U.S. patent application number 10/078968 was filed with the patent office on 2002-10-10 for insecticidal protein toxins from xenorhabdus.
Invention is credited to Bintrim, Scott B., Bowen, David J., Ciche, Todd A., Ensign, Jerald C., Fatig, Raymond O., Ffrench-Constant, Richard H., Orr, Gregory L., Petell, James K., Strickland, James A., Tenor, Jennifer L..
Application Number | 20020147148 10/078968 |
Document ID | / |
Family ID | 21939094 |
Filed Date | 2002-10-10 |
United States Patent
Application |
20020147148 |
Kind Code |
A1 |
Ensign, Jerald C. ; et
al. |
October 10, 2002 |
Insecticidal protein toxins from xenorhabdus
Abstract
Proteins from the genus Xenorhabdus are toxic to insects upon
oral exposure. These protein toxins can be applied to insect larvae
food and plants for insect control.
Inventors: |
Ensign, Jerald C.; (Madison,
WI) ; Bowen, David J.; (Oregon, WI) ; Tenor,
Jennifer L.; (Madison, WI) ; Ciche, Todd A.;
(Madison, WI) ; Petell, James K.; (Zionsville,
IN) ; Strickland, James A.; (Lebanon, IN) ;
Orr, Gregory L.; (Indianapolis, IN) ; Fatig, Raymond
O.; (Zionsville, IN) ; Bintrim, Scott B.;
(Carmel, IN) ; Ffrench-Constant, Richard H.;
(Madison, WI) |
Correspondence
Address: |
DOW AGROSCIENCES LLC
9330 ZIONSVILLE RD
INDIANAPOLIS
IN
46268
US
|
Family ID: |
21939094 |
Appl. No.: |
10/078968 |
Filed: |
February 19, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10078968 |
Feb 19, 2002 |
|
|
|
09514739 |
Feb 28, 2000 |
|
|
|
6379946 |
|
|
|
|
60045641 |
May 5, 1997 |
|
|
|
Current U.S.
Class: |
514/4.5 ;
514/21.2 |
Current CPC
Class: |
C07K 14/24 20130101;
Y10S 435/822 20130101; A01N 63/50 20200101; A01N 63/50 20200101;
A01N 63/20 20200101 |
Class at
Publication: |
514/12 |
International
Class: |
A01N 037/18 |
Claims
We claim:
1. A composition, comprising an effective amount of a Xenorhabdus
protein toxin having functional activity against an insect, said
protein toxin being derived from a protein having a native
molecular size of at least 100 kDa.
2. The composition of claim 1, wherein the Xenorhabdus toxin having
functional activity against an insect is produced by a purified
culture of Xenorhabdus nematophilus, Xenorhabdus poinarii,
Xenorhabdus bovienii, Xenorhabdus beddingii or Xenorhabdus
species.
3. The composition of claim 2, wherein said purified culture of
Xenorhabdus selected from the group consisting of S. carp, X. Wi,
X. nem, X. NH3, X. riobravis, GL 133B, DEX1, DEX2, DEX3, DEX4,
DEX5, DEX6, DEX7, DEX8, ILM037, ILM039, ILM070, ILM078, ILM079,
ILM080, ILM081, ILM082, ILM083, ILM084, ILM102, ILM103, ILM104,
ILM129, ILM133, ILM135, ILM138, ILM142, ILM143, GLX26, GLX40,
GLX166, SEX20, SEX76, and SEX180.
4. The composition of claim 1, wherein the toxin having functional
activity against an insect is produced by a purified culture of
Xenorhabdus strain designated S. carp, X. Wi, X. nem, X. NH3, X.
riobravis, GL 133B, DEX1, DEX2, DEX3, DEX4, DEX5, DEX6, DEX7, DEX8,
ILM037, ILM039, ILM070, ILM078, ILM079, ILM080, ILM081, ILM082,
ILM083, ILM084, ILM102, ILM103, ILM104, ILM129, ILM133, ILM135,
ILM138, ILM142, ILM143, GLX26, GLX40, GLX166, SEX20, SEX76, and
SEX180.
5. The composition of claim 4, wherein the toxin having functional
activity against an insect is a mixture of one or more toxins
produced from purified cultures of Xenorhabdus.
6. The composition of claim 3 wherein the toxin having functional
activity against an insect, said toxin being a mixture of one or
more toxins, is produced from said purified cultures of
Xenorhabdus, said purified cultures being selected from the group
consisting of S. carp, X. Wi, X. nem, X. NH3, X. riobravis, GL
133B, DEX1, DEX2, DEX3, DEX4, DEX5, DEX6, DEX7, DEX8, ILM037,
ILM039, ILM070, ILM078, ILM079, ILM080, ILM081, ILM082, ILM083,
ILM084, ILM102, ILM103, ILM104, ILM129, ILM133, ILM135, ILM138,
ILM142, ILM143, GLX26, GLX40, GLX166, SEX20, SEX76, and SEX180.
7. The composition of claim 1, wherein the insect is of the order
Coleoptera, Lepidoptera, Diptera, or Acarina.
8. The composition of claim 7, wherein the insect species from
order Coleoptera are selected from the group consisting of Corn
Rootworm, Boll Weevil, wireworms, pollen beetles, flea beetles,
seed beetles and Colorado potato beetle.
9. The composition of claim 7, wherein the insect species from
order Lepidoptera are selected from the group consisting of Beet
Armyworm, European Corn Borer, Tobacco Hornworm, Tobacco Budworm,
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.
10. The composition of claim 7, wherein the insect species from the
order Diptera are selected from the group consisting of pea midge,
carrot fly, cabbage root fly, turnip root fly, onion fly, crane
fly, house fly, and various mosquito species.
11. The composition of claim 7 wherein the insects species from the
order Acarina are selected from the group consisting of two-spotted
spider mites, strawberry spider mites, broad mites, citrus red
mite, European red mite, pear rust mite and tomato russet mite.
12. A substantially pure microorganism culture comprising of
Xenorhabdus strain selected from the group consisting of S. carp,
X. Wi, X. nem, X. NH3, X. riobravis, GL 133cn , ILM037, ILM039,
ILM070, ILM078, ILM079, ILM080, ILM081, ILM082, ILM083, ILM084,
ILM102, ILM103, ILM104, ILM129, ILM133, ILM135, ILM138, ILM142,
ILM143, GLX26, GLX40, GLX166, SEX20, SEX76, and SEX180.
13. A purified protein preparation comprising, a Xenorhabdus
protein with at least one subunit having an approximate molecular
weight between about 20 kDa to about 350 kDa; between about 130 kDa
to about 350 kDa; about 80 kDa to about 130 kDa; about 40 kDa to
about 80 kDa; or about 20 kDa to about 40 kDa.
14. The purified protein preparation of claim 13 comprising, a
native Xenorhabdus protein with at least one subunit having a
molecular weight of at least 100 kDa or greater.
15. A purified protein preparation comprising a protein containing
an amino acid sequence selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
16. A method of controlling an insect comprising, delivering to an
insect an effective amount of a protein toxin having functional
activity against an insect, wherein the protein is produced by a
purified bacterial culture of the genus Xenorhabdus and has an
native molecular weight of at least 100 kDa.
17. The method of claim 16, wherein the Xenorhabdus toxin having
functional activity against an insect is produced by a purified
culture of Xenorhabdus nematophilus, Xenorhabdus poinarii,
Xenorhabdus bovienii, Xenorhabdus beddingii or Xenorhabdus
species.
18. The method of claim 17, wherein said purified culture of
Xenorhabdus selected from the group consisting of S. carp, X. Wi,
X. nem, X. NH3, X. riobravis, GL 133B, DEX1, DEX2, DEX3, DEX4,
DEX5, DEX6, DEX7, DEX8, ILM037, ILM039, ILM070, ILM078, ILM079,
ILM080, ILM081, ILM082, ILM083, ILM084, ILM102, ILM103, ILM104,
ILM129, ILM133, ILM135, ILM138, ILM142, ILM143, GLX26, GLX40,
GLX166, SEX20, SEX76, and SEX180.
19. The method of claim 16, wherein the toxin having functional
activity against an insect is produced by a purified culture of
Xenorhabdus strain designated S. carp, X. Wi, X. nem, X. NH3, X.
riobravis, GL 133B, DEX1, DEX2, DEX3, DEX4, DEX5, DEX6, DEX7, DEX8,
ILM037, ILM039, ILM070, ILM078, ILM079, ILM080, ILM081, ILM082,
ILM083, ILM084, ILM102, ILM103, ILM104, ILM129, ILM133, ILM135,
ILM138, ILM142, ILM143, GLX26, GLX40, GLX166, SEX20, SEX76, and
SEX180.
20. The method of claim 16, wherein the toxin having functional
activity against an insect is a mixture of one or more toxins
produced from purified cultures of Xenorhabdus.
21. The method of claim 16, wherein the insect is of the order
Coleoptera, Lepidoptera, Diptera, or Acarina.
22. The method of claim 21, wherein the insect species from order
Coleoptera are selected from the group consisting of Corn Rootworm,
Boll Weevil, wireworms, pollen beetles, flea beetles, seed beetles
and Colorado potato beetle.
23. The method of claim 21, wherein the insect species from order
Lepidoptera are selected from the group consisting of Beet
Armyworm, European Corn Borer, Tobacco Hornworm, Tobacco Budworm,
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.
24. The method of claim 21, wherein the insect species from the
order Diptera are selected from the group consisting of pea midge,
carrot fly, cabbage root fly, turnip root fly, onion fly, crane
fly, house fly, and various mosquito species.
25. The method of claim 21, wherein the insect species from the
order Acarina are selected from the group consisting of two-spotted
spider mites, strawberry spider mites, broad mites, citrus red
mite, European red mite, pear rust mite and tomato russet mite.
26. A method of altering the toxin level or toxin composition
produced by Xenorhabdus strains comprising, modifying media
composition.
27. The method of claim 26 wherein said media composition is
modified by fermenting said Xenorhabdus in tryptic soy broth.
28. The method of claim 26 wherein said media composition is
modified by increasing ionic strength of said media.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims priority from a U.S.
Provisional Patent Application Serial No. 60/045,641 filed on May
5, 1997.
FIELD OF THE INVENTION
[0002] The present invention relates to toxins isolated from
bacteria and the use of said toxins as insecticides.
BACKGROUND OF THE INVENTION
[0003] In the past there has been interest in using biological
agents as an option for pest management. One such method explored
was the potential of insect control using certain genera of
nematodes. Nematodes, like those of the Steinernema and
Heterorhabditis genera, can be used as biological agents due in
part to their transmissible insecticidal bacterial symbionts of the
genera Xenorhabdus and Photorhabdus, respectively. Upon entry into
the insect, the nematodes release their bacterial symbionts into
the insect hemolymph where the bacteria reproduce and eventually
cause insect death. The nematode then develops and reproduces
within the cadaver. Bacteria-containing nematode progeny exit the
insect cadaver as infective juveniles which can then invade
additional larvae thus repeating the cycle leading to nematode
propagation. While this cycle is easily performed on a micro scale
in a laboratory setting, adaptation to the macro level, as needed
to be effective as a general use insecticide, is difficult,
expensive, and inefficient to produce, maintain, distribute and
apply.
[0004] In addition to biological approaches to pest management such
as nematodes, there are now pesticide control agents commercially
available that are naturally derived. These naturally derived
approaches can be as effective as synthetic chemical approaches.
One such naturally occurring agent is the crystal protein toxin
produced by the bacteria Bacillus thuringiensis (Bt) These protein
toxins have been formulated as sprayable insect control agents. A
more recent application of Bt technology has been to isolate and
transform into plants the genes that produce the toxins. Transgenic
plants subsequently produce the Bt toxins thereby providing insect
control, (see U.S. Patent Nos. 5,380,831; 5,567,600; and 5,567,862
to Mycogen in San Diego, Calif.).
[0005] Transgenic Bt plants are quite efficacious and usage is
predicted to be high in some crops and areas. This has caused a
concern that resistance management issues may arise more quickly
than with traditional sprayable applications. Thus, it would be
quite desirable to discover other bacterial sources distinct from
Bt which produce toxins that could be used in transgenic plant
strategies, or could be combined with Bts to produce insect
controlling transgenic plants.
[0006] It has been known in the art that bacteria of the genus
Xenorhabdus are symbiotically associated with the Steinernema
nematode. Unfortunately, as reported in a number of articles, the
bacteria only had pesticidal activity when injected into insect
larvae and did not exhibit biological activity when delivered
orally (see Jarosz J. et al. "Involvement of Larvicidal Toxins in
Pathogenesis of Insect Parasitism with the Rhabditoid Nematodes,
Steinernema Feltiae and Heterorhabditis Bacteriophora" Entomophaga
36 (3) 1991 361-368; Balcerzak, Malgorzata "Comparative studies on
parasitism caused by entomogenous nematodes, Steinernema feltiae
and Heterorhabditis bacteriophors I. The roles of the
nematode-bacterial complex, and of the associated bacteria alone,
in pathogenesis" Acta Parasitologica Polonica, 1991, 36(4),
175-181).
[0007] For the reasons stated above it has been difficult to
effectively exploit the insecticidal properties of the nematode or
its bacterial symbiont. Thus, it would be quite desirable to
discover proteinaceous agents derived from Xenorhabdus bacteria
that have oral activity so that the products produced therefrom
could either be formulated as a sprayable insecticide or the
bacterial genes encoding said proteinaceous agents could be
isolated and used in the production of transgenic plants. Until
applicants' invention herein there was no known Xenorhabdus species
or strains that produced protein toxin(s) having oral activity.
SUMMARY OF THE INVENTION
[0008] The native toxins are protein complexes that are produced
and secreted by growing bacterial cells of the genus Xenorhabdus.
The protein complexes, with a native molecular size ranging from
about 800 to 3000 kDa, can be separated by SDS-PAGE gel analysis
into numerous component proteins. The toxins exhibit significant
toxicity upon exposure to a number of insects. Furthermore, toxin
activity can be modified by altering media conditions. In addition,
the toxins have characteristics of being proteinaceous in that the
activity thereof is heat labile and sensitive to proteolysis.
[0009] The present invention provides an easily administered
functional protein.
[0010] The present invention also provides a method for delivering
insecticidal toxins that are functionally active and effective
against many orders of insects.
[0011] Objects, advantages, and features of the present invention
will become apparent from the following specification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a phenogram of Xenorhabdus strains as defined by
rep-PCR. The upper axis of FIG. 1 measures the percentage
similarity of strains based on scoring of rep-PCR products (i.e.,
0.0 [no similarity] to 1.0 [100% similarity]). At the right axis,
the numbers and letters indicate the various strains tested.
Vertical lines separating horizontal lines indicate the degree of
relatedness (as read from the extrapolated intersection of the
vertical line with the upper axis) between strains or groups of
strains at the base of the horizontal lines (e.g., strain DEX1 is
about 83% similar to strain X. nem).
DETAILED DESCRIPTION OF THE INVENTION
[0013] The present inventions are directed to discovery of a unique
class of functional protein toxins as defined herein produced by
bacteria of the genus Xenorhabdus, said toxins having oral toxicity
against insects. Xenorhabdus species/strains may be isolated from a
variety of sources. One such source is entomopathogenic nematodes,
more particularly nematodes of the genus Steinernema or from insect
cadavers infested by these nematodes. It is possible that other
sources could harbor Xenorhabdus bacteria that produce insecticidal
toxins having functional activity. Such sources in the environment
could be either terrestrial or aquatic based.
[0014] The genus Xenorhabdus is taxonomically defined as a member
of the Family Enterobacteriaceae, although it has certain traits
atypical of this family. For example, strains of this genus are
typically nitrate reduction negative, and catalase negative.
Xenorhabdus has only recently been subdivided to create a second
genus; Photorhabdus which is comprised of the single species
Photorhabdus luminescens (previously Xenorhabdus luminescens)
(Boemare et al., 1993 Int. J. Syst. Bacteriol. 43, 249-255). This
differentiation is based on several distinguishing characteristics
easily identifiable by the skilled artisan. These differences
include the following: DNA-DNA characterization studies; phenotypic
presence (Photorhabdus) or absence (Xenorhabdus) of catalase
activity; presence (Photorhabdus) or absence (Xenorhabdus) of
bioluminescence; the Family of the nematode host in that
Xenorhabdus is found in Steinernematidae and Photorhabdus is found
in Heterorhabditidae); as well as comparative, cellular fatty-acid
analyses (Janse et al. 1990, Lett. Appl. Microbiol 10, 131-135;
Suzuki et al. 1990, J. Gen. Appl. Microbiol., 36, 393-401). In
addition, recent molecular studies focused on sequence (Rainey et
al. 1995, Int. J. Syst. Bacteriol., 45, 379-381) and restriction
analysis (Brunel et al., 1997, App. Environ. Micro., 63, 574-580)
of 16S rRNA genes also support the separation of these two genera.
This change in nomenclature is reflected in this specification, but
in no way should a future change in nomenclature alter the scope of
the inventions described herein.
[0015] In order to establish that the strains disclosed herein were
comprised of Xenorhabdus strains, the strains were characterized
based on recognized traits which define Xenorhabdus species/strains
and differentiate them from other Enterobacteriaceae and
Photorhabdus species/strains. (Farmer, 1984 Bergey's Manual of
Systemic Bacteriology Vol. 1, pp. 510-511; Akhurst and Boemare
1988, J. Gen. Microbiol. 134, pp. 1835-1845; Boemare et al. 1993
int. J. Syst. Bacteriol. 43, pp. 249-255, which are incorporated
herein by reference). The expected traits for Xenorhabdus are the
following: Gram stain negative rods, organism size of
0.3-2.times.2-10 .mu.m, white to yellow/brown colony pigmentation,
presence of inclusion bodies, absence of catalase, inability to
reduce nitrate, absence of bioluminescence, ability to uptake dye
from medium, positive gelatin hydrolysis, growth on
Enterobacteriaceae selective media, growth temperature below
37.degree. C., survival under anaerobic conditions, and
motility.
[0016] Currently, the bacterial genus Xenorhabdus is comprised of
four recognized species, Xenorhabdus nematophilus, Xenorhabdus
poinarii, Xenorhabdus bovienii and Xenorhabdus beddingii (Brunel et
al., 1997, App. Environ. Micro., 63, 574-580). A variety of related
strains have been described in the literature (e.g., Akhurst and
Boemare 1988 J. Gen. Microbiol., 134, 1835-1845; Boemare et al.
1993 Int. J. Syst. Bacteriol. 43, pp. 249-255; Putz et al. 1990,
Appl. Environ. Microbiol., 56, 181-186, Brunel et al., 1997, App.
Environ. Micro., 63, 574-580, Rainey et al. 1995, Int. J. Syst.
Bacteriol., 45, 379-381). Numerous Xenorhabdus strains have i| been
characterized herein. Such strains and the characteristics thereof
are listed in Table 3 in the Examples. These strains have been
deposited with the Agricultural Research Service Patent Culture
Collection (NRRL) at 1815 North University Street Peoria, Ill.
61604 U.S.A. As can be seen in FIG. 1, these strains are diverse.
It is not unforeseen that in the future there may be other
Xenorhabdus species that will have some or all of the attributes of
the described species as well as some different characteristics
that are presently not defined as a trait(s) of Xenorhabdus.
However, the scope of the invention herein is to any Xenorhabdus
species or strains which produce proteins as described herein that
have functional activity as orally active insect control agents,
regardless of other traits and characteristics. Further included
within the inventions are the strains specified herein and any
mutants or phase variants thereof.
[0017] There are several terms that are used herein that have a
particular meaning and are as follows:
[0018] By "functional activity" it is meant herein that the protein
toxins function as orally active insect control agents, that the
proteins have a toxic effect, or are able to disrupt or deter
insect feeding which may or may not cause death of the insect. When
an insect comes into contact with an effective amount of toxin
derived from Xenorhabdus delivered via transgenic plant expression,
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.
[0019] By "native size" is meant the undenatured size of the
protein toxin or protein toxin subunit produced by the Xenorhabdus
strain of interest prior to any treatment or modification. Native
sizes of proteins can be determined by a variety of methods
available to the skilled artisan including but not limited to gel
filtration chromatography, agarose and polyacrylamide gel
electrophoresis, mass spectroscopy, sedimentation coefficients and
the like. Treatment or modifications to alter protein native size
can be performed by proteolysis, mutagenesis, gene truncation,
protein unfolding and other such techniques available to the
artisan skilled in the art of protein biochemistry and molecular
biology.
[0020] The protein toxins discussed herein are 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.
[0021] The term "toxic" or "toxicity" as used herein is meant to
convey that the toxins produced by Xenorhabdus have "functional
activity" as defined herein.
[0022] The term "Xenorhabdus toxin" is meant to include any protein
produced by a Xenorhabdus microorganism strain having functional
activity against insects, where the Xenorhabdus toxin could be
formulated as a sprayable composition, expressed by a transgenic
plant, formulated as a bait matrix, delivered via a baculovirus, a
plant RNA viral based system, or delivered by any other applicable
host or delivery system. It is also meant to include any sequence
of amino acids, polypeptides peptide fragment or other protein
preparation, whether derived in whole or in part from natural or
synthetic sources which demonstrates the ability to exhibit
functional activity as disclosed herein. Typically, a Xenorhabdus
toxin will be derived in whole or in part from a Xenorhabdus
bacterial source.
[0023] The term "Xenorhabdus toxin" is also meant to include
modified amino acid sequences, such as sequences which have been
mutated, truncated, increased and the like, as well as such
sequences which are partially or wholly artificially synthesized.
Xenorhabdus toxins and nucleic acid sequences encoding said toxins
may be obtained by partial or homogenous purification of bacterial
extracts, N-terminal or internal amino acid sequence information,
protein modeling, nucleic acid probes, antibody preparations, or
sequence comparison. Once a purified or partially purified
Xenorhabdus toxin is obtained, it may be used to obtain other
Xenorhabdus toxins by immunoprecipitation involving the formation
of an antigen:antibody immunocomplex thereby allowing recovery of
the new toxin which reacts thereto. Once the nucleic acid sequence
encoding a Xenorhabdus toxin is obtained, it may be employed in
probes for further screening or used in genetic engineering
constructs for transcription or transcription and translation in
host cells.
[0024] Fermentation broths from selected strains reported in Table
3 were used to examine the following: breadth of insecticidal
toxins having functional activity produced by the Xenorhabdus
genus, the functional spectrum of these toxins, and the protein
components of said toxins. 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, Lepidoptera, Diptera, and Acarina.
[0025] As with other bacterial toxins, the mutation rate of
bacteria in a population may result in the variation of the
sequence of toxin genes. Toxins of interest here are those which
produce proteins having functional activity against a variety of
insects upon exposure, as described herein. Preferably, the toxins
are active against Lepidoptera, Coleoptera, Diptera, 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 other protein toxins
produced by Xenorhabdus that have functional activity. These
homologous proteins may differ in sequence, but do not differ in
functional activity from those toxins described herein. Homologous
toxins are meant to include protein complexes of between 100 kDa to
3500 kDa and are comprised of at least one subunit, where a subunit
is a peptide which may or may not be the same as the other
subunit.
[0026] The toxins described herein are quite unique in that the
toxins have functional activity, which is key to developing an
insect management strategy. In 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. In such cases, 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 functional
toxin has potential application only in the laboratory.
[0027] The discovery that the functional protein toxins herein
exhibit their activity after oral ingestion or contact with the
toxins permits the development of an insect management plan based
solely on the ability to incorporate the protein toxins into the
insect diet. Such a plan could result in the production of insect
baits.
[0028] The Xenorhabdus toxins may be administered to insects in
both a purified and non-purified form. The toxins may also be
delivered in amounts from about 1 to about 1000 mg/liter of broth.
This may vary upon formulation condition, conditions of the
inoculum source, techniques for isolation of the toxin, and the
like. The toxins found herein can be administered as a sprayable
insecticide. Fermentation broth from Xenorhabdus can be produce,
diluted, or if needed, be concentrated about 100 to 1000-fold using
ultrafiltration or other techniques available to the skilled
artisan. Treatments can be applied with a syringe sprayer, a track
sprayer or any such equipment available to the skilled artisan
wherein the broth is applied to the plants. After treatments,
broths can be tested by applying the insect of choice to said
sprayed plant and can the be scored for damage to the leaves. If
necessary, adjuvants and photo-protectants can be added to increase
toxin-environmental half-life. In a laboratory setting, broth,
dilutions, or concentrates thereof can be applied using methods
available to the skilled artisan. Afterwards, the material can be
allowed to dry and insects to be tested are applied directly to the
appropriate plant tissue. After one week, plants can be scored for
damage using a modified Guthrie Scale (Koziel, M. G., Beland, G.
L., Bowman, C., Carozzi, N. B., Crenshaw, R., Crossland, L.,
Dawson, J., Desai, N., Hill, M., Kadwell, S., Launis, K., Lewis,
K., Maddox, D., McPherson, K., Meghji, M. Z., Merlin, E., Rhodes,
R., Warren, G. W., Wright, M. and Evola, S. V. 1993). In this
manner, broth or other protein containing fractions may confer
protection against specific insect pests when delivered in a
sprayable formulation or when the gene or derivative thereof,
encoding the protein or part thereof, is delivered via a transgenic
plant or microbe.
[0029] The toxins may be administered as a 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. Alternatively, the toxins may be
produced in bacteria or transgenic plants in the field or in the
insect by a baculovirus vector. Typically, insects will be exposed
to toxins by incorporating one or more of said toxins into the
food/diet of the insect.
[0030] Complete lethality to feeding insects is preferred, but is
not required to achieve functional activity. If an insect avoids
the toxin or ceases feeding, that avoidance will be useful in some
applications, even if the effects are sublethal or lethality is
delayed or indirect. For example, if insect resistant transgenic
plants are desired, the reluctance of insects to feed on the plants
is as useful as lethal toxicity to the insects since the ultimate
objective is protection of insect-induced plant damage rather than
insect death.
[0031] There are many other ways in which toxins can be
incorporated into an insect's diet. For example, it is possible to
adulterate the larval food source with the toxic protein by
spraying the food with a protein solution, as disclosed herein.
Alternatively, the purified protein could be genetically engineered
into an otherwise harmless bacterium, which could then be grown in
culture, and either applied to the food source or allowed to reside
in the soil in an area in which insect eradication was desirable.
Also, the protein could be genetically engineered directly into an
insect food source. For instance, the major food source for many
insect larvae is plant material. Therefore the genes encoding
Xenorhabdus toxins can be transferred to plant material so that
said plant material expresses the toxin of interest.
[0032] Transfer of the functional activity to plant or bacterial
systems requires nucleic acid sequences encoding the amino acid
sequences for the Xenorhabdus toxins integrated into a protein
expression vector appropriate to the host in which the vector will
reside. One way to obtain a nucleic acid sequence encoding a
protein with functional activity is to isolate the native genetic
material from the bacterial species or Xenorhabdus species which
produce the toxins, using information deduced from the toxin's
amino acid sequence, large portions of which are disclosed
herein.
[0033] There are also many different fermentation conditions that
can affect the amount or types of toxins produced by Xenorhabdus.
Several different factors can be varied by the skilled artisan to
optimize toxin production for increased or altered toxin activity.
Such factors include but are not limited to aeration of media,
temperature, media constituents such as phosphate, carbon sources,
minerals, vitamins, sugars, nitrogen sources, pH and the like.
Additional factors also include harvest time and the phase variant
of the bacteria used.
[0034] Once broth containing toxin has been produce, there are many
purification technique and chromatographic media available to the
person skilled in the art of protein biochemistry to allow
purification of Xenorhabdus toxins. After each and every step,
fractions can be assayed to find those particular fractions having
the functional activity of interest as described herein. For
example, protein toxins can be enriched in the broth by
centrifugation, membrane separation, and the like to form a highly
enriched, concentrated solution of toxin being predominantly
comprised of proteins having a native size greater than or equal to
100 kDa. The proteins can then fractionated by ion exchange
chromatography where upon they are separated based on overall ionic
charge. Again, fractions obtained therefrom can be assayed against
a variety of insects as described herein to find those fractions
having the protein toxins of interest. Said proteins can then be
separated based on native size using gel filtration-size exclusion
chromatography and the like. Typically, said fractions having
functional activity appear to elute from gel filtration columns in
a manner suggesting that the native toxin complex is about 500 kDa
to about 3,250 kDa, preferably about 750 kDa to about 3000 kDa,
with those in the range of about 800 kDa to about 1100 kDa being
most preferred. Fractions containing the toxins of interest can
then be further purified by using quantitative ion exchange,
quantitative gel filtration, hydrophobic chromatography,
isoelectric focusing and the like to again isolate highly enriched
and purified toxin fractions. The manner and order of protein
purification as described herein is exemplary only, thus other
techniques and approaches used by the skilled artisan to enrich and
isolate Xenorhabdus toxins are within the scope of this
invention.
[0035] When applied to SDS-PAGE analysis, fractions containing high
levels of Xenorhabdus toxin activity are shown to contain various
protein subunits as taught in the Examples herein. Typically, the
protein subunits are between about 20 kDa to about 350 kDa; between
about 130 kDa to about 300 kDa; between about 200 kDa to about 220
kDa; about 40 kDa to about 80 kDa; and about 20 kDa to about 40
kDa.
[0036] Given the few bands provided in the SDS-PAGE, immediate
efforts to obtain the corresponding amino acid and/or nucleic acid
sequences thereto are possible in accordance with methods familiar
to those skilled in the art. From such sequences, Xenorhabdus
toxins may be further confirmed with expression in controlled
systems, such as E. coli and the like. In addition, said sequences
allow the production of antibodies recognizing said toxins which
can then be used to : identify related Xenorhabdus toxin in other
bacterial systems using methods available to the skilled
artisan.
[0037] Amino acid sequences of fragments corresponding to partially
or fully purified protein preparations may be obtainable through
digestion with a protease, such as trypsin, and sequencing of
resulting peptide fragments. Amino acid are disclosed herein. Said
sequences can be used to design oligonucleotides using the genetic
code through reverse translation. DNA sequences can then be chosen
for use in Polymerase Chain Reactions (PCR) using genomic DNA
isolated from Xenorhabdus bacterial cells. The resulting
PCR-generated sequences can then be used as labeled probes in
screening genomic libraries. In this manner, the full length clones
corresponding to the Xenorhabdus toxin proteins seen on the
SDS-PAGE may be recovered if desired. Other Xenorhabdus toxin genes
may be obtained by screening genomic libraries from other
Xenorhabdus species and other bacteria in the family
Enterobacteriaceae.
[0038] The complete genomic sequence of a Xenorhabdus toxin may be
obtained by the screening of a genomic or cosmid library with a
probe. Probes can be considerably shorter than the entire gene
sequence, but should be at least about 10, preferably at least 15,
more preferably at least 20 or so nucleotides in length. Longer
oligonucleotides are also useful, up to the full length of the gene
encoding the polypeptide of interest. Both DNA and RNA can be used
as probes. In use, probes are typically labeled with .sup.32P,
biotinylated, and the like in a manner that allows for detection
thereof. Said probes are often incubated with single stranded DNA
from the source of which the gene is desired. Hybridization, or the
act of the probe binding to the DNA, is detected usually after
hybridization using nitrocellulose paper or nylon membranes by
means of the label on said probe. Hybridization techniques are well
known to the person skilled in the art of molecular biology. Thus
Xenorhabdus toxin genes may be isolated.
[0039] Other Xenorhabdus toxin genes or nucleic acid sequences are
obtainable from amino acid sequences provided herein. "Obtainable"
refers to those Xenorhabdus toxins and genes thereof which have
sufficiently similar sequences or "homology" to that of the native
sequences of this invention to provide a orally active functional
toxin. One skilled in the art will readily recognize that antibody
preparations, nucleic acid probes (DNA and RNA) and the like may be
prepared using the amino acid sequences disclosed herein and used
to screen and recover other Xenorhabdus toxin nucleic acid
sequences from other sources. Thus, sequences homologously related
to or derivations of Xenorhabdus toxins disclosed herein are
considered obtainable from the present invention.
[0040] "Homologously related" includes those nucleic acid and amino
acid sequences which are identical or conservatively substituted as
compared to the native sequence. Typically, a homologously related
nucleic acid sequence will show at least about 60% homology, and
more preferably at least about 70% homology to the probes created
from using the amino acid sequences disclosed herein and those
nucleic acid sequences obtained therefrom using those methods and
techniques as disclosed herein. Homology is determined upon
comparison of sequence information, e.g., nucleic acid or amino
acid or through hybridization reactions. Homology is also intended
to include conservative amino acid substitutions, which are will
known in the art. Conservative amino acid substitutions include
glutamic acid/aspartic acid; valine/isoleucine/leucine;
serine/threonine; arginine/lysine; glutamine/asparagine; or any
such substitution that results in no significant change in
functional activity of said toxin when compared to the native
toxin. Significant change as used herein is defined as at least a
50% change in activity based on molar amounts compared to said
native toxin.
[0041] It is within the scope of the invention as disclosed herein
that toxins may be truncated and still retain functional activity.
By "truncated toxin" is meant that a portion of a toxin protein may
be cleaved and yet still exhibit activity after cleavage. Cleavage
can be achieved by proteases inside or outside of the insect gut.
Furthermore, effectively cleaved proteins can be produced using
molecular biology techniques wherein the DNA bases encoding said
toxin are removed either through digestion with restriction
endonucleases or other techniques available to the skilled artisan.
After truncation, said proteins can be expressed in heterologous
systems such as E. coli, baculoviruses, plant-based viral systems,
yeast and the like and then placed in insect assays as disclosed
herein to determine activity. Truncated toxins have been
successfully produced with several insecticidal protein toxins in
that several proteins have been shown in the art to retain
functional activity while having less than the entire, full length
protein present. Said truncated proteins having insecticidal
activity include insect juvenile hormone esterase (U.S. Pat. No.
5,674,485 to the Regents of the University of California; and the
insecticidal toxin isolated from the bacterium Bacillus
thuringiensis (Adang et al., Gene 36:289-300 (1985) "Characterized
full-length and truncated plasmid clones of the crystal protein of
Bacillus thuringiensis subsp kurstaki HD-73 and their toxicity to
Manduca sexta)". As used herein, the term "Xenorhabdus toxin" is
also meant to include truncated versions thereof having functional
activity.
[0042] Recombinant constructs containing a nucleic acid sequence
encoding a Xenorhabdus toxin and heterologous nucleic acid
sequences of interest may be prepared. By heterologous is meant any
sequence which is not naturally found joined to the synthase
sequence. Hence, by definition, a sequence joined to sequence not
naturally found in a Xenorhabdus toxin is considered to be
heterologous.
[0043] Constructs may be designed to produce Xenorhabdus toxins in
either prokaryotic or eukaryotic cells. The expression of a
Xenorhabdus toxin in a plant cell is of special interest. Moreover,
the nucleic acid sequence encoding a Xenorhabdus toxin may be
integrated into a plant host genome. By transcribing and
translating a nucleic acid sequence encoding a Xenorhabdus toxin in
a plant host, said plant is expected to exhibit properties whereby
insects are discouraged from feeding. As stated herein, it is not
necessary for an functional agent to exhibit insect mortality to be
effective at controlling insects.
[0044] To obtain high expression of heterologous genes in plants it
may be preferred to reengineer said genes so that they are more
efficiently expressed in the cytoplasm of plant cells. Maize is one
such plant where it may be preferred to reengineer the heterologous
gene(s) prior to transformation to increase the expression level
thereof in said plant. Therefore, an additional step in the design
of genes encoding a Xenorhabdus toxin is the designed reengineering
of a heterologous gene for optimal expression.
[0045] One reason for the reengineering a Xenorhabdus toxin for
expression in maize is due to the non-optimal G+C content of the
native gene. For example, the very low G+C content of many native
bacterial gene(s) (and consequent skewing towards high A+T content)
results in the generation of sequences mimicking or duplicating
plant gene control sequences that are known to be highly A+T rich.
The presence of some A+T-rich sequences within the DNA of gene(s)
introduced into plants (e.g., TATA box regions normally found in
gene promoters) may result in aberrant transcription of the
gene(s). On the other hand, the presence of other regulatory
sequences residing in the transcribed mRNA (e.g., polyadenylation
signal sequences (AAUAAA), or sequences complementary to small
nuclear RNAs involved in pre-mRNA splicing) may lead to RNA
instability. Therefore, one goal in the design of genes encoding a
Xenorhabdus toxin for maize expression, more preferably referred to
as plant optimized gene(s), is to generate a DNA sequence having a
higher G+C content, and preferably one close to that of maize genes
coding for metabolic enzymes. Another goal in the design of the
plant optimized gene(s) encoding a Xenorhabdus toxin is to generate
a DNA sequence in which the sequence modifications do not hinder
translation.
[0046] The table below (Table 1) illustrates how high the G+C
content is in maize. For the data in Table 1, coding regions of the
genes were extracted from GenBank (Release 71) entries, and base
compositions were calculated using the MacVector.TM. program (IBI,
New Haven, Conn.). Intron sequences were ignored in the
calculations.
[0047] Due to the plasticity afforded by the redundancy of the
genetic code (i.e., some amino acids are specified by more than one
codon), evolution of the genomes in different organisms or classes
of organisms has resulted in differential usage of redundant
codons. This "codon bias" is reflected in the mean base composition
of protein coding regions. For example, organisms with relatively
low G+C contents utilize codons having A or T in the third position
of redundant codons, G or C in the third position. It is thought
that the presence of "minor" codons within a mRNA may reduce the
absolute translation rate of that mRNA, especially when the
relative abundance of the charged tRNA corresponding to the minor
codon is low. An extension of this is that the diminution of
translation rate by individual minor codons would be at least
additive for multiple minor codons. Therefore, mRNAs having high
relative contents of minor codons would have correspondingly low
translation rates. This rate would be reflected by subsequent low
levels of the encoded protein.
[0048] In reengineering genes encoding a Xenorhabdus toxin for
maize expression, the codon bias of the plant has been determined.
The codon bias for maize is the statistical codon distribution that
the plant uses for coding its proteins and the preferred codon
usage is shown in Table 2. After determining the bias, the percent
frequency of the codons in the gene(s) of interest is determined.
The primary codons preferred by the plant should be determined as
well as the second and third choice of preferred codons.
Afterwards, the amino acid sequence of the Xenorhabdus toxin of
interest is reverse translated so that the resulting nucleic acid
sequence codes for exactly the same protein as the native gene
wanting
1TABLE 1 Compilation of G + C contents of protein coding regions of
maize genes. Protein Class.sup.a Range % G + C Mean % G + C.sup.b
Metabolic Enzymes (76) 44.4-75.3 59.0 (.+-.8.0) Structural Proteins
(18) 48.6-70.5 63.6 (.+-.6.7) Regulatory Proteins (5) 57.2-68.9
62.0 (.+-.4.9) Uncharacterized Proteins (9) 41.5-70.3 64.3
(.+-.7.2) All Proteins (108) 44.4-75.3 60.8 (.+-.5.2) .sup.aNumber
of genes in class given in parentheses. .sup.bStandard deviations
given in parentheses. .sup.cCombined groups mean ignored in mean
calculation.
[0049] to be heterologously expressed. The new DNA sequence is
designed using codon bias information so that it corresponds to the
most preferred codons of the desired plant. The new sequence is
then analyzed for restriction enzyme sites that might have been
created by the modification. The identified sites are further
modified by replacing the codons with second or third choice with
preferred codons. Other sites in the sequence which could affect
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. In
addition to the doublets, G or C sequence blocks that have more
than about four residues that are the same can affect transcription
of the sequence. Therefore, these blocks are also modified by
replacing the codons of first or second choice, etc. with the next
preferred codon of choice.
[0050] It is preferred that the plant optimized gene(s) encoding a
Xenorhabdus toxin contain about 63% of first choice codons, between
about 22% to about 37% second choice codons, and between about 15%
to about 0% third choice codons, wherein the total percentage is
100%. Most preferred the plant optimized gene(s) contains about 63%
of first choice codons, at least about 22% second choice codons,
about 7.5% third choice codons, and about 7.5% fourth choice
codons, wherein the total percentage is 100%. The preferred codon
usage for engineering genes for maize expression are shown in Table
2. The method described above enables one skilled in the art to
modify gene(s) that are foreign to a particular plant so that the
genes are optimally expressed in plants. The method is further
illustrated in pending PCT application WO 97/13402, which is
incorporated herein by reference.
[0051] In order to design plant optimized genes encoding a
Xenorhabdus toxin, the amino acid sequence of said protein is
reverse translated into a DNA sequence utilizing a non-redundant
genetic code established from a codon bias table compiled for the
gene sequences for the particular plant, as shown in Table 2. The
resulting DNA sequence, which is completely homogeneous in codon
usage, is further modified to establish a DNA sequence that,
besides having a higher degree of codon diversity, also contains
strategically placed restriction enzyme recognition sites,
desirable base composition, and a lack of sequences that might
interfere with transcription of the gene, or translation of the
product mRNA.
[0052] In another aspect of the invention, genes encoding the
Xenorhabdus toxin are expressed from transcriptional units inserted
into the plant genome. Preferably, said transcriptional units are
recombinant vectors capable of stable integration into the plant
genome and selection of transformed plant lines expressing mRNA
encoding for said desaturase proteins are expressed either by
constitutive or inducible promoters in the plant cell. Once
expressed, the mRNA is translated into proteins, thereby
incorporating amino acids of interest into protein. The genes
encoding a Xenorhabdus toxin expressed in the plant cells can be
under the control of a constitutive promoter, a tissue-specific
promoter or an inducible promoter as described herein.
[0053] Several techniques exist for introducing foreign recombinant
vectors into plant cells, and for obtaining plants that stably
maintain and express the introduced gene. Such techniques include
acceleration of genetic material coated onto microparticles
directly into cells (U.S. Pat. No. 4,945,050 to Cornell and U.S.
Pat. No. 5,141,131 to DowElanco, now Dow AgroSciences, LLC) In
addition, plants may be transformed using Agrobacterium
2TABLE 2 Preferred amino acid codons for proteins expressed in
maize. Amino Acid Codon* Alanine GCC/GCG Cysteine TGC/TGT Aspartic
Acid GAC/GAT Glutamic Acid GAG/GAA Phenylalanine TTC/TTT Glycine
GGC/GGG Histidine CAC/CAT Isoleucine ATC/ATT Lysine AAG/AAA Leucine
CTG/CTC Methionine ATG Asparagine AAC/AAT Proline CCG/CCA Glutamine
CAG/CAA Arginine AGG/CGC Serine AGC/TCC Threonine ACC/ACG Valine
GTG/GTC Tryptophan TGG Tyrosine TAC/TAT Stop TGA/TAG *The first and
second preferred codons for maize.
[0054] technology, see U.S. Pat. No. 5,177,010 to University of
Toledo, U.S. Pat. No. 5 5,104,310 to Texas A&M, European Patent
Application 0131624B1, European Patent Applications 120516,
159418B1 and 176,112 to Schilperoot, U.S. Pat. Nos. 5,149,645,
5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot,
European Patent Applications 116718, 290799, 320500 all to Max
Planck, European Patent Applications 604662,627752 and U.S. Pat.
No.5,591,616 to Japan Tobacco, European Patent Applications
0267159, and 0292435 and U.S. Pat. No. 5,231,019 all to Ciba Geigy,
now Novartis, U.S. Pat. Nos. 5,463,174 and 4,762,785 both to
Calgene, and U.S. Pat. Nos. 5,004,863 and 5,159,135 both to
Agracetus. Other transformation technology includes whiskers
technology, see U.S. Pat. Nos. 5,302,523 and 5,464,765 both to
Zeneca. Electroporation technology has also been used to transform
plants, see WO 87/06614 to Boyce Thompson Institute, U.S. Pat Nos.
5,472,869 and 5,384,253 both to Dekalb, WO9209696 and WO9321335
both to Plant Genetic Systems. Furthermore, viral vectors can also
be used in produce transgenic plants expressing the protein of
interest. For example, monocotyledonous plant can be transformed
with a viral vector using the methods described in U.S. Pat No.
5,569,597 to Mycogen and Ciba-Giegy, now Novartis, as well as U.S.
Pat Nos. 5,589,367 and 5,316,931, both to Biosource. All of these
transformation patents and publications are incorporated herein by
reference.
[0055] As mentioned previously, the manner in which the DNA
construct is introduced into the plant host is not critical to this
invention. Any method which provides for efficient transformation
may be employed. For example, various methods for plant cell
transformation are described herein and include the use of Ti or
Ri-plasmids and the like to perform Agrobacterium mediated
transformation. In many instances, it will be desirable to have the
construct used for transformation bordered on one or both sides by
T-DNA borders, more specifically the right border. This is
particularly useful when the construct uses Agrobacterium
tumefaciens or Agrobacterium rhizogenes as a mode for
transformation, although T-DNA borders may find use with other
modes of transformation. Where Agrobacterium is used for plant cell
transformation, a vector may be used which may be introduced into
the host for homologous recombination with T-DNA or the Ti or Ri
plasmid present in the host. Introduction of the vector may be
performed via electroporation, tri-parental mating and other
techniques for transforming gram-negative bacteria which are known
to those skilled in the art. The manner of vector transformation
into the Agrobacterium host is not critical to w this invention.
The Ti or Ri plasmid containing the T-DNA for recombination may be
capable or incapable of causing gall formation, and is not critical
to said invention so long as the vir genes are present in said
host.
[0056] In some cases where Agrobacterium is used for
transformation, the expression construct being within the T-DNA
borders will be inserted into a broad spectrum vector such as pRK2
or derivatives thereof as described in Ditta et al., (PNAS USA
(1980) 77:7347-7351 and EPO 0 120 515, which are incorporated
herein by reference. Included within the expression construct and
the T-DNA will be one or more markers as described herein which
allow for selection of transformed Agrobacterium and transformed
plant cells. The particular marker employed is not essential to
this invention, with the preferred marker depending on the host and
construction used.
[0057] For transformation of plant cells using Agrobacterium,
explants may be combined and incubated with the transformed
Agrobacterium for sufficient time to allow transformation thereof.
After transformation, the agrobacteria are killed by selection with
the appropriate antibiotic and plant cells are cultured with the
appropriate selective medium. Once calli are formed, shoot
formation can be encourage by employing the appropriate plant
hormones according to methods well known in the art of plant tissue
culturing and plant regeneration. However, a callus intermediate
stage is not always necessary. After shoot formation, said plant
cells can be transferred to medium which encourages root formation
thereby completing plant regeneration. The plants may then be grown
to seed and said seed can be used to establish future generations.
Regardless of transformation technique, the gene encoding a
Xenorhabdus toxin is preferably incorporated into a gene transfer
vector adapted to express said gene in a plant cell by including in
the vector a plant promoter regulatory element, as well as 3'
non-translated transcriptional termination regions such as Nos and
the like.
[0058] In addition to numerous technologies for transforming
plants, the type of tissue which is contacted with the foreign
genes may vary as well. Such tissue would include but would not be
limited to embryogenic tissue, callus tissue types I, II, and III,
hypocotyl, meristem, root tissue and the like. Almost all plant
tissues may be transformed during dedifferentiation using
appropriate techniques described herein.
[0059] Another variable is the choice of a selectable marker.
Preference for a particular marker is at the discretion of the
artisan, but any of the following selectable markers may be used
along with any other gene not listed herein which could function as
a selectable marker. Such selectable markers include but are not
limited to aminoglycoside phosphotransferase gene of transposon Tn5
(Aph II) which encodes resistance to the antibiotics kanamycin,
neomycin and G418, as well as those genes which encode for
resistance or tolerance to glyphosate; hygromycin; methotrexate;
phosphinothricin (bialophos); imidazolinones, sulfonylureas and
triazolopyrimidine herbicides, such as chlorsulfuron; bromoxynil,
dalapon and the like.
[0060] In addition to a selectable marker, it may be desirous to
use a reporter gene. In some instances a reporter gene may be used
with or without a selectable marker. Reporter genes are genes which
are typically not present in the recipient organism or tissue and
typically encode for proteins resulting in some phenotypic change
or enzymatic property. Examples of such genes are provided in K.
Wising et al. Ann. Rev. Genetics, 22, 421 (1988), which is
incorporated herein by reference. Preferred reporter genes include
the beta-glucuronidase (GUS) of the uidA locus of E. coli, the
chloramphenicol acetyl transferase gene from Tn9 of E. coli, the
green fluorescent protein from the bioluminescent jellyfish
Aequorea victoria, and the luciferase genes from firefly Photinus
pyralis. An assay for detecting reporter gene expression may then
be performed at a suitable time after said gene has been introduced
into recipient cells. A preferred such assay entails the use of the
gene encoding beta-glucuronidase (GUS) of the uidA locus of E. coli
as described by Jefferson et al., (1987 Biochem. Soc. Trans. 15,
17-19) to identify transformed cells.
[0061] In addition to plant promoter regulatory elements, promoter
regulatory elements from a variety of sources can be used
efficiently in plant cells to express foreign genes. For example,
promoter regulatory elements of bacterial origin, such as the
octopine synthase promoter, the nopaline synthase promoter, the
mannopine synthase promoter; promoters of viral origin, such as the
cauliflower mosaic virus (35S and 19S), 35T (which is a
re-engineered 35S promoter, see PCT/US96/1682; WO 97/13402
published Apr. 17, 1997) and the like may be used. Plant promoter
regulatory elements include but are not limited to
ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu),
beta-conglycinin promoter, beta-phaseolin promoter, ADH promoter,
heat-shock promoters and tissue specific promoters. Other elements
such as matrix attachment regions, scaffold attachment regions,
introns, enhancers, polyadenylation sequences and the like may be
present and thus may improve the transcription efficiency or DNA
integration. Such elements may or may not be necessary for DNA
function, although they can provide better expression or
functioning of the DNA by affecting transcription, mRNA stability,
and the like. Such elements may be included in the DNA as desired
to obtain optimal performance of the transformed DNA in the plant.
Typical elements include but are not limited to Adh-intron 1,
Adh-intron 6, the alfalfa mosaic virus coat protein leader
sequence, the maize streak virus coat protein leader sequence, as
well as others available to a skilled artisan. Constitutive
promoter regulatory elements may also be used thereby directing
continuous gene expression in all cells types and at all times
(e.g., actin, ubiquitin, CaMV 35S, and the like). Tissue specific
promoter regulatory elements are responsible for gene expression in
specific cell or tissue types, such as the leaves or seeds (e.g.,
zein, oleosin, napin, ACP, globulin and the like) and these may
also be used.
[0062] Promoter regulatory elements may also be active during a
certain stage of the plants' development as well as active in plant
tissues and organs. Examples of such include but are not limited to
pollen-specific, embryo specific, corn silk specific, cotton fiber
specific, root specific, seed endosperm specific promoter
regulatory elements and the like. Under certain circumstances it
may be desirable to use an inducible promoter regulatory element,
which is responsible for expression of genes in response to a
specific signal, such as: physical stimulus (heat shock genes);
light (RUBP carboxylase); hormone (Em); metabolites; chemical; and
stress. Other desirable transcription and translation elements that
function in plants may be used. Numerous plant-specific gene
transfer vectors are known in the art.
[0063] One consideration associated with commercial exploitation of
transgenic plants is resistance management. This is of particular
concern with Bacillus thuringiensis toxins. There are numerous
companies commercially exploiting Bacillus thuringiensis and there
has been much concern about development of resistance to Bt toxins.
One strategy for insect resistance management would be to combine
the toxins produced by Xenorhabdus with toxins such as Bt,
vegetative insecticidal proteins from Bacillus stains (Ciba Geigy;
WO 94/21795) or other insect toxins. The combinations could be
formulated for a sprayable application or could be molecular
combinations. Plants could be transformed with Xenorhabdus genes
that produce insect toxins and other insect toxin genes such as
Bt.
[0064] European Patent Application 040024GAl describes
transformation of a plant with 2 Bts. This could be any 2 genes,
not just Bt genes. Another way to produce a transgenic plant that
contains more than one insect resistant gene would be to produce
two plants, with each plant containing an insect resistance gene.
These plants could then be backcrossed using traditional plant
breeding techniques to produce a plant containing more than one
insect resistance gene.
[0065] In addition to producing a transformed plant, there are
other delivery systems where it may be desirable to re-engineer the
bacterial gene(s). Along the same lines, a genetically engineered,
easily isolated protein toxin made by fusing together both a
molecule attractive to insects as a food source and the functional
activity of the toxin may be engineered and expressed in bacteria
or in 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.
[0066] 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 Xenorhabdus toxins. Infectious baculovirus harboring an
expression construct for the Xenorhabdus toxins could be introduced
into areas of insect infestation to thereby intoxicate or poison
infected insects.
[0067] Insect viruses, or baculoviruses, are known to infect and
adversely affect certain insects. The affect of the viruses on
insects is slow, and viruses do not immediately stop the feeding of
insects. Thus, viruses are not viewed as being optimal as insect
pest control agents. However, combining the Xenorhabdus toxin genes
into a baculovirus vector could provide an efficient way of
transmitting the toxins. In addition, since different baculoviruses
are specific to different insects, it may be possible to use a
particular toxin to selectively target particularly damaging insect
pests. A particularly useful vector for the toxins genes is the
nuclear polyhedrosis virus. Transfer vectors using this virus have
been described and are now the vectors of choice for transferring
foreign genes into insects. The virus-toxin gene recombinant may be
constructed in an orally transmissible form. Baculoviruses normally
infect insect victims through the mid-gut intestinal mucosa. The
toxin gene inserted behind a strong viral coat protein promoter
would be expressed and should rapidly kill the infected insect.
[0068] In addition to an insect virus or baculovirus or transgenic
plant delivery system for the protein toxins of the present
invention, the proteins may be encapsulated using Bacillus
thuringiensis encapsulation technology such as but not limited to
U.S. 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.
[0069] Plant RNA viral based systems can also be used to express
Xenorhabdus toxin. In so doing, the gene encoding a Xenorhabdus
toxin can be inserted into the coat promoter region of a suitable
plant virus which will infect the host plant of interest. The
Xenorhabdus toxin can then be expressed thus providing protection
of the plant from insect damage. Plant RNA viral based systems are
described in U.S. Pat No. 5,500,360 to Mycgoen Plant Sciences, Inc.
and U.S. Pat Nos. 5,316,931 and 5,589,367 to Biosource Genetics
Corp. which are incorporated herein by reference.
[0070] Standard and molecular biology techniques may be used to
clone and sequence the toxins described herein. Additional
information may be found in Sambrook, J., Fritsch, E. F., and
Maniatis, T. (1989), Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Press, which is incorporated herein by reference.
[0071] The following abbreviations are used throughout the
Examples: Tris=tris (hydroxymethyl) amino methane; SDS=sodium
dodecyl sulfate; EDTA=ethylenediaminetetraacetic acid,
IPTG=isopropylthio-B-galactoside,
X-gal=5-bromo-4-chloro-3-indoyl-B-D-galactoside,
CTAB=cetyltrimethylammon- ium bromide; kbp=kilobase pairs; DATP,
dCTP, dGTP, dTTP, I=2'-deoxynucleoside 5'-triphosphates of adenine,
cytosine, guanine, thymine, and inosine, respectively;
ATP=adenosine 5' triphosphate.
[0072] The particular embodiments of this invention are further
exemplified in the Examples. However, those skilled in the art will
readily appreciate that the specific experiments detailed are only
illustrative of the invention as described more fully in the claims
which follow thereafter.
EXAMPLE 1
Characterization of Xenorhabdus Strains
[0073] In order to establish that the collection described herein
consisted of Xenorhabdus isolates, strains were assessed in terms
of recognized microbiological traits that are characteristic of
phase I variants of Xenorhabdus and which differentiate it from
other Enterobacteriaceae and Photorhabdus spp. [Farmer, J. J. 1984.
Bergey's Manual of Systemic Bacteriology, vol 1. pp. 510-511. (ed.
Kreig N. R. and Holt, J. G.). Williams & Wilkins, Baltimore.;
Akhurst and Boemare, 1988, J. Gen. Microbiol. 134, 1835-1845; Forst
and Nealson, 1996. Microbiol. Rev. 60, 21-43]. These characteristic
traits were as follows: Gram stain negative rods; organism size of
0.3-2 .mu.m in width and 2-10 .mu.m in length with occasional
filaments (15-50 .mu.m) and spheroplasts; white to yellow/brown
colony pigmentation on nutrient agar; presence of crystalline
inclusion bodies; absence of catalase; negative for oxidase;
inability to reduce nitrate; absence of bioluminescence; ability to
take up dye from growth media; positive for protease production;
growth-temperature below 37.degree. C.; survival under anaerobic
conditions and positively motile (Table 3). Methods were checked
using reference Escherichia coli, Xenorhabdus and Photorhabdus
strains as controls. Overall results shown in Table 3 were
consistent with all strains being members of the family
Enterobacteriaceae and the genus Xenorhabdus.
[0074] A luminometer was used to establish the absence of
bioluminescence associated with Xenorhabdus strains. To measure the
presence or absence of relative light emitting units, broth from
each strain (cells and media) was measured at up to three time
intervals after inoculation in liquid culture (24, 48 and/or 72 h)
and compared to background luminosity (uninoculated media). Several
Photorhabdus strains were also tested as positive controls for
luminosity. Prior to measuring light emission from selected broths,
cell density was established by measuring A.sub.560 nm in a Gilford
Systems (Oberlin, Ohio) spectrophotometer using a sipper cell. The
resulting light emitting units were then normalized to cell
density. Aliquots of broths were placed into 96-well microtiter
plates (100 .mu.L each) and read in a Packard Lumicount luminometer
(Packard Instrument Co., Meriden Conn.). The integration period for
each sample was 0.1 to 1.0 sec. The samples were agitated in the
luminometer for 10 sec prior to taking readings. A positive test
was determined as being .gtoreq.3-fold background luminescence
(.about.1-5 relative light units). In addition, absence of colony
luminosity with some strains was confirmed with photographic film
overlays and visual analysis after visual adaptation in a
darkroom.
[0075] The Gram staining characteristics of each strain were
established with a commercial Gram-stain kit (BBL, Cockeysville,
Md.) in conjunction with Gram stain control slides (Fisher
Scientific, Pittsburgh, Pa.). Microscopic evaluation was then
performed using a Zeiss microscope (Carl Zeiss, Germany) 100.times.
oil immersion objective lens (with 10.times. ocular and 2.times.
body magnification). Microscopic examination of individual strains
for organism size, cellular description and inclusion bodies (the
latter two observations after logarithmic growth) was performed
using wet mount slides (10.times. ocular, 2.times. body and
40.times. objective magnification) and phase contrast microscopy
with a micrometer (Akhurst, R. J. and Boemare, N. E. 1990.
Entomopathogenic Nematodes in Biological Control (ed. Gaugler, R.
and Kaya, H.). pp. 75-90. CRC Press, Boca Raton, USA.; Baghdiguian
S., Boyer-Giglio M. H., Thaler, J. O., Bonnot G., Boemare N. 1993.
Biol. Cell 79, 177-185). Colony pigmentation was observed after
inoculation on Bacto nutrient agar, (Difco Laboratories, Detroit,
Mich.) prepared per label instructions. Incubation occurred at
28.degree. C. and descriptions were recorded after 5-7 days.
[0076] To test for the presence of catalase activity, 1 mL of
culture broth or a colony of the test organism on a small plug of
nutrient agar was placed into 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
(presumably oxygen) appeared immediately or within 5 sec. Negative
controls of uninoculated nutrient agar or culture broth and
hydrogen peroxide solution were also examined.
[0077] Theoxidase reaction of each strain was determined by rubbing
24 h colonies onto DrySlide Oxidase slides (Difco, Inc.; Detroit,
Mich.). Oxidase positive strains produce a dark purple color,
indicative of cytochrome oxidase C, within 20 sec after the
organism was rubbed against the slide. Failure to produce a dark
purple color indicated that the organism was oxidase negative.
[0078] To test for nitrate reduction, each culture was inoculated
into 10 mL of Bacto Nitrate Broth (Difco Laboratories, Detroit,
Mich.). After 24 h incubation at 28.degree. C., nitrite production
was tested by the addition of two drops of sulfanilic acid reagent
and two drops of alpha-naphthylamine reagent (Difco Manual, 10th
edition, Difco Laboratories, Detroit, Mich., 1984). The generation
of a distinct pink or red color indicated the formation of nitrite
from nitrate whereas the lack of color formation indicated that the
strain was nitrate reduction negative. In the latter case, finely
powdered zinc was added to further confirm the presence of
unreduced nitrate established by the formation of nitrite and the
resultant red color.
[0079] The ability of each strain to uptake dye from growth media
was tested with Bacto MacConkey agar containing the dye neutral
red; Bacto Tergitol-7 agar containing the dye bromothymol blue and
Bacto EMB Agar containing the dyes methylene blue and eosin-Y
(formulated agars from Difco Laboratories, Detroit, Mich., all
prepared according to label instructions). After inoculation on
these media, dye uptake was recorded upon incubation at 28.degree.
C. for 5 days. Growth on Bacto MacConkey and Bacto Tergitol-7 media
is characteristic for members of the family Enterobacteriaceae.
Motility of each strain was tested using a solution of Bacto
Motility Test Medium (Difco Laboratories, Detroit, Mich.) prepared
per label instructions. A butt-stab inoculation was performed with
each strain and positive motility was judged after incubation at
28.degree. C. by macroscopic observation of a diffuse zone of
growth spreading from the line of inoculation.
[0080] The production of protease was tested by observing
hydrolysis of gelatin using Bacto gelatin (Difco Laboratories,
Detroit, Mich.) plates made per label instructions. Cultures were
inoculated and the plates were incubated at 22.degree. C. for 3-5
days prior to assessment of gelatin hydrolysis. To assess growth at
different temperatures, agar plates [2% proteose peptone #3 with
two percent Bacto-Agar (Difco, Detroit, Mich.) in deionized water]
were streaked from a common source of inoculum. Plates were
incubated at 20, 28 and 37.degree. C. for 5 days. The incubator
temperatures were checked with an electronic thermocouple and
metered to insure valid temperature settings.
[0081] Oxygen requirements for Xenorhabdus strains were tested in
the following manner. A butt-stab inoculation into fluid
thioglycolate broth medium (Difco, Detroit, Mich.) 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
resazurin was used to indicate the presence of medium oxygenation
or the aerobiosis zone (Difco Manual, 10th edition, Difco
Laboratories, Detroit, Mich.). 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.
[0082] The diversity of Xenorhabdus strains was measured by
analysis of PCR (Polymerase Chain Reaction) mediated genomic
fingerprinting using genomic DNA from each strain. This technique
is based on families of repetitive DNA sequences present throughout
the genome of diverse bacterial species (reviewed by Versalovic,
J., Schneider, M., D E Bruijn, F. J. and Lupski, J. R. 1994.
Methods Mol. Cell. Biol., 5, 25-40). Three of these, repetitive
extragenic palindromic sequence (REP), enterobacterial repetitive
intergenic consensus (ERIC) and the BOX element, are thought to
play an important role in the organization of the bacterial genome.
Genomic organization is believed to be shaped by selection and the
differential dispersion of these elements within the genome of
closely related bacterial strains can be used to discriminate
between strains (e.g. Louws, F. J., Fulbright, D. W., Stephens, C.
T. and D E 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.
[0083] To isolate genomic DNA from strains, cell pellets were
resuspended in TE buffer (10 or 50 mM Tris-HCl, 1 or 50 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 min at 15,000 .times.g. The
resulting pellet was resuspended in 5.7 mL of TE and 300 .mu.L of
10% SDS and 60 .mu.L 20 mg/ml proteinase K (Gibco BRL Products,
Grand Island, N.Y.) were added. This mixture was incubated at
37.degree. C. for 1 h, about 10 mg of lysozyme was added, and the
mixture was then incubated for an additional 30 to 45 min. One mL
of SM NaCl and 800 .mu.L of CTAB/NaCl solution (10% w/v CTAB, 0.7 M
NaCl) were then added and the mixture was incubated 10 to 20 min at
65.degree. C., and in some cases, gently agitated, then incubated
and agitated for an additional 20 min to aid in clearing of the
cellular material. An equal volume of chloroform/isoamyl alcohol
solution ( 24:1, v/v) was added, mixed gently then centrifuged. Two
extractions were performed with an equal volume of
phenol/chloroform/isoamyl alcohol (PCI; 50:49:1). Genomic DNA was
precipitated with 0.6 volume of isopropanol. Precipitated DNA was
removed with a sterile plastic loop or glass rod, washed twice with
70% ethanol, dried
3TABLE 3 Taxonomic Traits of Xenorhabdus Strains Strain A* B C D E
F G H I J.sup..sctn. K L M N O P Q R S. carp -.sup.' + - rd S + - -
+ + W + + + + + + - - X. Wi - + - rd S + - - + + W + + + + + + - -
X. nem - + - rd S + - - + + W + + + + + + - - X. NH3 - + - rd S + -
- + + W + + + + + + - - X. riobravis - + - rd S + - - + + W + + + +
+ + - - DEX1 - + - rd S + - - + + W + + + + + + - - DEX6 - + - rd S
+ - - + + W + + + + + + - - ILM037 - + - rd S + - - + + C + + + + +
+ - - ILM039 - + - rd S + - - + + W + + + + + + - - ILM070 - + - rd
S + - - + + W + + + + + + - - ILM078 - + - rd S + - - + + W + + + +
+ + - - ILM079 - + - rd S + - - + + C + + + + + + - - ILM080 - + -
rd S + - - + + W + + + + + + - - ILM081 - + - rd S + - - + + W + +
+ + + + - - ILM082 - + - rd S + - - + + W + + + + + + - - ILM083 -
+ - rd S + - - + + W + + + + + + - - ILM084 - + - rd S + - - + + W
+ + + + + + - - ILM102 - + - rd S + - - + + C + + + + + + - -
ILM103 - + - rd S + - - + + C + + + + + + - - ILM104 - + - rd S + -
- + + C + + + + + + - - ILM129 - + - rd S + - - + + Y + + + + + + -
- ILM133 - + - rd S + - - + + Y + + + + + + - - ILM135 - + - rd S +
- - + + Y + + + + + + - - ILM138 - + - rd S + - - + + Y + + + + + +
- - ILM142 - + - rd S + - - + + Y + + + + + + - - ILM143 - + - rd S
+ - - + + Y + + + + + + - - GLX26 - + - rd S + - - + + C + + + + +
+ - - GLX40 - + - rd S + - - + + C + + + + + + - - GLX166 - + - rd
S + - - + + C + + + + + + - - SEX20 - + - rd S + - - + + C + + + +
+ + - - SEX76 - + - rd S + - - + + C + + + + + + - - SEX180 - + -
rd S + - - + + C + + + + + + - - GL133B - + - rd S + - - + + Y + +
+ + + + - - DEX2 - + - rd S + - - + + W + + + + + + - - DEX3 - + -
rd S + - - + + Y + + + + + + - - DEX4 - + - rd S + - - + + W + + +
+ + + - - DEX5 - + - rd S + - - + + W + + + + + + - - DEX7 - + - rd
S + - - + + W + + + + + + ND - DEX8 - + - rd S + - - + + W + + + +
+ + ND - *A = Gram's stain, B = Crystalline inclusion bodies, C =
Bioluminescence, D = Cell form, E = Motility, F = Nitrate
reduction, G = Presence of catalase, H = Gelatin hydrolysis, I =
Dye uptake, J = Pigmentation on Nutrient Agar, K = Growth on EMB
agar, L = Growth on MacConkey agar, M = Growth on Tergitol-7 agar,
N = Facultative anaerobe, O = Growth at 20.degree. C., P = Growth
at 28.degree. C., Q = Growth at 37.degree. C., R = oxidase.
.dagger.+= positive for trait, - = negative for trait; rd = rod, S
= sized within Genus descriptors, ND = not determined .sctn.W =
white, C = cream, Y = yellow.
[0084] and dissolved in 2 mL of STE (10 mM Tris-HCl pH8.0, 10 mM
NaCl, 1 mM EDTA). The DNA was then quantitated at A.sub.260 nm. In
a second method, 0.01 volumes of RNAase A (50 .mu.g/mL final) was
added and incubated at 37.degree. C. for 2 h. The sample was then
extracted with an equal volume of PCI. The samples were then
precipitated with 2 volumes of 100% ethanol and collected as
described above. Samples were then air dried and resuspended in
250-1000 .mu.L of TE.
[0085] To perform rep-PCR analysis of Xenorhabdus genomic DNA, the
following primers were used: REP1R-I; 5'-IIIICGICGICATCIGGC-3' and
REP2-I; 5'-ICGICTTATCIGGCCTAC-3'. PCR was performed using the
following 25 .mu.L reaction: 7.75 .mu.L H.sub.2O, 2.5 .mu.L
10.times. LA buffer (PanVera Corp., Madison, Wis.), 16 .mu.L DNTP
mix (2.5 mM each), 1 .mu.L of each primer at 50 pM/.mu.L, 1 .mu.L
DMSO, 1.5 .mu.L genomic DNA (concentrations ranged from 0.075-0.480
.mu.g/.mu.L) and 0.25 .mu.L TaKaRa EX Taq (PanVera Corp., Madison,
Wis.). The PCR amplification was performed in a Perkin Elmer DNA
Thermal Cycler (Norwalk, Conn.) using the following conditions:
95.degree. C. for 7 min then [94.degree. C. for 1 min, 44.degree.
C. for 1 min, 65.degree. C. for 8 min] for 35 cycles; followed by
65.degree. C. for 15 min. After cycling, 25 .mu.L of reaction was
added to 5 .mu.L of 6.times. gel loading buffer (0.25% bromophenol
blue, 40% w/v sucrose in H.sub.2O). A 15.times.20 cm 1%-agarose gel
was then run in TBE buffer (0.09 M Tris-borate, 0.002 M EDTA) using
8 .mu.L of each reaction. The gel was run for approximately 16 h at
45 V. Gels were then stained in 20 .mu.g/mL ethidium bromide for 1
h and destained in TBE buffer for approximately 3 h. Polaroid.RTM.
photographs of the gels were then taken under UV illumination.
[0086] The presence or absence of bands at specific sizes for each
strain was scored from the photographs using RFLP scan Plus
software (Scanalytics, Billerica, Mass.) and entered as a
similarity matrix in the numerical taxonomy software program,
NTSYS-pc (Exeter Software, Setauket, N.Y.). Controls of E. coli
strain HE101 and Xanthomonas oryzae pv. oryzae assayed under the
same conditions produced PCR fingerprints corresponding to
published reports (Versalovic, J., Koeuth, T. and Lupski, J. R.
1991. Nucleic Acids Res. 19, 6823-6831; Vera Cruz, C. M.,
Halda-Alija, L., Louws, F., Skinner, D. Z., George, M. L., Nelson,
R. J., D E Bruijn, F. J., Rice, C. and Leach, J. E. 1995. Int. Rice
Res. Notes, 20, 23-24.; Vera Cruz, C. M., Ardales, E. Y., Skinner,
D. Z., Talag, J., Nelson, R. J., Louws, F. J., Leung, H., Mew, T.
W. and Leach, J. E. 1996. Phytopathology 86, 1352-1359). The data
from Xenorhabdus strains were then analyzed with a series of
programs within NTSYS-pc; SIMQUAL (Similarity for Qualitative data)
to generate a matrix of similarity coefficients (using the Jaccard
coefficient) and SAHN (Sequential, Agglomerative, Heirarchical and
Nested) clustering using the UPGMA method (Unweighted Pair-Group
Method with Arithmetic Averages) which groups related strains and
can be expressed as a phenogram (FIG. 1). 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. A resulting normalized Mantel statistic (r) was generated
which was a measure of the goodness of fit for a cluster analysis
(r=0.8-0.9 representing a very good fit). In our case r=0.9,
indicated an excellent fit. Therefore, strains disclosed herein
were determined to be comprised of a diverse group of easily
distinguishable strains representative of the Xenorhabdus
genus.
[0087] Strains disclosed herein were deposited before application
filing with the following International Deposit Authority:
Agricultural Research Service Patent Culture Collection (NRRL),
National Center for Agricultural Utilization Research, ARS-USDA,
1815 North University St., Peoria, Ill. 61604. The following
strains , with NRRL designations were deposited Apr. 29, 1997: S.
Carp (NRRL-B-21732); X. Wi (NRRL-B-21733); X. nem (NRRL-B-21734);
X. NH3 (NRRL-B-21735); X. riobravis (NRRL-B-21736); GL 133B
(NRRL-B-21737); DEX1 (NRRL-B-21738); DEX2 (NRRL-B-21739); DEX3
(NRRL-B-21740); DEX4 (NRRL-B-21741); DEX 5 (NRRL-B-21742); and DEX
6 (NRRL-B-21743). The remaining strains disclosed herein were
deposited with NRRL on Apr. 30, 1998. In all, thirty-nine (39)
strains were deposited.
EXAMPLE 2
Functional Utility of Toxin(S) Produced by Various Xenorhabdus
Strains
[0088] "Storage" cultures of the various Xenorhabdus strains were
produced by inoculating 175 mL of 2% Proteose Peptone #3 (PP3)
(Difco Laboratories, Detroit, Mich.) liquid medium with a phase I
variant colony in a 500 mnL tribaffled flask with a Delong neck
covered with a Kaput closure. After inoculation, flasks were
incubated for between 24-72 h at 28.degree. C. on a rotary shaker
at 150 rpm. Cultures were then transferred to a sterile bottle
containing a sterile magnetic stir bar and then over-layered with
sterile mineral oil to limit exposure to air. Storage cultures were
kept in the dark at room temperature. These cultures were then used
as inoculum sources for the fermentation of each strain. Phase I
variant colonies were also stored frozen at -70.degree. C. for use
as an inoculum source. Single, phase I colonies were selected from
PP3 plates containing bromothymol blue (0.0025%) and placed in 3.0
mL PP3 and grown overnight on a rotary shaker (150 rpm) at
28.degree. C. Glycerol (diluted in PP3) was then added to achieve a
final concentration of 20% and the cultures were frozen in aliquots
at -70.degree. C. For culture inoculation, a portion of the frozen
aliquot was removed aseptically and streaked on PP3 containing
bromothymol blue for reselection of phase I colonies.
[0089] Pre-production "seed" flasks or cultures were produced by
either inoculating 2 mL of an oil over-layered storage culture or
by transferring a phase I variant colony into 175 mLt sterile
medium in a 500 mL tribaffled flask covered with a Kaput closure.
Typically, following 16 h incubation at 28.degree. C. on a rotary
shaker at 150 rpm, seed cultures were transferred into production
flasks. Production flasks were usually inoculated by adding
.about.1% of the actively growing seed culture to sterile PP3 or
tryptic soy broth (TSB, Difco Laboratories, Detroit Mich.). For
small-scale productions, flasks were inoculated directly with a
phase I variant colony. Production of broths occurred in 500 mL
tribaffled flasks covered with a Kaput closure. Production flasks
were agitated at 28.degree. C. on a rotary shaker at 150 rpm.
Production fermentations were terminated after 24-72 h.
[0090] Following appropriate incubation, broths were dispensed into
sterile 1.0 L polyethylene bottles, spun at 2600 .times.g for 1 h
at 10.degree. C. and decanted from the cell and debris pellet.
Broths were then filter sterilized or further broth clarification
was achieved with a tangential flow microfiltration device (Pall
Filtron, Northborough, Mass.) using a 0.5 .mu.M open-channel
poly-ether sulfone (PES) membrane filter. The resulting broths were
then concentrated (up to 10-fold) using a 10,000 or 100,000 MW
cut-off membrane, M12 ultra-filtration device (Amicon, Beverly
Mass.) or centrifugal concentrators (Millipore, Bedford, Mass. and
Pall Filtron, Northborough, Mass.) with a 10,000 or 100,000 MW pore
size. In the case of centrifugal concentrators, broths were spun at
2000 .times.g for approximately 2 h. The membrane permeate was
added to the corresponding retentate to achieve the desired
concentration of components greater than the pore size used.
Following these procedures, broths were used for biochemical
analysis or biological assessment. Heat inactivation of processed
broth samples was achieved by heating 1 mL samples at 100.degree.
C. in a sand-filled heat block for 10-20 min.
[0091] Broth(s) and toxin complex(es) from different Xenorhabdus
strains were useful for reducing populations of insects and were
used in a method of inhibiting an insect population which comprised
applying to a locus of the insect an effective insect inactivating
amount of the active described. A demonstration of the breadth of
functional activity observed from broths of a selected group of
Xenorhabdus strains fermented as described above is shown in Table
4. It is possible that improved or additional functional activities
could be detected with these strains through increased
concentration of the broth or by employing different fermentation
methods as disclosed herein. Consistent with the activity being
associated with a protein, the functional activity showed heat
lability and/or was present in the high molecular weight retentate
(greater than 10 kDa and predominantly greater than 100 kDa) after
concentration of the broth.
[0092] Culture broth(s) from diverse Xenorhabdus strains showed
differential functional activity (mortality and/or growth
inhibition) against a number of insects. More specifically,
activity was 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. The broths and
purified toxin complex(es) were also active against tobacco
budworm, tobacco hornworm, corn earworm and European corn borer
which are members of the order Lepidoptera. Other typical members
of this order are beet armyworm, cabbage looper, black cutworm,
codling moth, clothes moth, Indian mealmoth, leaf rollers, cabbage
worm, bagworm, Eastern tent caterpillar, sod webworm and fall
armyworm. Activity was also seen against mosquito larvae which are
members of the order Diptera. Other members of the order Diptera
are, pea midge, carrot fly, cabbage root fly, turnip root fly,
onion fly, crane fly and house fly and various mosquito species.
Activity with broth(s) was also seen against two-spotted spider
mite which is a member of the order Acarina which includes
strawberry spider mites, broad mites, citrus red mite, European red
mite, pear rust mite and tomato russet mite.
[0093] Activity against corn rootworm larvae was tested as follows.
Xenorhabdus culture broth(s) (10.times. concentrated, filter
sterilized), PP3 or TSB (10.times. concentrated), purified toxin
complex(es) or 10 mM sodium phosphate buffer , pH 7.0, were applied
directly to the surface (about 1.5 cm.sup.2) of artificial diet
(Rose, R. I. and McCabe, J. M. 1973. J. Econ. Entomol. 66, 398-400)
in 40 .mu.L aliquots. Toxin complex was diluted in 10 mM sodium
phosphate buffer, pH 7.0. The diet plates were allowed to air-dry
in a sterile flow-hood and the wells were infested with single,
neonate Diabrotica undecimpunctata howardi (Southern corn rootworm,
SCR) hatched from surface sterilized eggs. Plates were sealed,
placed in a humidified growth chamber and maintained at 27.degree.
C. for the appropriate period (3-5 days). Mortality and larval
weight determinations were then scored. Generally, 8-16 insects per
treatment were used in all studies. Control mortality was generally
less than 5%.
[0094] Activity against boll weevil (Anthomonas grandis) was tested
as follows. Concentrated (10.times.) Xenorhabdus broths or control
medium (PP3) were applied in 60 .mu.L aliquots to the surface of
0.35 g of artificial diet (Stoneville Yellow lepidopteran diet) and
allowed to dry. A single, 12-24 h boll weevil larva was placed on
the diet, the wells were sealed and held at 25.degree. C., 50%
relative humidity (RH) for 5 days. Mortality and larval weights
were then assessed. Control mortality ranged between 0-25%.
[0095] Activity against mosquito larvae was tested as follows. The
assay was conducted in a 96-well microtiter plate. Each well
contained 200 .mu.L of aqueous solution (10.times.concentrated
Xenorhabdus culture broth(s), control medium (2% PP3) and about 20,
1-day old larvae (Aedes aegypti). There were 6 wells per treatment.
The results were read at 24 h after infestation. No control
mortality was observed.
[0096] Activity against lepidopteran larvae was tested as follows.
Concentrated (1.times.) Xenorhabdus culture broth(s), control
medium (PP3 or TSB), purified toxin complex(es) or 10 mM sodium
phosphate buffer, pH 7.0 were applied directly to the surface
(.about.1.5 cm.sup.2) of standard artificial lepidopteran diet
(Stoneville Yellow diet) in 40 .mu.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. European corn borer
(Ostrinia nubilalis), fall armyworm (Spodoptera frugiperda), corn
earworm (Helicoverpa zea) and tobacco hornworm (Manduca sexta) eggs
were obtained from commercial sources and hatched in-house whereas
tobacco budworm (Heliothis virescens) and beet armyworm (Spodoptera
exigua) larvae were supplied internally. Following infestation with
larvae, diet plates were sealed, placed in a humidified growth
chamber and maintained in the dark at 27.degree. C. for the
appropriate period. Mortality and weight determinations were scored
at day 5. Generally, 16 insects per treatment were used in all
studies. Control mortality generally ranged from 0-12.5%.
[0097] Activity against two-spotted spider mite (Tetranychus
urticae) was determined as follows. Young squash plants were
trimmed to a single cotyledon and sprayed to run-off with 10.times.
concentrated broth(s) or control medium (PP3). After drying, plants
were infested with a mixed population of spider mites and held at
room temperature and humidity for 72 hr. Live mites were then
counted to determine levels of control.
EXAMPLE 3
Functional Activity of Highly Purified Toxin Proteins from
Xenorhabdus Strain X. riobravis
[0098] Functional toxin protein was purified from fermentation
broth of Xenorhabdus strain X. riobravis as described herein. This
toxin was tested against neonate larvae of five insect species,
Southern corn rootworm, European cornborer, Tobacco hornworm, Corn
earworm and Tobacco budworm following the methods described in
Example 2. The results are seen in Table 5. All species showed
growth inhibitory and/or lethal effects after five days when
presented with toxin at a dose of 440 ng toxin/cm.sup.2 diet.
4TABLE 4 Observed Functional Spectrum of Broths From Different
Xenorhabdus Strains Xenorhabdus strain Sensitive* Insect Species S.
carp 1**, 2, 3, 4, 5, 6, 7 X. riobravis 1, 2, 3, 5, 6, 7 X. NH3 1,
2, 3, 6 X. Wi 1, 2, 3, 5, 6, 7 X. nem 3, 5, 6 DEX1 1, 2, 3, 6 DEX6
1, 2, 3, 4, 5, 6 ILM037 1, 4 ILM039 4 ILM070 4, 8 ILM078 3, 4
ILM079 3 ILM080 3 ILM081 3 ILM082 3 ILM083 3 ILM084 3 ILM102 1, 2,
4 ILM103 1, 3, 4, 8 ILM104 3, 4, 8 ILM116 1, 4 ILM129 1, 4 ILM133
1, 4 ILM135 1, 2, 4 ILM138 4 ILM142 1, 2, 3, 4, 8 ILM143 4 GLX26 8
GLX40 3, 8 GLX166 4 SEX20 1, 4, 8 SEX76 1, 4 SEX180 4 GL 133B 4
DEX2 6, 7 DEX3 3, 6 DEX4 6, 7 DEX5 3, 6 DEX7 3 DEX8 3 * =
.gtoreq.25% mortality and/or growth inhibition vs. control ** = 1;
Tobacco budworm, 2; European corn borer, 3; Tobacco hornworm, 4;
Southern corn rootworm, 5; Boll weevil, 6; Mosquito, 7; Two-spotted
spider mite, 8; Corn earworm
[0099]
5TABLE 5 Effect of Highly Purified X. riobravis Toxin on Various
Insect Species S. corn European Tobacco Corn Tobacco Treatment
rootworm cornborer hornworm earworm budworm X. 19/46* 75/61 75/75
25/95 13/98 riobravis *Value are the % mortality/% growth
inhibition corrected for control effects.
EXAMPLE 4
Effect of Different Culture Media on Functional Activity of
Fermentation Broths from Selected Xenorhabdus Strains
[0100] Several different culture media were used to further
optimize conditions for detection of functional activity in the
fermentation broths of several Xenorhabdus strains. GL133B, X.
riobravis, X. Wi, DEX8 and DEX1 were grown in PP3, TSB and PP3 plus
1.25% NaCl (PP3S) as described herein. Broths were then prepared as
described herein and assayed against neonate Tobacco hornworm to
determine any changes in insecticidal activity. In both
experimental cases (condition A which is PP3 vs. TSB; and condition
B which is PP3 vs. PP3S), the functional activity of fermentations
in PP3S and/or TSB were improved as compared to simultaneous PP3
fermentations (Table 6). In certain cases, activity was uncovered
which was not apparent with PP3 fermentations. The functional
activity produced under condition A and condition B was shown to be
heat labile and retained by high molecular weight membranes
(>100,000 kDa). Addition of NaCl to broth after bacterial growth
was complete did not increase toxin activity indicating that the
increased functional activity observed was not due to increase NaCl
concentration in the media but instead due to increased toxin.
[0101] The increased activity observed with X. riobravis fermented
in PP3S was further investigated by partial purification of
toxin(s) from fermentations in PP3 and PP3S as described herein.
Consistent with observations using culture broth, the active
fraction(s) from PP3S broth (obtained from anion exchange and
size-exclusion chromatography as described herein) contained
increased biological activity, protein concentration and a more
complex protein pattern as determined by SDS-PAGE analysis.
6TABLE 6 The Effect of Different Culture Media on Functional
Potency of Selected Xenorhabdus Fermentation Broths Condition A
Condition B Strains PP3 TSB PP3 PP3S GL133B -* - - + X. riobravis +
+++ + +++ X. Wi + +++ + +++ DEX8 - + - - DEX6 + ++ + +++ Control -
- - - *+ = 25-50% mortality, ++ = 51-75% mortality, +++ = >76%
mortality, - = <25% mortality
EXAMPLE 5
Xenorhabdus Strains X.nem, X. riobravis, and X. Wi: Purification,
Characterization and Activity
[0102] The protocol, as follows, was established based on purifying
those fractions having the most activity against EYE Tobacco
Hornworm (Manduca sexta) , hereinafter THW, as determined in
bioassays (see Example 2). Typically, 4-20 L of Xenorhabdus culture
that had been grown in PP3 broth being filtered, as described
herein, were received and concentrated using an Amicon spiral ultra
filtration cartridge Type SlYlGO attached to an Amicon M-12
filtration device (Amicon Inc., Beverly, Mass.). The retentate
contained native proteins wherein the majority consisted of those
having molecular sizes greater than 100 kDa, whereas the flow
through material contained native proteins less than 100 kDa in
size. The majority of the activity against THW was contained in the
100 kDa retentate. The retentate was then continually diafiltered
with 10 mM sodium phosphate (pH=7.0) until the filtrate reached an
A.sub.280 <0.100. Unless otherwise stated, all procedures from
this point were performed in buffer defined as 10 mM sodium
phosphate (pH 7.0). The retentate was then concentrated to a final
volume of about 0.20 L and then filtered using a 0.45 .mu.m sterile
filtration unit (Corning, Corning, N.Y.).
[0103] The filtered material was loaded at 7.5 mL/min 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 (PerSeptive
Biosystems, Framingham, Mass.). After loading, the column was
washed with buffer until an A.sub.280 nm <0.100 was achieved.
Proteins were then eluted from the column at 2.5 mL/min using
buffer with 0.4 M NaCl for 20 min for a total volume of 50 mL. The
column was then washed using buffer with 1.0 M NaCl at the same
flow rate for an additional 20 min (final volume=50 ml). Proteins
eluted with 0.4 M and 1.0 M NaCl were placed in separate dialysis
bags (SPECTRA/POR Membrane MWCO: 2,000; Spectrum, Houston, Tex.)
and allowed to dialyze overnight at 4.degree. C. in 12 L buffer. In
some cases, the 0.4 M fraction was not dialyzed but instead was
immediately desalted by gel filtration (see below). The majority of
activity against THW was contained in the 0.4 M fraction.
[0104] The 0.4 M fraction was further purified by application of 20
mL to a Pharmacia XK 26/100 column that had been prepacked with
Sepharose CL4B (Pharmacia) using a flow rate of 0.75 mL/min.
Fractionation of the 0.4 M fraction on the Sepharose CL4B column
yielded four to five distinct peaks when purifying X. nem and X.
Wi. Proteins from strain X. riobravis, while having a distinct peak
equivalent to the void volume, also had a very broad, low
absorbance region ranging from ca. 280 min to ca. 448 min of the
800 min run. Typically, two larger absorbance peaks were observed
after 450 min and before 800 min. Active fractions from X. Wi and
X. nem typically eluted at about 256 min to 416 min of a 800 min
run
[0105] Fractions were pooled based on A.sub.280 nm peak profile and
concentrated to a final volume of 0.75 ml using a Millipore
ULTRAFREE-15 centrifugal filter device Biomax-50K NMWL membrane
(Millipore Inc., Bedford, Mass.) or concentrated by binding to a
Pharmacia MonoQ HR10/10 column, as described herein. Protein
concentrations were determined using a BioRad Protein Assay Kit
(BioRad, Hercules, Calif.) with bovine gamma globulin as a
standard.
[0106] The native molecular weight of the THW toxin complex was
determined using a Pharmacia HR 16/50 column that had been
prepacked with Sepharose CL4B in said phosphate buffer. The column
was then calibrated using proteins of known molecular size thereby
allowing for calculation of the toxin complex approximate native
molecular size. As shown in Table 7, the molecular size of the
toxin complex were as follows: 1500.+-.530 kDa for strain X. nem;
1000.+-.350 kDa for strain X. riobravis; 3290 kDa+1150 kDa for
strain X. Wi; 980.+-.245 for strain ILM078; 1013.+-.185 for strain
DEX6; and 956.+-.307 for strain ILM080. A highly purified fraction
of X. Wi, said fraction being purified via ion exchange, gel
filtration, ion exchange, hydrophobic interaction chromatography,
and ion exchange chromatography as disclosed herein was then
analyzed for size using quantitative gel filtration. This material
was found to have a native molecular size of 1049.+-.402 kDa (Table
7).
[0107] Proteins found in the toxin complex were examined for
individual polypeptide size using SDS-PAGE analysis. Typically, 20
.mu.g protein of the toxin complex from each strain was loaded onto
a 2-15% polyacrylamide gel (Integrated Separation Systems, Natick,
Mass.) and electrophoresed at 20 mA in SDS-PAGE buffer (BioRad).
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). Gels were then
rinsed with water for 15 min and scanned using a Molecular Dynamics
PERSONAL LASER DENSITOMETER (Sunnyvale, Calif.). Lanes were
quantitated and molecular sizes were calculated as compared to
BioRad high molecular weight standards, which ranged from 200-45
kDa.
[0108] Sizes of individual polypeptides comprising the THW toxin
complex from each strain are listed in Table 8. The sizes of the
individual polypeptides ranged from 32 kDa to 330 kDa. Each of X.
Wi, X. nem, X. riobravis, ILM080, ILM078, and DEX6 strains had
polypeptides comprising the toxin complex that were in the 160-330
kDa range, the 100-160 kDa range, and the 50-80 kDa range. These
data indicate that the toxin 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 with subunits ranging from 23 kDa to 330
kDa.
EXAMPLE 5
Sub-fractionation of Xenorhabdus Toxin Complex from X. riobravis
and X. Wi
[0109] For subfractionation, about 10 mg of the Xenorhabdus protein
toxin complex of X. riobravis was isolated as described above and
was applied to a Pharmacia MonoQ HR 10/10 column equilibrated with
10 mM phosphate buffer, pH 7.0 at a flow rate of 2 mL/min. The
column was washed with said buffer until the absorbance at 280 nm
returned to baseline. Proteins bound to the column were eluted with
a linear gradient of 0 to 1.0 M NaCl in said buffer at 2 mL/min for
1 h. Two mL fractions were collected and subjected to analysis by
bioassay against THW as described herein. Peaks of activity were
determined by examining a 2-fold dilution of each fraction in THW
bioassays. A peak of activity against THW was observed that eluted
at about 0.3-0.4 M NaCl. The fractions having activity against THW
were pooled and analyzed by SDS-PAGE gel electrophoresis. It was
observed that there were four predominant peptides having the
approximate sizes of 220 kDa, 190 kDa, 130 kDa, and 54 kDa.
[0110] The peptides described above were electrophoresed on a 4-20%
SDS-PAGE (Integrated Separation Systems) and transblotted to
PROBLOTT PVDF membranes (Applied Biosystems, Foster City, Calif.) .
Blots were sent for amino acid analysis and N-terminal amino acid
sequencing at Harvard MicroChem and Cambridge ProChem,
respectively. The amino terminal sequence of the 220 kDa protein is
entered herein as SEQ ID NO:4.
[0111] For sub-fractionation experiments with X. Wi, ca. 10 mg
toxin was applied to a MonoQ HR 10/10 column equilibrated with 10
mM phosphate buffer, pH 7.0 at a flow rate of 2 mL/min. The column
was washed with said buffer until the A.sub.280 nm returned to
baseline. Proteins bound to the column were eluted with a linear
gradient of 0 to 1.0 M NaCl in said buffer at 2 mL/min for 1 h. Two
mL fractions were collected and subjected to analysis by bioassay
against THW as described herein. At least two major detectable
peaks at A.sub.280 nm were observed. The majority of functional THW
activity that was observed eluted at about 0.10-0.25 M NaCl. The
fractions having activity against THW were pooled and analyzed by
gel electrophoresis. By SDS-PAGE it was observed that there were up
to eight predominant peptides having the approximate sizes of 330
kDa, 320 kDa, 270 ka, 220 kDa, 200 kDa, 190 kDa, 170 kDa, 130 kla,
91 kDa, 76 kDa, 55 kDa and 36 kDa.
[0112] The peak THW pooled activity fraction was applied to
phenyl-sepharose HR 5/5 column. Solid (NH.sub.4).sub.2SO.sub.4
added to a final concentration of 1.7 M. The solution was then
applied onto the column equilibrated with 1.7 M
(NH.sub.4).sub.2SO.sub.4 in 50 mM potassium phosphate buffer, pH 7,
at 1 mL/min. Proteins bound to the column were then eluted with a
linear gradient of 1.7 M (NH.sub.4).sub.2SO.sub.4, 50 mM potassium
phosphate, pH 7.0 to 10 mM potassium phosphate, pH 7.0 at 0.5
mL/min for 60 min. After THW bioassays, it was determined that the
peak activity eluted at an A.sub.280 nm between 40 min to ca. 50
min. Fractions were dialyzed overnight against 10 mM sodium
phosphate buffer, pH 7.0. By SDS-PAGE it was observed that there
were up to six predominant peptides having the approximate sizes of
270 kDa, 220 kDa, 170 kDa, 130 kDa, and 76 kDa.
[0113] The peptides from THW active fractions from either 5/5 or
10/10 phenyl-sepharose column were electrophoresed on a 4-20%
SDS-PAGE gel (Integrated Separation Systems) and transblotted to
PROBLOTT PVDF membranes (Applied Biosystems, Foster City, Calif.).
Blots were sent for amino acid analysis and N-terminal amino acid
sequencing at Harvard MicroChem and Cambridge ProChem,
respectively. The N-terminal amino acid sequences for 130 kDa (SEQ
ID NO:1), 76 kDa (SEQ ID NO:2), 48 kDa (SEQ ID NO:5) and 38 kDa
(SEQ ID NO:3) peptides are entered herein.
[0114] Insect bioassays were performed using either toxin complex
or THW phenyl-sepharose purified fractions. Functional activity (at
least 20% mortality) and/or growth inhibition (at least 40%) was
observed for fall armyworm, beet armyworm, tobacco hornworm,
tobacco budworm, European corn borer, and southern corn rootworm.
In toxin complex preparations tested, higher activity was observed
against tobacco hornworm and tobacco budworm than against southern
corn rootworm larvae. The insect activity of X. Wi toxin complex
and any additionally purified fractions were shown to be heat
sensitive.
7TABLE 7 Characterization of a Toxin Complex From Xenorhabdus
Strains. STRAIN TOXIN COMPLEX SIZE.sup.a X. Wi 3290 kDa .+-. 1150
kDa X. Wi 1049 kDa .+-. 402 kDa (Highly Purified) X. nem 1010 kDa
.+-. 350 kDa X. riobravis 1520 kDa .+-. 530 kDa ILM 078 980 kDa
.+-. 245 kDa ILM 080 1013 kDa .+-. 185 kDa DEX6 956 kDa .+-. 307
kDa .sup.aNative molecular weight determined using a Pharmacia
HR16/50 column packed with Sepharose CL4B. Highly purified X. Wi
was from a fraction isolated from a Mono Q 5/5 column.
[0115]
8TABLE 8 Molecular Sizes of Peptides in Toxin Complex from
Xenorhabdus Strains in kDa. X. Wi X. nem X. riobravis ILM 080 ILM
078 DEX 6 330 220 220 200 203 201 320 190 190 197 200 181 270 170
100 173 173 148 220 150 96 112 150 138 200 140 92 106 144 128 190
85 85 90 106 119 170 79 79 80 80 90 130 65 65 74 62 75 91 56 56 61
58 65 76 50 50 60 54 59 55 42 47 58 50 55 49 38 42 55 45 45 46 31
38 53 41 43 29 34 48 37 40 26 31 46 32 36 26 43 32 23 42 40
EXAMPLE 6
Production, Isolation, and Characterization of Xenorhabdus Strain
X. carpocapsae
[0116] A 1% inoculum of an overnight culture of the isolate X.
carpocapsae, also known as X. carp, was added to a 125 mL flask
containing 25 mL PP3 and incubated for 72 h at 28.degree. C. on a
rotary shaker at 250 rpm. Afterwards, the cultures were centrifuged
for 20 min at 10,000 .times.g as described herein followed by
filtration of the supernatant using a 0.2 .mu.m membrane filter. A
15 mL sample of the supernatant was then added to an Ultrafree-15
100,000 NMWL centrifugal filter device (Millipore, Mass.) and
centrifuged at 2000 .times.g. The retentate was washed 2.times.
with 100 mM KPO.sub.4, pH 6.9, and then resuspended in 1.0 mL of
the same. Proteins were analyzed by SDS-PAGE as disclosed herein
using a 10% resolving gel and 4% stacking gel with sizes calibrated
using BioRad prestained standards (Hercules, Calif.). Gels were
electrophoresed at 40V for 16 h at 15.degree. C. and then stained
with Colloidal Blue from Novex, Inc., (San Diego, Calif.).
[0117] For additional separations, samples were applied to a
BIO-SEP S4000 column (Phenomenex, Torrance, Calif.), 7.5 mm I.D.,
60 cm CML under an isocratic system using 100 mM KPO.sub.4 pH 6.9.
Total amount loaded per sample was 250-500 .mu.g protein. Fractions
were collected in 3 groups depending on protein size (size
exclusion chromatography) as follows: proteins greater than 1,000
kDa; proteins being 800-1,000 kDa; and proteins less than 800,000
kDa. The 800,000.about.1,000,000 Da fraction was selected for
further analysis.
[0118] The 800-1000 kDa fractions, which had the most functional
activity, were pooled and concentrated using a 100,000 NMWL
centrifugal filter devices (Millipore, Bedford, Mass). Each pooled
retentate fraction was washed 2.times. and resuspended in 300 .mu.L
of 100 mM KPO.sub.4 pH 6.9. The protein concentrations were
determined using the bicinchoninic acid protein assay reagent kit
(Pierce, Rockford, Ill.). Proteins in this fraction were analyzed
by SDS-PAGE as described herein and found to have many proteins of
different sizes. This material was then further separated on a DEAE
column whereby proteins were eluted with increasing salt
concentrations. Those fractions having the most activity were then
examined again via SDS-PAGE and were found to be comprised of 4
predominate proteins having sizes as follows: 200, 190, 175 and 45
kDa. The active fraction from the DEAE step was passed through a
HPLC gel filtration column as described above (BioSep S4000) and
the toxic activity against Manduca sexta was found to be contained
within a fraction having native proteins >800 kDa. Resolution of
this fraction via SDS-PAGE revealed only one protein, said protein
having a denatured size of 200 kDa. These data suggest that the 200
kDa protein is responsible for the Manduca sexta functional
activity (see below) and is possibly found as a tetramer in the
culture broth.
[0119] Bioassays were performed as follows. Eggs of M. sexta were
purchased from Carolina Biological Supply Co. The eggs were hatched
and reared on fresh wheat germ diet (ICN, CA) while incubated at
25.degree. C. in a 16 h light/8 h dark photocycle incubator. Oral
toxicity data were determined by placing twelve M. sexta larva onto
a piece of insect food containing 300 .mu.g ultrafiltration
retentate obtained as described above. Observations were made over
5 days. For the HPLC-size exclusion chromatography fractions, 20
.mu.g total protein were applied to wheat germ diet. Experiment was
repeated in duplicate.
Sequence CWU 1
1
5 1 12 PRT Xenorhabdus Wi 1 Asn Gln Asn Val Glu Pro Ser Ala Gly Asp
Ile Val 1 5 10 2 8 PRT Xenorhabdus Wi 2 Ser Gln Asn Val Tyr Arg Tyr
Pro 1 5 3 7 PRT Xenorhabdus Wi 3 Met Thr Lys Gln Glu Tyr Leu 1 5 4
11 PRT Xenorhabdus Wi 4 Met Tyr Ser Thr Ala Val Leu Leu Asn Lys Ile
1 5 10 5 12 PRT Xenorhabdus Wi UNSURE (11) 5 Ala Gly Phe Gln Leu
Asn Glu Tyr Ser Thr Xaa Gly 1 5 10
* * * * *