determined during N-termmal ammo acid sequence analysis of this peptide] predicts a size of 30 ammo acids Using the size of this peptide to designate the C-termmus of the TcaB x coding region [Glu at position 604 of SEQ ID NO: 28] , the derived size of TcaB x is determined to be 604 am o acids or 68,463 Daltons, more in agreement with experimental observations.
  • TcaB n peptide coding region of 1686 base pairs yields a protein of 562 ammo acids (disclosed herein as SEQ ID NO 30) with predicted mass of 60,789 Daltons, which corresponds well with the observed 61 kDa
  • a potential ribosome binding site (bases 3682-3687) is found 48 bp downstream of the stop codon for the tcaB open reading frame
  • bases 3694-3726 is found a sequence encoding the N-terminus of pepti ⁇ e TcaC, (disclosed as SEQ ID NO.2)
  • the open reading frame initiated by this N-termmal peptide continues uninterrupted to base 6005 (2361 base pairs, disclosed herein as the first 2361 base pairs of SEQ ID NO.31)
  • a gene ( tcaC) encoding the entire TcaC peptide, (apparent size about 165 kDa, about 1500 ammo acids) would comprise about 4500 bp
  • SUBST ⁇ UTE SHEET (RULE 26) 3.3 kbp EcoR I fragment of pDAB2004.lies adjacent to the 5 kbp EcoR I fragment represented in pDAB2002.
  • the 2361 base pair open reading frame discovered in pDAB2002 continues uninterrupted for another 2094 bases in pDAB2004 [disclosed herein as base pairs 2362 to 4458 of SEQ ID NO: 31] .
  • DNA sequence analysis using the parent cosmid 26A5 DNA as template confirmed the continuity of the open reading frame.
  • the open reading frame ( tcaC SEQ ID NO: 31) comprises 4455 base pairs, and encodes a protein (TcaC) of 1485 amino acids [disclosed herein as SEQ ID NO: 32] .
  • the calculated molecular size of 166,214 Daltons is consistent with the estimated size of the TcaC peptide (165 kDa) , and the derived amino acid sequence matches exactly that disclosed for the TcaC N- terminal sequence [SEQ ID NO: 2] .
  • the lack of an amino acid sequence corresponding to SEQ ID NO: 17; used to design the degenerate oligonucleotide primer pool in the discovered sequence indicates that the generation of the PCR ® products found in isolates GZ4 and HB14, which were used as probes in the initial library screen, were fortuitously generated by reverse-strand priming by one of the primers in the degenerate pool. Further, the derived protein sequence does not include the internal fragment disclosed herein as SEQ ID NO: 18. These sequences reveal that plasmid pDAB2004 contains the complete coding region for the TcaC peptide.
  • SEQ ID NO: 25 Further analysis of SEQ ID NO: 25 reveals the end of an open reading frame (bases 1-43), which encodes the final 13 amino acids of the TcaA i ⁇ :L peptide, disclosed herein as SEQ ID NO: 35. Only 24 bases separate the end of the TcaA i3 _i coding region and the start of the TcaBi coding region. Included within the 24 bases are sequences that may serve as a ribosome binding site. Although possible, it is not likely that a Photorhabdus gene promoter is encoded within this short region.
  • genomic region tea which includes three long open reading frames [ tcaA (SEQ ID NO:33), tcaB (SEQ ID NO:25, bases 65-36334), and tcaC (SEQ ID NO:31) , which is separated from the end of tcaB by only 59 bases] is regulated as an operon, with transcription initiating upstream of the start of the tcaA gene (SEQ ID NO:33), and resulting in a polycistronic messenger RNA.
  • tcaA SEQ ID NO:33
  • tcaB SEQ ID NO:25, bases 65-36334
  • tcaC SEQ ID NO:31
  • This example describes a method used to identify DNA clones that contain the TcbA xl peptide-encoding genes, the isolation of the gene, and the determination of its partial DNA base sequence.
  • TcbAn polypeptide of the insect active preparation is about 206 kDa.
  • the ammo acid sequence of the N-terminus of this peptide is disclosed as SEQ ID NO:l.
  • Four pools of degenerate oligonucleotide primers ("Forward primers"- TH-4, TH-5, TH-6, and TH-7) were synthesized to encode a portion of this ammo acid sequence, as described in Example 8, and are shown below
  • Acid Phe lie Gin Gly Tyr Ser Asp Leu Phe TH-4 5'-TT(T/C) ATI CA(A/G) GGI TA(T/C) TCI GA(T/C) CTI TT-
  • a primary (“a”) and a secondary (“b”) sequence of an internal peptide preparation have been determined and are disclosed herein as SEQ ID NO: 23 and SEQ ID NO: 24, respectively
  • SEQ ID NO: 23 and SEQ ID NO: 24 respectively
  • Four pools of degenerate oligonucleotides (“Reverse Primers”: TH-8, TH-9, TH-10 and TH-11) were similarly designed and synthesized to encode the reverse complement of sequences that encode a portion of the peptide of SEQ ID NO: 23, as shown below.
  • 10X GeneAmp" PCR Buffer II is composed of 100 mM Tris-HCl, pH 8.3; and 500 mM KC1.
  • the tubes were heated to 80 °C for 2 minutes and allowed to cool.
  • a solution containing 10X GeneAmp" PCR Buffer II, DNA template, and A pliTaq ® DNA polymerase were added.
  • final reaction conditions were: 10 mM Tris-HCl, pH 8.3; 50 mM KC1; 2.5 mM MgCl 2 ; 200 ⁇ M each in dATP, dCTP, dGTP, dTTP; 1.25 mM in a single
  • Forward primer pool 1.25 ⁇ M in a single Reverse primer pool, 1.25 units of AmpliTaq" DNA polymerase, and 170 ng of template DNA.
  • SUBST ⁇ UTE SHEET (RULE 26) pools. Reactions with annealing at .65°C had no amplification products visible following agarose gel electrophoresis. Reactions having a 60°C annealing regime and containing primers TH-5+TH-10 produced an amplification product that had a mobility corresponding to 2.9 kbp. A lesser amount of the 2.9 kbp product was produced under these conditions with primers TH-7+TH-10. When reactions were annealed at 55°C, these primer pairs produced more of the 2.9 kbp product, and this product was also produced by primer pairs TH- 5+TH-8 and TH-5+TH-11. Additional very faint 2.9 kbp bands were seen in lanes containing amplification products from primer pairs TH-7 plus TH-8, TH-9, TH-10, or TH-11.
  • TcbA ⁇ internal peptides are disclosed herein as SEQ ID NO: 21 and SEQ ID NO: 22.
  • degenerate oligonucleotides Reverse primers TH-17 and TH-18 were made corresponding to the reverse complement of sequences that encode a portion of the amino acid sequence of these peptides.
  • oligonucleotides TH-18 and TH-17 were used in an amplification experiment with Photorhabdus l uminescens W-14 DNA as template and primers TH-4, TH-5, TH-6, or TH-7 as the 5'- (Forward) primers. These reactions amplified products of approximately 4 kbp and 4.5 kbp, respectively. These DNAs were transferred from
  • a sequencing primer (TH-21, 5'- CCGGGCGACGTTTATCTAGG-3 ' ) was designed to reverse complement bases
  • the 2.9 kb gel-purified PCR fragment was labeled with 32 P using the Boehrmger Mannheim High Prime labeling kit as described m Example 8. Filters containing remnants of approximately 800 colonies from the cosmid library were screened as described previously (Example 8) , and positive clones were streaked for isolated colonies and rescreened. Three clones (8A11, 25G8, and 26D1) gave positive results through several screening and characterization steps. No hybridization of the TcbA x -spec ⁇ fic probe was ever observed with any of the four cosmids identified in Example 8 , and which contain the tcaB and tcaC genes .
  • cosmids 8All, 25G8 , and 26D1 were digested with restriction enzymes Bgl II, EcoR I or Hind III (either alone or combination with one another) , and the fragments were separated on an agarose gel and transferred to a nylon membrane as described in Example 8
  • the membrane was hybridized with 32 P- labeled probe prepared from the 4.5 kbp fragment (generated by amplification of Photorhabdus genomic DNA with primers TH-5+TH-17) .
  • the patterns generated from cosmid DNAs 8A11 and 26D1 were identical to those generated with similarly-cut genomic DNA on the same membrane. It is concluded that cosmids 8A11 and 26D1 are accurate representations of the genomic TcbA xx encoding locus.
  • cosmid 25G8 has a single Bgl II fragment which is slightly larger than the genomic DNA. This may result from positioning of the insert withm the vector.
  • the membrane hybridization analysis of cosmid 26D1 revealed that the 4.5 kbp probe hybridized to a single large EcoR I fragment (greater than 9 kbp) This fragment was gel purified and ligated into the EcoR I site of pBC KS (+) as described in Example 8, to generate plasmid pBC-Sl/Rl.
  • the partial DNA sequence of the insert DNA of this plasmid was determined by "primer walking" from the flanking vector sequence, using procedures described in Example 8. Further sequence was generated by extension from new oligonucleotides designed from the previously determined sequence.
  • the sequence of the DNA fragment identified as SEQ ID NO: 11 encodes a protein whose derived amino acid sequence is disclosed herein as SEQ ID NO: 12.
  • the TcbA xx N- terminal peptide (SEQ ID NO.-l; Phe lie Gin Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala) is encoded as amino acids 88-100.
  • the TcbA xx internal peptide TcbA xx -PT81 (a) (SEQ ID NO: 23) is encoded as amino acids 1065-1077
  • TcbA xx -PT81 (b) (SEQ ID NO:24) is encoded as amino acids 1571-1592.
  • the internal peptide TcbA xx -PT56 (SEQ ID NO:22) is encoded as amino acids 1474-1488
  • the internal peptide TcbA xx -PT103 (SEQ ID NO: 21) is encoded as amino acids 1614-1639. It is obvious that this gene is an authentic clone encoding the TcbA xx peptide as isolated from insecticidal protein preparations of Photorhabdus luminescens strain W-14.
  • the protein isolated as peptide TcbA xx is derived from cleavage of a longer peptide.
  • TcbA is the precursor protein for TcbA xx .
  • the tcaB and tcaC genes are closely linked and may be transcribed as a single mRNA (Example 8) .
  • the tcbA gene is borne on cosmids that apparently do not overlap the ones harboring the tcaB and tcaC cluster, since the respective genomic library screens identified different cosmids.
  • comparison of the amino sequences encoded by the tcaB and tcaC genes with the tcbA gene reveals a substantial degree of homology.
  • the amino acid conservation Protein Alignment Mode of MacVectorTM Sequence
  • TcbA peptide (SEQ ID NO:12) comprises 2505 ammo acids, a total of 684 ammo acids (27%) at the C- proximal end of it is homologous to the TcaB x or TcaB lx peptides, and the homologies are arranged colmear to the arrangement of the putative TcaB preprotem (SEQ ID NO:26) .
  • a sizeable gap in the TcbA homology coincides with the junction between the TcaB ⁇ and
  • TcaB xl portions of the TcaB preprotem are evolutionarily related, and it is proposed that they share some common functio (s) Photorhabdus .
  • Protease Inhibition and Classification Assays Protease assays were performed using FITC-casem dissolved water as substrate (0.08% final assay concentration) . Proteolysis reactions were performed at 25°C for 1 h in the appropriate buffer with 25 ⁇ l of Photorhabdus broth (150 ⁇ l total reaction volume) . Samples were also assayed the presence and absence of dithiothreitol After incubation, an equal volume of 12% t ⁇ chloroacetic acid was added to precipitate undigested protein.
  • protease inhibitors used mcluded E-64 (L-trans-expoxysacc ylleucylamido [4- , -guanidmo] - butane), 3,4 dichloroisoc ⁇ umarm, Leupeptin, pepstatm, amastat , ethylenediammetetraacetic acid (EDTA) and 1,10 phenanthrolme.
  • Protease assays performed over a pH range revealed that indeed protease (s) were present which exhibited maximal activity at about pH 8.0 (Table 17) . Addition of DTT did not have any effect on protease activity.
  • SUBST ⁇ UTE SHEET (RULE 26) gel electrophoresis (SDS-PAGE) laced with gelatin as described m Schmidt, T.M., Bleakley, B. and Nealson, K.M. 1988.
  • SDS running gels (5.5 x 8 cm) were made with 12.5 % polyacrylamide (40% stock solution of acrylamide/bis-acrylamide; Sigma Chemical Co., St. Louis, MO) into which 0.1% gelatin final concentration (Biorad EIA grade reagent; Richmond CA) was incorporated upon dissolving m water.
  • SDS-stackmg gels (1.0 x 8 cm) were made with 5% polyacrylamide, also laced with 0.1% gelatin.
  • 2.5 ⁇ g of protein to be tested was diluted 0.03 ml of SDS-PAGE loading buffer without dithiothreitol (DTT) and loaded onto the gel.
  • DTT dithiothreitol
  • Proteins were electrophoresed in SDS running buffer (Laemmli, U.K. 1970 Nature 227, 680) at 0° C and at 8 mA After electrophoresis was complete, the gel was washed for 2 h in 2.5% (v/v) Triton X- 100. Gels were then incubated for 1 h at 37 °C in 0 1 M glycme (pH 8.0). After incubation, gels were fixed and stained overnight with 0.1% amido black in methanol -acetic acid- water (30.10-60, vol . /vol . /vol . ; Sigma Chemical Co.). Protease activity was visualized as light areas against a dark, amido black stained background due to proteolysis and subsequent diffusion of incorporated gelatin. At least three distinct bands produced by proteolytic activity at 58-, 41-, and 38 kDa were observed
  • Casein with different protease concentrations at 37°C for 0-10 mm A unit of activity was arbitrarily defined as the amount of enzyme needed to produce 1000 fluorescent units/mm and specific activity was defined as umts/mg of protease.
  • the proteases of three day W-14 Photorhabdus broth were purified as follows: 4.0 liters of broth were concentrated using an Amicon spiral ultra filtration cartridge Type SIYIOO attached to an Amicon M-12 filtration device.
  • the flow-through material having native proteins less than 100 kDa in size (3.8 L) was concentrated to 0.375 L using an Amicon spiral ultra filtration cartridge Type S1Y10 attached to an Amicon M-12 filtration device.
  • the retentate material contained proteins ranging in size from 10-100 kDa.
  • Proteins to be ammo-terminal sequenced were blotted onto PVDF membrane following purification, infra . , (ProBlottTM Membranes, Applied Biosyste s, Foster City, CA) , visualized with 0.1% amido black, excised, and sent to Cambridge Prochem,- Cambridge, MA, for sequencing.
  • SUBST ⁇ UTE SHEET (RULE 26) were DV-GSEKANEKLK (SEQ ID NO: 45) and DSGDDDKVTNTDIHR (SEQ ID NO: 44), respectively.
  • TcbA x peptide SEQ ID NO:l
  • Genomic DNA was isolated from the Photorhabdus l uminescens strain W-14 grown in Grace's insect tissue culture medium The bacteria were grown in 5 ml of culture medium in a 250 ml Erlenmeyer flask at 28 °C and 250 rpm for approximately 24 hours Bacterial cells from 100 ml of culture medium were pelleted at 5000 x g for 10 minutes. The supernatant was discarded, and the cell pellets then were used for the genomic DNA isolation.
  • the genomic DNA was isolated using a modification of the CTAB method described in Section 2.4.3 of Ausubel [ supra . ) The section entitled “Large Scale CsCl prep of bacterial genomic DNA” was followed through step 6. At this point, an additional chloroform/isoamyl alcohol (24:1) extraction was performed followed by a phenol/chloroform/isoamyl (25:24:1) extraction step and a final chloroform/isoamyl/alcohol (24:1) extraction. The DNA was precipitated by the addition of a 0.6 volume of isopropanol . The precipitated DNA was hooked and wound around the end of a bent glass rod, dipped briefly into 70% ethanol as a final wash, and dissolved m 3 ml of TE buffer.
  • the DNA concentration estimated by optical density at 280/260 nm, was approximately 2 mg/ml.
  • genomic DNA was partly digested with Sau3 Al . Then NaCl density gradient centrifugation was used to size fractionate the partially digested DNA fragments. Fractions containing DNA
  • Stable monoclonal antibody-producing hybrido a cell lines were recovered after spleen cells from unimmunized mouse were fused with a stable myeloma cell line. Monoclonal antibodies were recovered from the hybndomas . Separately, polyclonal antibodies were created by taking native agarose gel purified band 1 (see Example 1) protein which was then used to immunize a New Zealand white rabbit. The protein was prepared by excising the band from the native agarose gels, briefly heating the gel pieces to 65°C to melt the agarose, and immediately emulsifying with adjuvant.
  • SUBST ⁇ UTE SHEET (RULE 26) from the deduced ammo acid sequence created by the open reading frame of SEQ ID NO: 11. This can be confirmed by referring to SEQ ID NO: 12, which is the deduced ammo acid sequence created by SEQ ID NO: 11.
  • the second region of toxin peptides contains the segments referred to above as TcaB x , TcaB lx and TcaC. Following the screening of the library with the polyclonal antisera, this second region of toxm genes was identified by several clones which produced different size proteins, all of which cross-reacted with the polyclonal antibody on an immunoblot and were also found to share DNA homology on a Southern Blot. Sequence comparison revealed that they belonged to the gene complex designated TcaB and TcaC above.
  • SUBST ⁇ UTE SHEET (RULE 26) 131 bases separate the stop codon of tccB and the tccC (bases 4930- 4932 of SEQ ID NO: 58) .
  • the physical map is presented in Fig. 6B .
  • the deduced ammo acid sequence from the tccA open reading frame indicates that the gene encodes a protein of 105,459 Da. This protein was designated TccA (SEQ ID NO: 57)
  • the first 12 ammo acids of this protein match the N-term al sequence obtained from a 108 kDa protein, SEQ ID NO: 8, previously identified as part of the toxm complex.
  • the deduced amino acid sequence from the tccB open reading frame indicates that this gene encodes a protein of 175,716 Da.
  • This protein was designated TccB (SEQ ID NO: 59) .
  • the first 11 ammo acids of this protein match the N-termmal sequence obtained from a protein with estimated molecular weight of 185 kDa, SEQ ID NO: 7.
  • TccB protein is related to the proteins identified as TcbA SEQ ID NO: 12; 37% similarity and 28% identity, TcdA SEQ ID NO:47, 35% similarity and 28% ⁇ dent ⁇ ty, and TcaB SEQ ID NO:26; 32% similarity and 26% identity (using the GAP algorithm Wisconsin Package Version 9 0, Genetics Computer Group (GCG) Madison Wisconsin)
  • GAP algorithm Wisconsin Package Version 9 Genetics Computer Group (GCG) Madison Wisconsin
  • cell density Prior to measuring light emission from the various broths, cell density was established by measuring light absorbance (560 nM) in a Gilford Systems (Oberlm, OH) spectrophotometer using a sipper cell. Appropriate dilutions were then made (to normalize optical density to 1.0 unit) before measuring luminosity Aliquots of the diluted broths were then placed into cuvettes (300 ⁇ l each) and read in a Bio-Orbit 1251 Lummometer (Bio-Orbit Oy, Twiku, Finland) . The integration period for each sample was 45 seconds. The samples were continuously mixed (spun in baffled cuvettes) while being read to provide oxygen availability. A positive test was determined as being > 5-fold background luminescence (about 5-10 units) .
  • colony luminosity was detected with photographic film overlays and visually, after adaptation in a darkroom.
  • the Gram's staining characteristics of each strain were established with a commercial Gram's stain kit (BBL, Cockeysville, MD) used m conjunction with Gram's stain control slides (Fisher Scientific, Pittsburgh, PA) . Microscopic evaluation was then performed using a Zeiss microscope (Carl Zeiss, Germany) 100X oil immersion objective lens (with 10X ocular and 2X body magnification) .
  • SUBST ⁇ UTE SHEET (RULE 2S) inoculation on Bacto nutrient agar, .(Difco Laboratories, Detroit, MI) prepared as per label instructions. Incubation occurred at 28 °C and descriptions were produced after 5-7 days.
  • a colony of the test organism was removed on a small plug from a nutrient agar plate and placed into the bottom of a glass test tube.
  • One ml of a household hydrogen peroxide solution was gently added down the side of the tube. A positive reaction was recorded when bubbles of gas (presumptive oxygen) appeared immediately or with 5 seconds. Controls of unmoculated nutrient agar and hydrogen peroxide solution were also examined.
  • each culture was inoculated into 10 ml of Bacto Nitrate Broth (Difco Laboratories, Detroit, MI). After 24 hours incubation at 28°C, nitrite production was tested by the addition of two drops of sulfanilic acid reagent and two drops of alpha-naphthylamme reagent (see Difco Manual, 10th edition, Difco Laboratories, Detroit, MI, 1984) .
  • protease was tested by observing hydrolysis of gelatin using Bacto gelatin (Difco Laboratories, Detroit, MI) plates made as per label instructions. Cultures were inoculated and the plates were incubated at 28°C for 5 days. To assess growth at different temperatures, agar plates [2% proteose peptone #3 with two percent Bacto-Agar (Difco, Detroit, MI) in deionized water] were streaked from a common source of inoculum. Plates were sealed with Nesco" film and incubated at 20, 28 and 37°C for up to three weeks. Plates showing no growth at 37°C showed no cell viability after transfer to a 28°C incubator for one week.
  • Oxygen requirements for Photorhabdus strains were tested m the following manner. A butt -stab inoculation into fluid thioglycolate broth medium (Difco, Detroit, MI) was made. The tubes were incubated at room temperature for one week and cultures were then examined for type and extent of growth. The indicator resazurm demonstrates the level of medium oxidation or the aerobiosis zone (Difco Manual, 10th edition, Difco Laboratories, Detroit, MI). Growth zone results obtained for the Photorhabdus strains tested were consistent with those of a facultative anaerobic microorganism.
  • MIS Microbial Identification System
  • the computer compares the sample fatty acid methyl esters to a microbial fatty acid library and against a calibration mix of known fatty acids. As selected by the contract laboratory, strains were grown for 24 hours at 28°C on trypticase soy agar prior to analysis. Extraction of samples was performed by the contract lab as per standard FAME methodology. There was no direct identification of the strains to any luminescent bacterial group other than Photorhabdus. When the cluster analysis was performed, which compares the fatty acid profiles of a group of isolates, the strain fatty acid profiles were related at the genus level .
  • Genomic organization is believed to be shaped by selection and the differential dispersion of these elements withm the genome of closely related bacterial strains can be used to discriminate these strains (e.g., Louws, F J., Fulb ⁇ ght, D. W., Stephens, C. T. and DE Bruijn, F. J. 1994. Appl. Environ. Micro 60, 2286-2295) .
  • Rep-PCR utilizes oligonucleotide primers complementary to these repetitive sequences to amplify the variably sized DNA fragments lying between them. The resulting products are separated by electrophoresis to establish the DNA "fingerprint" for each strain.
  • SUBST ⁇ UTE SHEET (RULE 26) final volume of 10 ml and 12 ml of 5. M NaCl was then added. This mixture was centrifuged 20 mm. at 15,000 x g. The resulting pellet was resuspended m 5.7 ml of TE and 300 ⁇ l of 10% SDS and 60 ⁇ l 20 mg/ml protemase K (Gibco BRL Products, Grand Island, NY) were added. This mixture was incubated at 37 °C for 1 hr, approximately 10 mg of lysozy e was then added and the mixture was incubated for an additional 45 mm.
  • Precipitated DNA was removed with a glass rod, washed twice with 70% ethanol, dried and dissolved in 2 ml of STE (10 mM Tris-HCl pH8.0, 10 mM NaCl, 1 mM EDTA) . The DNA was then quantitated by optical density at 260 nm.
  • Tris-borate 0.002 M EDTA
  • the gel was run for approximately 16 hours at 45v. Gels were then stained in 20 ⁇ g/ml ethidium bromide for 1 hour and destamed in TBE buffer for approximately 3 hours. Polaroid photographs of the gels were then taken under UV illumination.
  • SUBST ⁇ UTE SHEET (RULE 26) uniform inoculum sources for a given fermentation of each strain. Additionally, overlaying the post-log seed culture with sterile mineral oil, adding a sterile magnetic stir bar for future resuspension and storing the culture the dark, at room temperature provided long-term preservation of inoculum in a toxin- competent state. The production broths were inoculated by adding 1% of the actively growing seed culture to fresh 2% PP3 media (e.g., 1.75 ml per 175 ml fresh media).
  • Whatman GF/D 2.7 ⁇ M retention
  • GF/B 1.0 ⁇ M retention
  • the broth (s) and toxm complex (es) from different Photorhabdus strains are useful for reducing populations of insects and were used in a method of inhibiting an insect population which comprises applying to a locus of the insect an effective insect inactivating amount of the active described.
  • a demonstration of the breadth of msecticidal activity ODserved from broths of a selected group of Photorhabdus strains fermented as described above is shown in Table 20. It is possible that additional msecticidal activities could be detected with these strains through increased concentration of the broth or by employing different fermentation methods. Consistent with the activity being associated with a protein, the msecticidal activity of all strains tested was heat labile (see above )
  • Culture broth (s) from diverse Photorhabdus strains show differential msecticidal activity (mortality and/or growth inhibition, reduced adult emergence) against a number of insects. More specifically, the activity is seen against corn rootworm larvae and boll weevil larvae which are members of the insect order Coleoptera .
  • Other members of the Coleoptera include wireworms, pollen beetles, flea beetles, seed beetles and Colorado potato beetle Activity is also observed against aster leafhopper and corn plant hopper, which are members of the order Homoptera
  • Other members of the Homoptera include planthoppers , pear psylla, apple sucker, scale insects, whiteflies, spittle bugs as well as numerous host specific aphid species
  • the broths and purified toxm comple (es) are also active against tobacco budworm, tobacco hornworm and European corn borer which are members of the order Lepidoptera
  • Other typical members of this order are beet armyworm, cabbage looper, black cutworm, corn earworm, codling moth, clothes moth, Indian mealmoth, leaf rollers, cabbage worm, cotton bollworm, bagworm, Eastern tent caterpillar, sod webworm and fall armyworm Activity is also seen against fruitfly and mosquito larvae which are members of the order Diptera .
  • Activity against mosquito larvae was tested as follows The assay was conducted m a 96-well microtiter plate. Each well contained 200 ⁇ l of aqueous solution (10-fold concentrated Photorhabdus culture broth(s) , control medium (2% Proteose Peptone #3), 10 mM sodium phosphate buffer, toxin complex(es) @ 0.23 mg/ml or H2O) and approximately 20, 1-day old larvae [Aedes aegypti ) .
  • the reservoir for the active/ "food” solution is made by making 2 holes in the center of the bottom portion of a 35X10 mm Petri dish. A 2 inch Parafilm M square is placed across the top of the dish and secured with an "0" ring. A 1 oz. plastic cup is then infested with approximately 7 hoppers and the reservoir is placed on top of the cup, Parafilm down. The test solution is then added to the reservoir through the holes.
  • SUBST ⁇ TUTE SHEET Activity against two-spotted spider mite [ Tetranychu ⁇ urticae) was determined as follows. Young squash plants were trimmed to a single cotyledon and sprayed to run-off with 10-fold concentrated broth(s), control medium (2% Proteose Peptone #3), purified toxin complex (es) , 10 mM sodium phosphate buffer, pH 7.0. After drying, the plants were infested with a mixed population of spider mites and held at lab temperature and humidity for 72 hr. Live mites were then counted to determine levels of control .
  • the protocol is similar to that developed for the purification of W-14 and was established based on purifying those fractions having the most activity against Southern corn root worm (SCR), as determined bioassays (see Example 13)
  • SCR Southern corn root worm
  • 4- 20 L of broth that had been filtered, as described in Example 13 were received and concentrated using an Amicon spiral ultra filtration cartridge Type SIYIOO attached to an Amicon M-12 filtration device.
  • the retentate contained native proteins consisting of molecular sizes greater than 100 kDa, whereas the flow through material contained native proteins less than 100 kDa m size. The majority of the activity against SCR was contained in the 100 kDa retentate.
  • the filtered material was loaded at 7.5 ml/mm onto a Pharmacia HR16/10 column which had been packed with PerSeptive Biosystem Poros ® 50 HQ strong anion exchange matrix equilibrated in buffer using a PerSeptive Biosystem Sprint ® HPLC system After loading, the column was washed with buffer until an A28O ⁇ 0-100 was achieved.
  • SUBST ⁇ UTE SHEET (RULE 26) pooled based on A28O peak profile and concentrated to a final volume of 0.75 ml using a Millipore Ultrafree ® - 15 centrifugal filter device Biomax-50K NMWL membrane. Protein concentrations were determined using a Biorad Protein Assay Kit with bovine gamma globulin as a standard.
  • the native molecular weight of the SCR toxin complex was determined using a Pharmacia HR 16/50 that had been prepacked with Sepharose CL4B in buffer. The column was then calibrated using proteins of known molecular size thereby allowing for calculation of the toxin approximate native molecular size. As shown in Table 21, the molecular size of the toxin complex ranged from 777 kDa with strain Hb to 1,900 kDa with strain WX-14. The yield of toxin complex also varied, from strain WX-12 producing 0.8 mg/L to strain Hb, which produced 7.0 mg/L.
  • Proteins found in the tox complex were examined for individual polypeptide size using SDS-PAGE analysis. Typically, 20 mg protein of the toxin complex from each strain was loaded onto a 2-15% polyacrylamide gel (Integrated Separation Systems) and electrophoresed at 20 mA in Biorad SDS-PAGE buffer. After completion of electrophoresis, the gels were stained overnight in Biorad Coomassie blue R-250 (0.2% in methanol: acetic acid: water; 40:10:40 v/v/v) . Subsequently, gels were destained in methanol .-acetic acid: water; 40:10:40 (v/v/v).
  • the gels were then rinsed with water for 15 min and scanned using a Molecular Dynamics Personal Laser Densitometer ® . Lanes were quantitated and molecular sizes were calculated as compared to Biorad high molecular weight standards, which ranged from 200-45 kDa. Sizes of the individual polypeptides comprising the SCR toxin complex from each strain are listed in Table 22. The sizes of the individual polypeptides ranged from 230 kDa with strain WX-1 to a size of 16 kDa, as seen with strain WX-7.
  • strain Hb Every strain, with the exception of strain Hb, had polypeptides comprising the toxin complex that were in the 160-230 kDa range, the 100-160 kDa range, and the 50-80 kDa range. These data indicate that the toxm complex may vary in peptide composition and components from strain to strain, however, in all cases the toxin attributes appears to consist of a large, oligomeric protein complex.
  • the toxm complexes purified from strains Hm and H9 were tested for activity against a variety of insects, with the toxm complex from strain W-14 for comparison.
  • the assays were performed as described in Example 13.
  • the toxm complex from all three strains exhibited activity against tobacco bud worm, European corn borer, Southern corn root worm, and aster leafhopper.
  • the toxm complex from strains Hm and W- 14 also exhibited activity against two-spotted spider mite
  • the toxin complex from W-14 exhibited activity against mosquito larvae
  • the Photorhabdus protein tox complex was isolated as described in Example 14. Next, about 10 mg toxm was applied to a MonoQ 5/5 column equilibrated with 20 mM Tris-HCl, pH 7.0 at a flow rate of lml/min. The column was washed with 20 mM Tris-HCl, pH 7.0 until the optical density at 280 nm returned to baseline absorbance . The proteins bound to the column were eluted with a linear gradient of 0 to 1.0 M NaCl m 20 mM Tris-HCl, pH 7.0 at 1 ml/min for 30 min. One ml fractions were collected and subjected to Southern corn rootworm (SCR) bioassay (see Example 13).
  • SCR Southern corn rootworm
  • Peaks of activity were determined by a series of dilutions of each fraction in SCR bioassays. Two activity peaks against SCR were observed and were named A (eluted at about 0.2-0.3 M NaCl) and B (eluted at 0.3-0.4 M NaCl) . Activity peaks A and B were pooled separately and both peaks were further purified using a 3 -step procedure described below.
  • Solid (NH-j. ⁇ 2S ⁇ 4 was added to the above protein fraction to a final concentration of 1.7 M. Proteins were then applied to a phenyl-Superose 5/5 column equilibrated with 1.7 M (NH4)2S04 in 50 mM potassium phosphate buffer, pH 7 at 1 ml/mm. Proteins bound to the column were eluted with a linear gradient of 1.7 M (NH4)2S ⁇ 4, 0% ethylene glycol, 50 mM potassium phosphate, pH 7.0 to 25% ethylene glycol, 25 mM potassium phosphate, pH 7.0 (no (NH4)2 ⁇ 4) at 0.5 ml/mm. Fractions were dialyzed overnight against 10 mM sodium phosphate buffer, pH 7.0. Activities m each fraction against SCR were determined by bioassay.
  • the final purified protein by the above 3 -step procedure from peak A was named toxm A and the final purified protein from peak B was named toxm B .
  • both toxm A and toxin B contained two major (> 90% of total Commassie stained protein) peptides: 192 kDa (named Al and Bl, respectively) and 58 kDa (named A2 and B2 , respectively)
  • Both toxm A and toxm B revealed only one major band in native PAGE, indicating Al and A2 were subunits of one protein complex, and Bl and B2 were subunits of one protein complex
  • the native molecular weight of both toxm A and tox B were determined to be 860 kDa by gel filtration chromatography.
  • the relative molar concentrations of Al to A2 was judged to be a 1 to 1 equivalence as determined by densiometric analysis of SDS-PAGE gels. Similarly, Bl and B2 peptides were present at the same molar concentration.
  • Tox A and toxm B were electrophoresed m 10% SDS-PAGE and transblotted to PVDF membranes. Blots were sent for ammo acid analysis and N-termmal ammo acid sequencing at Harvard MicroChe and Cambridge ProChem, respectively.
  • the N-termmal amino sequence of Bl was determined to be identical to SEQ ID N0:1, the TcbA xl region of the tcbA gene (SEQ ID NO: 12, position 87 to 99)
  • a unique N-termmal sequence was obtained for peptide B2 (SEQ ID NO -.40)
  • the N-termmal ammo acid sequence of peptide B2 was identical to the TcbA xll region of the derived ammo acid sequence
  • SUBST ⁇ UTE SHEET (RULE 26) for the tcbA gene (SEQ ID NO: 12, position 1935 to 1945) . Therefore, the B toxm contained predominantly two peptides, TcbAn and Tc Ain, that were observed to be derived from the same gene product, TcbA.
  • the N-terminal sequence of A2 (SEQ ID NO: 41) was unique in comparison to the TcbA llx peptide and other peptides.
  • the A2 peptide was denoted TcdA n ⁇ (see Example 17) .
  • SEQ ID NO: 6 was determined to be a mixture of amino acid sequences SEQ ID NO: 40 and 41. Peptides Al and A2 were further subjected to internal am o acid sequencing.
  • tox A 10 ⁇ g was electrophoresized in 10% SDS-PAGE and transblotted to PVDF membrane. After the blot was stained with amido black, peptides Al and A2 , denoted TcdA 1;L and TcdA , respectively, were excised from the blot and sent to Harvard MicroChem and Cambridge ProChem. Peptides were subjected to trypsin digestion followed by HPLC chromatography to separate individual peptides. N-termmal ammo acid analysis was performed on selected tryptic peptide fragments .
  • TcdAn-PK71, SEQ ID NO.-38 and TcdA xl -PK44, SEQ ID NO: 39 Two internal am o acid sequences of peptide Al (TcdAn-PK71, SEQ ID NO.-38 and TcdA xl -PK44, SEQ ID NO: 39) were found to have significant homologies with deduced ammo acid sequences of the TcbAn region of the tcbA gene (SEQ ID NO: 12) .
  • N-termmal sequence SEQ ID NO: 41
  • TcdA 1; ⁇ l -PK57, SEQ ID NO:42 and Tc A lu - PK20, SEQ ID NO.43 also showed significant homology with deduced ammo acid sequences of TcbA n ⁇ region of the tcbA gene (SEQ ID NO: 12) .
  • the toxm complex has at least two ⁇ act ⁇ ve protein toxm complexes against SCR; toxin A and tox B.
  • Tox A and toxm B are similar in their nat£ve ⁇ and subunits molecular weight, however, their peptide compositions are different.
  • Tox A contained peptides TcdA lx and TcdA lu as the major peptides and the toxm B contains TcbAn and TcbAn I as the major peptides.
  • the Photorhabdus protein toxm complex was isolated as described above. Next, about 50 mg toxm was applied to a MonoQ 10/10 column equilibrated with 20 mM Tris-HCl, pH 7 0 at a flow rate of 2 ml/mm. The column was washed with 20 mM Tris-HCl, pH7.0
  • Fractions were dialyzed overnight against 10 M sodium phosphate buffer, pH 7.0.
  • fractions reacted with pAb TcaBn-syn antibody above determined by Western analysis were combined and applied to a Mono Q 5/5 column equilibrated with 20 mM Tris-HCl, pH 7.0 at lml/mm.
  • the proteins bound to the column were eluted at lml/mm by a linear gradient of 0 to IM NaCl m 20 mM Tris-HCl, pH 7 0 for 30 mm.
  • the final purified protein fraction contained 6 major peptides examined by SDS-PAGE. 165 kDa, 90 kDa, 64 kDa, 62 kDa, 58 kDa, and 22 kDa.
  • SUBST ⁇ UTE SHEET (RULE 26) fraction were determined to be 100 ng and 500 ng against SCR and ECB, respectively.
  • the above peptides were blotted to PVDF membranes and blots were sent for ammo acids analysis and 5 ammo acid long N-termmal sequencing at Harvard MicroChem and Cambridge ProChem, respectively.
  • the N-termmal ammo acid sequence of the 165 kDa peptide was determined to be identical to peptide TcaC (SEQ ID 2, position 1 to 5) .
  • the N-termmal ammo acid sequence of the 90 kDa peptide was determined to be TcaA lx region of the derived ammo acid sequence for the tcaA gene (SEQ ID NO 33, position 254 to 258) .
  • the N-termmal ammo acid sequence of 64 kDa peptide was determined to be identical to peptide TcaB x (SEQ ID 3, position 1 to 5) .
  • the N-termmal ammo acid sequence of the 62 kDa peptide was determined to be TcaA region of the derived ammo acid sequence for the tcaA gene (SEQ ID NO 33, position 489 to 493) .
  • N-termmal ammo acid sequence of 58 kDa peptide was determined to be identical to peptide TcaB u (SEQ ID 5, position 1 to 5) .
  • N-termmal ammo acid sequence of the 22 kDa peptide (SEQ ID NO 62) was determined to be TcaA region, denoted TcaA lv , of the derived ammo acid sequence for the tcaA gene (SEQ ID NO 34, position 98 to 102) It is noted that all tcaA, tcaB, and tcaC genes reside in the same tea operon (Fig. 6A) .
  • TcbAn an d TcbA xll originate from the single gene product TcbA (Example 15) .
  • TcbA peptide to TcbAn an d TcbA m is presumably by the action of
  • Photorhabdus protease (s), and most likely, the metalloproteases described in Example 10.
  • TcbA peptide was present in toxm B complex as a major component, m addition to peptides TcbAn and TcbA ⁇ - Identical procedures, described for the purification of toxm B complex (Example 15) , were used to enrich peptide TcbA from tox complex fraction of W-14 broth.
  • the final purified material was analyzed in a 4-20% gradient SDS-PAGE and ma j or peptides were quantified by densitometry .
  • TcbA, TcbAn and TcbA lu comprised 58%, 36%, and 6%, respectively, of total protein
  • the identities of these peptides were confirmed by their respective molecular sizes in SDS-PAGE and Western blot analysis using monospecific antibodies
  • the native molecular weight of this fraction was determined to be 860 kDa.
  • the cleavage of TcbA was evaluated by treating the above purified material with purified 38 kDa and 58 kDa W-14 Photorhabdus metalloproteases (Example 10) , and tryps as a control enzyme (Sigma, MO).
  • the standard reaction consisted 17.5 ⁇ g the above purified fraction, 1.5 unit protease, and 0.1 M Tris buffer, pH 8.0 in a total volume of 100 ⁇ l .
  • protease was omitted.
  • the reaction mixtures were incubated at 37°C for 90 mm.
  • 20 ⁇ l was taken and boiled with SDS-PAGE sample buffer immediately for electrophoresis analysis m a 4-20% gradient SDS-PAGE.
  • SUBST ⁇ UTE SHEET (RULE 26) specifically process peptide TcbA into peptides TcbAn and TcbA lu
  • Protease treated and untreated control of the remaining 80 ⁇ l reaction mixture were serial diluted with 10 mM sodium phosphate buffer, pH 7 0 and analyzed by SCR bioassay By comparing activity in several dilution, it was determined that the 38 kDa protease treatment increased SCR msecticidal activity approximately 3 to 4 fold. The growth inhibition of remaining insects in the protease treatment was also more severe than control (Table 24) .
  • guts were homogenized in a microcentrifuge tube containing 100 ⁇ l sterile water. The tube was then centrifuged at 14,000 rpm for 10 minutes and the pellet discarded. The supernatant was stored at a -70°C freezer until use.
  • tox B The processing of tox B by insect gut was evaluated by treating the above purified toxm B with the SCR gut content collected.
  • the reaction consisted 40 ⁇ g tox B (1 mg/ml), 50 ⁇ l
  • SUBST ⁇ UTE SHEET (RULE 26) SCR gut content, and 0. IM Tris buffer, pH 8.0 in a total volume of 100 ⁇ l .
  • SCR gut content was omitted.
  • the reaction mixtures were incubated at 37°C for overnight.
  • 10 ⁇ l was withdraw and boiled with equal volume 2x SDS-PAGE sample buffer for SDS-PAGE analysis.
  • the remaining 90 ⁇ l reaction mixture was serial diluted with 10 mM sodium phosphate buffer, pH 7.0 and analyzed by SCR bioassay.
  • SDS-PAGE analysis indicated in SCR gut content treatment, peptide TcbA was digested completely into smaller peptides.
  • Y C or T
  • K G or T
  • R A or G
  • M A or C
  • PCR Polymerase Cham Reactions (PCR) were performed essentially as described in Example 8, using as forward primers P2.3.6.CB or P2.3.5, and as reverse primers P2.79.R.1 or P2.79R.CB, in all forward/reverse combinations, using Photorhabdus W-14 genomic DNA as template.
  • P2.79.3 were used as forward primers, and P2.3.5R, P2.3.5RI, and P2.3R.CB were used as reverse primers in all forward/reverse combinations. Only in the reactions containing P2 3.6. CB as the forward primers combined w th P2.79.R.1 or P2.79R.CB as the reverse primers was a non-artifactual amplified product seen, of estimated size (mobility on agarose gels) of 2500 base pairs. The order of the primers used to obtain this amplification product indicates that the peptide fragment TcdAn-PTlll lies ammo-proximal to the peptide fragment TcdA lx -PT79.
  • the 2500 bp PCR products were ligated to the plasmid vector pCRTMII (Invitrogen, San Diego, CA) according to the supplier's instructions, and the DNA sequences across the ends of the insert fragments of two isolates (HS24 and HS27) were determined using the supplier's recommended primers and the sequencing methods described previously. The sequence of both isolates was the same.
  • New primers were synthesized based on the determined sequence, and used to prime additional sequencing reactions to obtain a total of 2557 bases of the insert [SEQ ID NO:36]
  • Translation of the partial peptide encoded by SEQ ID No: 36 yields the 845 ammo acid sequence disclosed as SEQ ID NO: 37
  • Protein homology analysis of this portion of the TcdAn peptide fragment reveals substantial ammo acid homology ( (68% similarity, and 53% identity using the Wisconsin Package Version 8.0, Genetics Computer Group (GCG) , Madison, WI ) to residues 542 to 1390 of protein TcbA [SEQ ID NO: 12] or (60% similarity, and 54% identity using the Wisconsin Package Version
  • SUBST ⁇ UTE SHEET (RULE 26) Two pools of degenerate oligonucleot.ides, designed to encode the amino acid sequences described as SEQ ID NO: 39 (Table 28) and SEQ ID NO:41 (Table 27) , and the reverse complements of those sequences, were synthesized as described in Example 8, and their DNA sequences .
  • PCR Polymerase Chain Reactions
  • forward primers Al.44.1 or Al.44.2 and reverse primers A2.3R or A2.4R, m all forward/reverse combinations, using Photorhabdus W-14 genomic DNA as template.
  • primers A2.1 or A2.2 were used as forward primers
  • A1.44.1R, and A1.44.2R were used as reverse primers in all forward/reverse combinations.
  • the order of the primers used to obtain this amplification product indicates that the peptide fragment TcdA 1L -PK44 lies ammo-proximal to the 58 kDa peptide fragment of TcdA lu
  • the 1400 bp PCR products were ligated to the plasmid vector pCR ,M II according to the supplier's instructions.
  • the DNA sequences across the ends of the insert fragments of four isolates were determined using primers similar in sequence to the supplier's recommended primers and using sequencing methods described previously.
  • the nucleic acid sequence of all isolates differed as expected in the regions corresponding to the degenerate primer sequences, but the ammo acid sequences deduced from these data were the same as the actual amino acid sequences for the peptides determined previously, (SEQ ID NOS: 41 and 39) .
  • SUBST ⁇ UTE SHEET (RULE 26) DNA sequence determination of the cloned EcoR I fragments revealed an uninterrupted reading frame of 7551 base pairs (SEQ ID NO:46) , encoding a 282.9 kDa protein of 2516 ammo acids (SEQ ID NO: 47) .
  • TcdAn SEQ ID N0S:17, 18, 37, 38 and 39
  • TcdA peptide N-terminus SEQ ID NO: 41
  • TcdAm TcdAm internal peptides
  • SEQ ID NO:47 shows, starting at position 89, the sequence disclosed as SEQ ID NO: 13, which is the N-termmal sequence of a peptide of size approximately 201 kDa, indicating that the initial protein produced from SEQ ID NO: 46 is processed in a manner similar to that previously disclosed for SEQ ID NO: 12.
  • the protein s further cleaved to generate a product of size 209.2 kDa, encoded by SEQ ID NO:48 and disclosed as SEQ ID NO-.49 (TcdAn peptide), and a product of size 63.6 kDa, encoded by SEQ ID NO: 50 and disclosed as SEQ ID NO: 51 (TcdAm peptide)
  • TcdAm peptide a product of size 63.6 kDa, encoded by SEQ ID NO: 50 and disclosed as SEQ ID NO: 51
  • the msecticidal activity identified as toxm A (Example 15) derived from the products of SEQ ID NO: 46, as exemplified by the full-length protein of 282.9 kDa disclosed as SEQ ID NO: 47, is processed to produce the peptides disclosed as SEQ ID NOS: 49 and 51.
  • Example 15 the msecticidal activity identified as tox B (Example 15) derives from the products of SEQ ID NO: 11, as exemplified by the 280 6 kDa protein disclosed as SEQ ID NO.12. This protein is proteolytically processed to yield the 207 6 kDa peptide disclosed as SEQ ID NO: 53, which is encoded by SEQ ID NO: 52, and the 62.9 kDa peptide having N-termmal sequence disclosed as SEQ ID NO: 40, and further disclosed as SEQ ID NO: 55, which is encoded by SEQ ID NO: 54.
  • SUBST ⁇ JTE SHEET (RULE 26) NO: 55 (derived from SEQ ID NO: 12) have 76% similarity and 64% identity using the Wisconsin Package Version 8.0, Genetics Computer Group (GCG) , Madison, WI or 71% similarity and 64% identity using version 9.0 of the program.
  • Photorhabdus toxm(s) The ability of Photorhabdus toxm(s) to reduce plant damage caused by insect larvae was demonstrated by measuring leaf damage caused by European corn borer [ Ostrinia nubilalis) infested onto maize plants treated with Photorhabdus broth. Fermentation broth from Photorhabdus strain W-14 was produced and concentrated approximately 10-fold using ultraflltration (10,000 MW pore-size) as described in Example 13. The resulting concentrated broth was then filter sterilized using 0.2 micron nitrocellulose membrane filters. A similarly prepared sample of uninoculated 2% proteose peptone #3 was used for control purposes.
  • Maize plants (an mbred line) were grown from seed to vegetative stage 7 or 8 in pots containing a soilless mixture in a greenhouse (27°C day,- 22°C night, about 50%RH, 14 hr day-length, watered/fertilized as needed) .
  • test plants were arranged in a randomized complete block design (3 reps/treatment, 6 plants/treatment) in a greenhouse with temperature about 22°C day; 18°C night, no artificial light and with partial shading, about 50%RH and watered/fertilized as needfc ⁇
  • Treatments uninoculated media and concentrated Photorhabdus broth
  • a syringe sprayer 2.0 mis applied from directly (about 6 inches) over the whorl
  • one group of plants received no treatment.
  • Means with different letters are statistically different (p ⁇ 0.05 or p ⁇ 0.1) .
  • a series of plasmids were constructed to express the tcbA gene of Photorhabdus W-14 in Escherichia coli .
  • a list of the plasmids is shown in Table 30. A brief description of each construction follows as well as a summary of the E. coli expression data obtained.
  • Example 9 a large EcoR I fragment which hybridizes to the TcbA xl probe is described. This fragment was subcloned into pBC
  • the tcbA gene was PCR amplified from plasmid pDAB2025 using the following primers,- 5' primer ⁇ SlAc51) 5' TTT AAA CCA TGG GAA ACT CAT TAT CAA GCA CTA TC 3' and 3' primer (SlAc31) 5' TTT AAA GCG GCC GCT TAA CGG ATG GTA TAA CGA ATA TG 3' PCR was performed using a TaKaRa LA PCR kit from PanVera (Madison, WI) in the following reaction: 57.5 microliters water, 10 microliters 10X LA buffer, 16 microliters dNTPs (2.5 mM each stock solution), 20 microliters each primer at 10 pmoles/ microliters, 300 ng of the plasmid pDAB2025 containing the W-14 tcbA gene and one microliter of TaKaRa LA Taq polymerase.
  • the cycling conditions were 98°C/20 sec, 68°C/5 mm, 72°C/10 min for 30 cycles.
  • a PCR product of the expected about 7526 bp was isolated in a 0.8% agarose gel m TBE (100 mM Tris, 90 mM boric acid, 1 mM EDTA) buffer and purified using a Qiaex II kit from Qiagen (Chatsworth, CA) .
  • the purified tcbA gene was digested with Neo I and Wot T and ligated into the baculovirus transfer vector pAcGP67B (PharM gen (San Diego, CA) ) and transformed into DH5 ⁇ E. coli .
  • the resulting recombinant is called pDAB2026
  • the tcbA gene was then cut from pDAB2026 and transferred to pET27b to create plasmid pDAB2027.
  • a missense mutation the tcbA gene was repaired m pDAB2027.
  • the repaired tcbA gene contains two changes from the sequence shown m Sequence ID NO: 11, an A>G at 212 changing an asparagme 71 to serine 71 and a G>A at 229 changing an alanme 77 to threon e 77. These changes are both upstream of the proposed TcbA ⁇ :L N " terminus .
  • DDAB202B Construction of DDAB202B
  • the tcbA coding region of pDAB2027 was transferred to vector pET15b. This was accomplished using shotgun ligations, the DNAs were cut with restriction enzymes Neo I and Xho I The resulting recombinant is called pDAB2028.
  • tcbA in E. coli was obtained by modification of the methods previously described by Studier et al . (Studier, F.W., Rosenberg, A., Dunn, J., and Dubendorff, J., (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol . , 185. 60-89.) Competent E. coli cells strain BL2KDE3) were transformed with plasmid pDAB2028 and plated on LB agar containing 100 ⁇ g/mL ampicillm and 40 mM glucose The transformed cells were plated to a density of several hundred isolated colonies/plate.
  • IPTG IPTG
  • E. coli cultures expressing TcbA peptides were processed as follows Cells were harvested by centrifugation at 17,000 x G and the media was decanted and saved in a separate container
  • SUBST ⁇ JTE SHEET (RULE 26) The media was concentrated about 8x using the M12 (Amicon, Beverly MA) filtration system and a 100 kD molecular mass cut-off filter. The concentrated media was loaded onto an anion exchange column and the bound proteins were eluted with 1.0 M NaCl. The 1.0 M NaCl elution peak was found to cause mortality against Southern corn rootworm (SCR) larvae Table 30). The 1.0 M NaCl fraction was dialyzed against 10 mM sodium phosphate buffer pH 7.0, concentrated, and subjected to gel filtration on Sepharose CL-4B (Pharmacia, Piscataway, NJ) .
  • the region of the CL-4B elution profile corresponding to calculated molecular weight (about 900 kDa) as the native W-14 tox complex was collected, concentrated and bioassayed against larvae.
  • the collected 900 kDa fraction was found to have msecticidal activity (see Table 31 below) , with symptomology similar to that caused by native W-14 tox complex
  • This fraction was subjected to Protemase K and heat treatment, the activity both cases was either eliminated or reduced, providing evidence that the activity is prote aceous m nature
  • the active fraction tested immunologically positive for the TcbA and TcbA ul peptides in immunoblot analysis when tested with an anti-TcbAm monoclonal antibody Table 31
  • the cellular debris was pelleted by centrifugation at 25,000 x g and the cell supernatant was decanted, passed through a 0.2 micron filter and subjected to anion exchange chromatography using a Pharmacia 10/10 column packed with Poros HQ 50 beads
  • the bound proteins were eluted by performing a NaCl gradient of 0.0 to 1.0 M Fractions containing the TcbA protein were combined and concentrated using a 50 kDa concentrator and subjected to gel filtration chromatography using Pharmacia CL-4B beaded matrix.
  • TcbA oligomer molecular mass of approximately 900 kDa
  • the fractions containing TcbA oligomer, molecular mass of approximately 900 kDa, were collected and subjected to anion exchange chromatography using a Pharmacia Mono Q 10/10 column equilibrated with 20 mM Tris buffer pH 7.3
  • a gradient of 0.0 to 1.0 M NaCl was used to elute recombinant TcbA protein.
  • the recombinant TcbA fraction was found to cause SCR mortality in bioassay experiments similar to those in Table 31.
  • a second set of expression constructions were prepared and tested for expression of the TcbA protein toxm.
  • the plasmid pDAB2028 contains the tcbA coding region m the commercial vector pET15 (Novagen, Madison, WI),
  • SUBST ⁇ JTE SHEET (RULE 26) encodes an ampicillm selection marker.
  • the plasmid pDAB2030 was created to express the tcbA coding region from a plasmid which encodes a kanamycm selection marker. This was done by cutting pET27 (Novagen, Madison, WI) a kanamycm selection plasmid, and pDAB2028 with Xba I and Xho I . This releases the entire multiple cloning site, including the tcbA coding region from plasmid pDAB2028.
  • the two cut plasmids were mixed and ligated Recombinant plasmids were selected on kanamycm and those containing the pDAB2028 fragment were identified by restriction analysis.
  • the new recombinant plasmid is called pDAB2030.
  • a plasmid containing the mutated tcbA coding region (pDAB2030) was digested with iVco I and Not I and purified away from the 1670 bp fragment m a 0.8% agarose with Qiaex II (Qiagen, Chatsworth, CA) . The corrected Neo I /Pin Al fragment was then ligated into pDAB2030 The ligated DNA was transformed into DH5 ⁇ E. coli . A clone was isolated, sequenced and found to be correct. This plasmid, containing the corrected tcbA coding region, is called pDAB2031
  • the expression plasmids pDAB2025 and pDAB2027-2031 all rely on the Bacte ⁇ ophage T7 expression system An additional vector system was used for bacterial expression of the tcbA gene and its derivatives
  • the expression vector Trc99a (Pharmacia Biotech, Piscataway, NJ) contains a strong trc promoter upstream of a multiple cloning site with a 5' Neo I site which is compatible with the tcbA coding region from pDAB2030 and 2031
  • the plasmid does not have a compatible 3 ! site Therefore, the Hind III site of Trc99a was cut and made blunt by treatment with T4 DNA
  • SUBST ⁇ UTE SHEET (RULE 26) polymerase Boehringer Mannheim, Indianapolis, IN
  • the vector plasmid was then cut by Neo I followed by treatment with alkaline phosphatase.
  • the plasmids pDAB2030 and pDAB2031 were each cut with Xho I (cuts at the 3' end of the tcbA coding region) followed by treatment with T4 DNA polymerase to blunt the ends .
  • the plasmids were then cut with Neo I, the DNAs were extracted with phenol, ethanol precipitated and resuspended in buffer.
  • Trc99a and pDAB2030 and pDAB2031 plasmids were mixed separately, ligated and transformed into DH5 ⁇ cells and plated on LB media containing ampicillin and 50 mM glucose. Recombinant plasmids were identified by restriction digestion.
  • the new plasmids are called pDAB2033 (contains the tcbA coding sequence with the two mutations in tcbA- ⁇ ) and pDAB2034 (contains the corrected version of tcbA from pDAB2031) .
  • Plasmid pDAB2032 An Expression Plasmid for tSh ⁇ u ⁇ m
  • a plasmid encoding the TcbA ⁇ A j ⁇ portion of TcbA was created in a similar way as plasmid pDAB2031.
  • a PCR product was generated using TH42 (5' TAG GTC TCC ATG GCT TTT ATA CAA GGT TAT AGT GAT CTG 3') and TH50 (5' ACC GTC TTC TTT ACG ATC AGT G 3') primers and plasmid pDAB2025 as template. This yielded a product of 1521 bp having an initiation codon at the beginning of the coding sequence of tcbA ⁇ i_ .
  • This PCR product was isolated in a 1% agarose gel and purified.
  • the purified product was cloned into pCR2.1 as above and a correct clone was identified by DNA sequence analysis.
  • This clone was digested with Neo I and Pin Al, a 1414 bp fragment was isolated in a 1% agarose gel and ligated into the Neo I and Pin Al sites of plasmid pDAB2030 and transformed into DH5 E. coli .
  • This new plasmid, designed to express in E. coli is called pDAB2032.
  • tcbA and tcbA ⁇ i ⁇ ii from Plasmids pDAB2030.
  • pDAB2031 and pDAB2032 Expression of tcbA in E. coli from plasmids pDAB2030, pDAB2031 and pDAB2032 was as described herein, except expression of tcbAnA-ii -i was done in E. coli strain HMS174(DE3) (Novagen, Madison, I) .
  • the plasmid pDAB2033 was transformed into BL21 cells (Novagen, Madison, WI) and plated on LB containing 100 micrograms/mL ampicillin and 50 mM glucose. The plates were spread such that several hundred well separated colonies were present on each plate following incubation at either 30°C or 37°C overnight. The colonies were scraped from the plates and suspended m LB containing 100 micrograms/mL ampicillm, but no glucose. Typical culture volume was 250 mL m a single 1 L baffle bottom flask. The cultures were induced when the culture reached a density of 0.3-0.6 OD600 n . Most often this density was achieved immediately after suspension of the cells from the plates and did not require a growth period in liquid media.
  • Method 1 cells were induced with 1 mM IPTG at 37°C. The cultures were shaken at 200 rpm on a platform shaker for 5 hours and harvested.
  • Method 2 The cultures were induced with 25 micromolar IPTG at 30°C and shaken at 200 rpm for 15 hours at either 20°C or 30°C. The cultures were stored at 4°C until used for purification.
  • TcbA and TcbA 11 A 111 were as described herein. Results of several representative E. coli expression experiments are shown Table 32. All materials shown in Table 32 were purified from the media fraction of the cultures. The predicted native molecular weight is approximately 900 kD as described herein The purity of the samples, the amount of TcbA relative to contaminating proteins, varied with each preparation.
  • Toxms isolated from W-14 broth were purified as described m Example 15.
  • the TcaB protein toxm was pretreated with proteases (Example 16) that had been isolated from W-14 broth as previously described (Example 15) .
  • Protein molecular mass was determined using matrix-assisted laser desorption lomzation time- of-flight mass spectroscopy, hereinafter MALDI-TOF, on a VOYAGER BIOSPECTROMETRY workstation with DELAYED EXTRACTION technology (PerSeptive Biosystems, Frammgham, MA).
  • the protein of mte r re " s rrGO-500 pmoles m 5 ⁇ l) was mixed with 1 ⁇ l of acetonit ⁇ le and dialyzed for 0.5 to 1 h on a Millipore VS filter having a pore size of 0.025 ⁇ M (Millipore Corp. Bedford, MA). Dialysis was performed by floating the filter on water (shinny side up) followed by adding protem-acetonitrile mixture as a droplet to the surface of the filter. After dialysis, the dialyzed protein removed using a pipette and was then mixed with a matrix consisting of s apinic acid and trifluoroacetic acid according to manufacturers instructions. The protein and matrix were allowed to co-crystallize on a about 3 cm 2 gold-plated sample plate
  • SUBST ⁇ UTE SHEET (RULE 26) 90.0%; guide wire voltage of 0.010%; linear mode; and a pulse delay time of 350 ns .
  • Protein mass analysis data are shown m Table 33.
  • the data obtained from MALDI-TOF was compared to that hypothesized from gene sequence information and as previously determined by SDS-PAGE
  • TcbAn Protease Generated 201 000 Da 216 , 614 Da' 215 , 123 Da' 210 , 391 Da' 208 , 680 Da'
  • SUBST ⁇ UTE SHEET (RULE 26) sequences, i.e., regions that might be antigenic sites. This method combined information from hydrophilicity, surface probability, and backbone flexibility predictions along with the secondary structure predictions order to produce a composite prediction of the surface contour of a protein. The scores for each of the analyses were normalized to a value between -1 0 and
  • the antigenicity index value was obtained for the entire sequence of the target peptide. From each peptide, an area covering 19 or more ammo acids that showed a high antigenicity index from the original sequence was re-analyzed to determine the antigenicity index of the subpeptide without the flanking residues This re-analysis was necessary because the antigenicity index of a peptide could be influenced by the flanking ammo acid residues. If the isolated subpeptide sequence did not maintain a high antigenicity index, a new region was chosen and the analysis was repeated
  • Each selected subpeptide sequence was aligned and compared to all seven target peptide sequences using MacVector TM alignment program. If a selected subpeptide sequence showed identity (greater than 20%) to another target peptide, a new 19 or more ammo acid region was isolated and re-analyzed. Unique subpeptide sequences covering 19 or more ammo acid showing high antigenicity index were selected from all target peptides.
  • Pre-cast SDS-polyacrylamide gels with 4-20% gradient were used Between 1 to 8 ⁇ g of protein was applied to each gel well Electrophoresis was performed and the protein was electroblotted onto Hybond-ECL nitrocellulose membrane (Amersham International) The membrane was blocked with 10% milk in TBST (25 mM Tris HCl pH 7.4, 136 mM NaCl, 2.7 mM KCl, 0 1% Tween 20) for one hour at room temperature Each rabbit serum was diluted in 10% milk/TBST to 1:500. Other dilutions between 1.50 to 1 1000 were also used. The serum was added to the membrane and placed on a platform rocker for at least one hour.
  • TBST 25 mM Tris HCl pH 7.4, 136 mM NaCl, 2.7 mM KCl, 0 1% Tween 20
  • the membrane was washed thoroughly with the blocking solution or TBST.
  • a 1.2000 dilution of secondary antibodies goat anti -mouse IgG conjugated to horse radish peroxidase,- BioRad Laboratories
  • n 10% milk/TBST was applied to the membrane placed on a platform rocker for one hour
  • the membrane was subsequently washed with excess amount of TBST.
  • SUBST ⁇ UTE SHEET (RULE 26) detection of the protein was performed by using an ECL (Enhanced Chemiluminescence) detection kit (Amersham International) .
  • a luminometer was used to establish the bioluminescence associated with these Photorhabdus strains.
  • the broths from each strain were measured at three time intervals after inoculation in liquid culture (24, 48, 72 hr) and compared to background luminosity (uninoculated media) .
  • Several Xenorhabdus strains were tested as negative controls for luminosity.
  • cell density Prior to measuring light emission from the various broths, cell density was established by measuring light absorbance (560 nM) in a Gilford Systems (Oberl , OH) spectrophotometer using a sipper cell. The resulting light emitting units could then be normalized to density of cells.
  • SUBST ⁇ UTE SHEET (RULE 26) label instructions. Incubation occurred at 28 °C and descriptions were produced after 5 days.
  • a colony of the test organism was removed on a small plug from a nutrient agar plate and placed into the bottom of a glass test tube.
  • One ml of a household hydrogen peroxide solution was gently added down the side of the tube. A positive reaction was recorded when bubbles of gas (presumptive oxygen) appeared immediately or withm 5 seconds . Controls of uninoculated nutrient agar and hydrogen peroxide solution were also examined.
  • each culture was inoculated into 10 ml of Bacto Nitrate Broth (Difco Laboratories, Detroit, MI). After 24 hours incubation with gentle agitation at 28 °C, nitrite production was tested by the addition of two drops of sulfanilic acid reagent and two drops of alpha-naphthylamme reagent (see Difco Manual, 10th edition, Difco Laboratories, Detroit, MI, 1984) The generation of a distinct pink or red color indicates the formation of nitrite from nitrate whereas the lack of color formation indicates that the strain is nitrate reduction negative.
  • the indicator resazurin demonstrates the presence of medium oxygenation or the aerobiosis zone (Difco Manual, 10th edition, Difco Laboratories, Detroit, MI). Growth zone results obtained for the Photorhabdus strains tested were consistent with those of a facultative anaerobic microorganism. In the case of unclear results, the final agar concentration of fluid thioglycolate broth medium was raised to 0.75% and the growth characteristics rechecked.
  • SUBST ⁇ UTE SHEET (RULE 26) element are thought to play an important role m the organization of the bacterial genome. Genomic organization is believed to be shaped by selection and the differential dispersion of these elements withm the genome of closely related bacterial strains can be used to discriminate these strains (e.g., Louws, F. J. ,
  • Rep-PCR utilizes oligonucleotide primers complementary to these repetitive sequences to amplify the variably sized DNA fragments lying between them. The resulting products are separated by electrophoresis to establish the DNA "fingerprint" for each strain.
  • TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to a final volume of 10 ml and 12 ml of 5 M NaCl was then added. This mixture was centrifuged 20 mm. at 15,000 x g. The resulting pellet was resuspended in 5.7 ml of TE and 300 ⁇ l of 10% SDS and 60 ⁇ l 20 mg/ml protemase K (Gibco BRL Products, Grand Island, NY) were added. This mixture was incubated at 37°C for 1 hr, approximately 10 mg of lysozyme was then added and the mixture was incubated for an additional 45 mm.
  • Precipitated DNA was removed with a glass rod, washed twice with 70% ethanol, dried and dissolved m 2 ml of STE (10 mM Tris -HCl pH8.0, 10 mM NaCl, 1 mM EDTA) . The DNA was then quantitated by optical density at 260 nm.
  • SUBSTTTUTE SHEET (RULE 26) amplification was performed in a Perkm Elmer DNA Thermal Cycler (Norwalk, CT) using the following conditions. 95°C/7 min. then 35 cycles of; 94°C/1 mm.,44°C/l mm., 65°C/8 mm., followed by 15 mm. at 65°C. After cycling, the 25 ⁇ l reaction was added to 5 ⁇ l of 6X gel loading buffer (0.25% bromophenol blue, 40% w/v sucrose in
  • Tris-borate 0.002 M EDTA
  • the gel was run for approximately 16 hours at 45v. Gels were then stained in 20 ⁇ g/ml ethidium bromide for 1 hour and destamed m TBE buffer for approximately 3 hours. Polaroid photographs of the gels were then taken under UV illumination.
  • the COPH (cophenetic values) and MXCOMP (matrix comparison) programs were used to generate a cophenetic value matrix and compare the correlation between this and the original matrix upon which the clustering was based.
  • "Seed" flasks or cultures were produced by either inoculating 2 mis of an oil overlayered storage culture or by transferring a primary variant colony into 175 ml sterile medium in a 500 ml tribaffled flask covered with a Kaput closure (The use of other inoculum sources is also possible.)
  • the seed culture was transferred into production flasks
  • Production flasks were usually inoculated by adding about 1% of the actively growing seed culture to sterile 2% PP3 medium (e.g 2.0 ml per 175 ml sterile medium) Production of broths occurred in 500 ml tribaffled flasks covered with a Kaput Production flasks were agitated at 28 °C on a rotary shaker at 150 rpm.
  • the resulting broths were then concentrated (up to 10-fold) using a 10,000 or 100,000 MW cut-off membrane, M12 ultra-flltration device (Amicon, Beverly MA) or centrifugal concentrators (Millipore, Bedford, MA and Pall Filtron, Northborough, MA) with a 13,000 or
  • SUBST ⁇ UTE SHEET 100,000 MW pore size
  • the broth was spun at 2000xg for approximately 2 hr
  • the membrane permeate was added to the corresponding retentate to achieve the desired concentration of components greater than the pore size used.
  • Photorhabdus strains fermented as described above is shown m Table 36. It is possible that improved or additional msecticidal activities could be detected with these strains through increased concentration of the broth or by employing different fermentation methods Consistent with the activity being associated with a protein, the msecticidal activity of all strains tested was heat labile .
  • Culture broth (s) from diverse Photorhabdus strains show differential msecticidal activity (mortality and/or growth inhibition) against a number of insects. More specifically, the activity is seen against corn rootworm which is a member of the insect order Coleoptera Other members of the Coleoptera include boll weevils, wireworms, pollen beetles, flea beetles, seed beetles and Colorado potato beetle
  • the broths and purified toxm complex (es) are also active against tobacco budworm, tobacco hornworm and European corn borer which are members of the order Lepidoptera.
  • SUBST ⁇ JTE SHEET Activity against corn rootworm larvae was tested as follows. Photorhabdus culture broth(s) (10 fold concentrated, filter sterilized), 2% Proteose Peptone #3 (10 fold concentrated), purified toxm complex(es), 10 mM sodium phosphate buffer, pH 7.0 were applied directly to the surface (about 1 5 cm 2 ) of artificial diet (Rose, R. I. and McCabe, J. M. 1973. J Econ Entomol 66, 398-400) in 40 ⁇ l aliquots.
  • Toxm complex was diluted m 10 mM sodium phosphate buffer, pH 7.0
  • the diet plates were allowed to air-dry in a sterile flow-hood and the wells were infested with single, neonate Diabrotica undeeimpunetata howardi (Southern corn rootworm, SCR) hatched from surface sterilized eggs
  • the plates were sealed, placed in a humidified growth chamber and maintained at 27°C for the appropriate period (3-5 days) Mortality and larval weight determinations were then scored Generally 16 insects per treatment were used all studies Control mortality was generally less than 5%.
  • Activity against lepidopteran larvae was tested as follows Concentrated (10-fold) Photorhabdus culture broth(s) , control medium (2% Proteose Peptone #3), purified toxm complex(es), ⁇ o mM sodium phosphate buffer, pH 7.0 were applied directly to the surface (about 1.5 cm 2 ) of standard artificial lepidopteran diet (Stoneville Yellow diet) in 40 ⁇ l aliquots. The diet plates were allowed to air-dry in a sterile flow-hood and each well was infested with a single, neonate larva.
  • SUBST ⁇ JTE SHEET (RULE 26) air-dry in a sterile flow-hood and each well was infested with a single, CO- anesthetized first instar German cockroach [ Blatella germanica) . Following infestation, the diet plates were sealed, placed in a humidified growth chamber and maintained in the dark at 27°C for the appropriate period. Mortality and weight determinations were scored at day 5. Control mortality less than 10%.

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Abstract

L'invention porte sur des protéines isolées à partir du genre Photorhabdus et qui sont toxiques pour les insectes lors d'une exposition. On a découvert des Photorhabdus luminescens (anciennement Xenorhabdus luminescens) dans des échantillons cliniques mammaliens sous forme de symbiote bactérienne de nématodes entomopathogènes du genre Heterorhabditis. Ces toxines protéiniques peuvent être appliquées aux aliments de larves d'insectes ainsi qu'à des plantes, ou peuvent être obtenues par génie génétique dans ceux-ci, en vue d'éliminer les insectes.
PCT/US1997/007657 1995-11-06 1997-05-05 TOXINES PROTEINIQUES INSECTICIDES ISOLEES A PARTIR DE $i(PHOTORHABDUS) Ceased WO1998008932A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
IL12859097A IL128590A0 (en) 1996-08-29 1997-05-05 Insecticidal protein toxins from photorhabdus
CA002263819A CA2263819A1 (fr) 1996-08-29 1997-05-05 Toxines proteiniques insecticides isolees a partir de photorhabdus
AU28299/97A AU2829997A (en) 1996-08-29 1997-05-05 Insecticidal protein toxins from (photorhabdus)
EP97922696A EP0970185A4 (fr) 1996-08-29 1997-05-05 Toxines proteiniques insecticides isolees a partir de photorhabdus
JP10511612A JP2000515024A (ja) 1996-08-29 1997-05-05 ホトラブダス由来殺虫性タンパク質毒素
PL97332033A PL332033A1 (en) 1995-11-06 1997-05-05 Insecticidal proteinous toxins obtained from photorhabdus
BR9711441-3A BR9711441A (pt) 1996-08-29 1997-05-05 Toxinas de proteìnas inseticidas provenientes de photorhabdus
SK246-99A SK24699A3 (en) 1996-08-29 1997-05-05 Insecticidal protein toxins from photorhabdus

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US08/705,484 1996-08-28
US70548496A 1996-08-29 1996-08-29
US74369996A 1996-11-06 1996-11-06
PCT/US1996/018003 WO1997017432A1 (fr) 1995-11-06 1996-11-06 Toxines proteiques insecticides provenant de photorhabdus
US08/743,699 1996-11-06
KEPCT/US96/18003 1996-11-06

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AR007133A1 (es) 1999-10-13
KR20000037116A (ko) 2000-07-05
JP2000515024A (ja) 2000-11-14
TR199901126T2 (xx) 1999-07-21
IL128590A0 (en) 2000-01-31
EP0970185A4 (fr) 2003-02-26
AU2829997A (en) 1998-03-19
SK24699A3 (en) 2000-04-10
EP0970185A1 (fr) 2000-01-12
TW509722B (en) 2002-11-11

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