Disclosed and claimed are novel nucleotide primers for the identification of genes encoding toxins active against nematodes and coleopterans. The primers are useful in PCR techniques to produce gene fragments which are characteristic of genes encoding these toxins. The primers are also useful as nucleotide probes to detect the toxins-encoding genes. The subject invention also concerns novel isolates, toxins, and genes useful in the control of plant pests.

BACKGROUND OF THE INVENTION 
The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, 
spore-forming bacterium characterized by parasporal crystalline protein 
inclusions. These inclusions often appear microscopically as distinctively 
shaped crystals. The proteins can be highly toxic to pests and specific in 
their toxic activity. Certain B.t. toxin genes have been isolated and 
sequenced, and recombinant DNA-based B.t. products have been produced and 
approved for use. In addition, with the use of genetic engineering 
techniques, new approaches for delivering these B.t. endotoxins to 
agricultural environments are under development, including the use of 
plants genetically engineered with endotoxin genes for insect resistance 
and the use of stabilized intact microbial cells as B.t. endotoxin 
delivery vehicles (Gaertner et al., 1988). Thus, isolated B.t. endotoxin 
genes are becoming commercially valuable. 
Until the last ten years, commercial use of B.t. pesticides has been 
largely restricted to a narrow range of lepidopteran (caterpillar) pests. 
Preparations of the spores and crystals of B. thuringiensis subsp. 
kurstaki have been used for many years as commercial insecticides for 
lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1 
produces a crystalline .delta.-endotoxin which is toxic to the larvae of a 
number of lepidopteran insects. 
In recent years, however, investigators have discovered B.t. pesticides 
with specificities for a much broader range of pests. For example, other 
species of B.t., namely israelensis and morrisoni (a.k.a. tenebrionis, 
a.k.a. B.t. M-7, a.k.a. B.t. san diego), have been used commercially to 
control insects of the orders Diptera and Coleoptera, respectively 
(Gaertner, 1989). See also Couch, 1980 and Beegle, 1978. Krieg et al., 
1983, describe Bacillus thuringiensis var. tenebrionis, which is 
reportedly active against two beetles in the order Coleoptera. These are 
the Colorado potato beetle, Leptinotarsa decemlineata, and Agelastica 
alni. 
Recently, new subspecies of B.t. have been identified, and genes 
responsible for active .delta.-endotoxin proteins have been isolated 
(Hofte and Whiteley, 1989). Hofte and Whiteley classified B.t. crystal 
protein genes into four major classes. The classes were CryI 
(Lepidoptera-specific), CryII (Lepidoptera- and Diptera-specific), CryIII 
(Coleoptera-specific), and CryIV (Diptera-specific). The discovery of 
strains specifically toxic to other pests has been reported. (Feitelson et 
al., 1992). CryV has been proposed to designate a class of toxin genes 
that are nematode-specific. 
The cloning and expression of a B.t. crystal protein gene in Escherichia 
coli has been described in the published literature (Schnepf and Whiteley, 
1981). U.S. Pat. No. 4,448,885 and U.S. Pat. No. 4,467,036 both disclose 
the expression of B.t. crystal protein in E. coli. U.S. Pat. Nos. 
4,990,332; 5,039,523; 5,126,133; 5,164,180; and 5,169,629 are among those 
which disclose B.t. toxins having activity against lepidopterans. U.S. 
Pat. Nos. 4,797,276 and 4,853,331 disclose B. thuringiensis strain 
tenebrionis which can be used to control coleopteran pests in various 
environments. U.S. Pat. No. 4,918,006 discloses B.t. toxins having 
activity against dipterans. U.S. Pat. No. 5,151,363 and U.S. Pat. No. 
4,948,734 disclose certain isolates of B.t. which have activity against 
nematodes. Other U.S. patents which disclose activity against nematodes 
include 5,093,120; 5,236,843; 5,262,399; 5,270,448; 5,281,530; 5,322,932; 
5,350,577; 5,426,049; and 5,439,881. As a result of extensive research and 
investment of resources, other patents have issued for new B.t. isolates 
and new uses of B.t. isolates. See Feitelson et al., 1992 for a review. 
However, the discovery of new B.t. isolates and new uses of known B.t. 
isolates remains an empirical, unpredictable art. 
Regular use of chemical control of unwanted organisms can select for 
chemical resistant strains. Chemical resistance occurs in many species of 
economically important insects and has also occurred in nematodes of 
sheep, goats, and horses. The development of chemical resistance 
necessitates a continuing search for new control agents having different 
modes of action. The subject invention pertains specifically to materials 
and methods for the identification of B.t. toxins active against nematodes 
or coleopteran pests. Of particular interest among the coleopteran pests 
is the corn rootworm. 
In recent times, the accepted methodology for control of nematodes has 
centered around the drug benzimidazole and its congeners. The use of these 
drugs on a wide scale has led to many instances of resistance among 
nematode populations (Prichard et al., 1980; Coles, 1986). There are more 
than 100,000 described species of nematodes. 
A small number of research articles have been published about the effects 
of delta endotoxins from B. thuringiensis species on the viability of 
nematode eggs. Bottjer, Bone and Gill, (1985) have reported that B.t. 
kurstaki and B.t. israelensis were toxic in vitro to eggs of the nematode 
Trichostrongylus colubriformis. In addition, 28 other B.t. strains were 
tested with widely variable toxicities. Ignoffo and Dropkin, 1977, have 
reported that the thermostable toxin from Bacillus thuringiensis (beta 
exotoxin) was active against a free-living nematode, Panagrellus redivivus 
(Goodey); a plant-parasitic nematode, Meloidogyne incognita (Chitwood); 
and a fungus-feeding nematode, Aphelenchus avena (Bastien). Beta exotoxin 
is a generalized cytotoxic agent with little or no specificity. Also, 
Ciordia and Bizzell (1961) gave a preliminary report on the effects of B. 
thuringiensis on some cattle nematodes. 
There are a number of beetles that cause economic damage. Corn rootworms 
include species found in the genus Diabrotica (e.g., D. undecimpunctata 
undecimpunctata, D. undecimpunctata howardii, D. longicornis, D. virgifera 
and D. balteata). Corn rootworms cause extensive damage to corn and 
curcubits. Approximately $250 million worth of insecticides are applied 
annually to control corn rootworms alone in the United States. Even with 
insecticide use, rootworms cause about $750 million worth of crop damage 
each year, making them the most serious corn insect pest in the Midwest. 
Current methods for controlling corn rootworm damage in corn are limited to 
the use of crop rotation and insecticide application. However, economic 
demands on the utilization of farmland restrict the use of crop rotation. 
In addition, an emerging two-year diapause (or overwintering) trait of 
Northern corn rootworms is disrupting crop rotations in some areas. 
The use of insecticides to control corn rootworm and other coleopteran 
pests also has several drawbacks. Continual use of insecticides has 
allowed resistant insects to evolve. Insecticide use often raises 
environmental concerns such as contamination of soil and of both surface 
and underground water supplies. Working with insecticides may also pose 
hazards to the persons applying them. 
At the present time there is a need to have more effective means to control 
the many nematodes and coleopterans that cause considerable damage to 
susceptible hosts and crops. Advantageously, such effective means would 
employ specific biological agents. 
Bacillus thuringiensis toxins which are active against nematodes and corn 
rootworm are now known. Isolating responsible toxin genes has been a slow 
empirical process. Carozzi et al., 1991 describe methods for identifying 
toxin genes. This report does not disclose or suggest the specific primers 
and probes of the subject invention for nematode-active and corn 
rootworm-active toxin genes. U.S. Pat. No. 5,204,237 describes specific 
and universal probes for the isolation of B.t. toxin genes. This patent, 
however, does not describe the probes and primers of the subject 
invention. 
BRIEF SUMMARY OF THE INVENTION 
The subject invention concerns materials and methods useful in the control 
of pests and, particularly, plant pests. Specifically, the subject 
invention provides new toxins useful for the control of nematodes. Certain 
isolates and toxins of the subject invention can also be used to control 
coleopteran pests, including corn rootworm. The subject invention further 
provides nucleotide sequences which encode these toxins. The subject 
invention further provides nucleotide sequences useful in the 
identification and characterization of genes which encode pesticidal 
toxins. The subject invention further provides new Bacillus thuringiensis 
isolates having pesticidal activities. 
In one embodiment, the subject invention concerns unique nucleotide 
sequences which are useful primers in PCR techniques. The primers produce 
gene fragments which are characteristic of genes encoding nematode-active 
toxins and, thus, can be used in the identification and isolation of 
specific toxin genes. 
In specific embodiments, the invention concerns the following nucleotide 
sequences which can be used to identify genes encoding toxins: 
1. A forward primer designated V3 whose nucleotide sequence is 
GATCGTMTWGARTTRTTCC (SEQ ID NO. 1); 
2. A forward primer designated V5 whose nucleotide sequence is 
AAAGTNGATGCMTTATCWGATGA (SEQ ID NO. 2); 
3. A forward primer designated V7 whose nucleotide sequence is 
ACACGTATAHDGTTTFCTGG (SEQ ID NO. 3); 
4. A reverse primer designated .DELTA.V5' whose nucleotide sequence is 
TCATCWGATAAKGCATCNAC (SEQ ID NO. 4); and 
5. A reverse primer designated .DELTA.V8' whose nucleotide sequence is 
TGGACGDTCTTCAMKAATTTCYAAA (SEQ ID NO. 5). 
In one embodiment of the subject invention, B.t. isolates can be cultivated 
under conditions resulting in high multiplication of the microbe. After 
treating the microbe to provide single-stranded genomic nucleic acid, the 
DNA can be contacted with the primers of the invention and subjected to 
PCR amplification. Characteristic fragments of toxin-encoding genes will 
be amplified by the procedure, thus identifying the presence of the 
toxin-encoding gene(s). In a particularly preferred embodiment, the primer 
pair V7-.DELTA.V8' is used to identify genes encoding nematicidal B.t. 
toxins. 
Another important aspect of the subject invention is the use of the 
disclosed nucleotide sequences as probes to detect genes encoding B.t. 
toxins which are active against nematodes. The probes may be RNA or DNA. 
The probe will normally have at least about 10 bases, more usually at 
least about 18 bases, and may have up to about 50 bases or more, usually 
not having more than about 200 bases if the probe is made synthetically. 
However, longer probes can readily be utilized, and such probes can be, 
for example, several kilobases in length. The probe sequence is designed 
to be at least substantially complementary to a gene encoding a toxin of 
interest. The probe need not have perfect complementary to the sequence to 
which it hybridizes. The probes may be labelled utilizing techniques which 
are well known to those skilled in this art. 
Further aspects of the subject invention include the genes and isolates 
identified using the methods and nucleotide sequences disclosed herein. 
The genes thus identified will encode a toxin active against nematodes. 
Similarly, the isolates will have activity against these pests. 
New pesticidal B.t. isolates of the subject invention include PS32B, PS49C, 
PS52E3, PS54G2, PS101CC3, PS178D4, PS185L2, PS197P3, PS242B6, PS242G4, 
PS242H10, PS242K17, PS244A2, and PS244D1. 
A further aspect of the subject invention is the discovery of new 
pesticidal activities for known B.t. isolates and toxins. Specifically 
exemplified herein is the discovery that B.t. isolates PS86Q3 and PS201T6, 
and toxins therefrom, can be used for the control of nematodes. In a 
preferred embodiment, the product of the 86Q3a gene, and fragment thereof; 
are used to control nematode pests. 
Toxins with activity against corn rootworm are also an aspect of the 
subject invention. 
In a preferred embodiment, the genes described herein which encode 
pesticidal toxins are used to transform plants in order to confer pest 
resistance upon said plants. Such transformation of plants can be 
accomplished using techniques well known to those skilled in the an and 
would typically involve modification of the gene to optimize expression of 
the toxin in plants.

DETAILED DISCLOSURE OF THE INVENTION 
The subject invention concerns materials and methods for the control of 
pests. In specific embodiments, the subject invention pertains to new 
Bacillus thuringiensisisolates and toxins which have activity against 
nematodes. Certain of the toxins also have activity against coleopteran 
pests. The subject invention further concerns novel genes which encode 
these pesticidal toxins and novel methods for identifying and 
characterizing B.t. genes which encode toxins with useful properties. 
In one embodiment, the subject invention concerns materials and methods 
including nucleotide primers and probes for isolating and identifying 
Bacillus thuringiensis (B.t.) genes encoding protein toxins which are 
active against nematode pests. The nucleotide sequences described herein 
can also be used to identify new pesticidal B.t. isolates. The invention 
further concerns the genes, isolates, and toxins identified using the 
methods and materials disclosed herein. 
It is well known that DNA possesses a fundamental property called base 
complementary. In nature, DNA ordinarily exists in the form of pairs of 
anti-parallel strands, the bases on each strand projecting from that 
opposite strand. The base adenine (A) on one strand will always be opposed 
to the base thymine (T) on the other strand, and the base guanine (G) will 
be opposed to the base cytosine (C). The bases are held in apposition by 
their ability to hydrogen bond in this specific way. Though each 
individual bond is relatively weak, the net effect of many adjacent 
hydrogen bonded bases, together with base stacking effects, is a stable 
joining of the two complementary strands. These bonds can be broken by 
treatments such as high pH or high temperature, and these conditions 
result in the dissociation, or "denaturation", of the two strands. If the 
DNA is then placed in conditions which make hydrogen bonding of the bases 
thermodynamically favorable, the DNA strands will anneal, or "hybridize", 
and reform the original double stranded DNA. If carried out under 
appropriate conditions, this hybridization can be highly specific. That 
is, only strands with a high degree of base complementary will be able to 
form stable double stranded structures. The relationship of the 
specificity of hybridization to reaction conditions is well known. Thus, 
hybridization may be used to test whether two pieces of DNA are 
complementary in their base sequences. It is this hybridization mechanism 
which facilitates the use of probes of the subject invention to readily 
detect and characterize DNA sequences of interest. 
Polymerase Chain Reaction (PCR) is a repetitive, enzymatic, primed 
synthesis of a nucleic acid sequence. This procedure is well known and 
commonly used by those skilled in this art (see Mullis, U.S. Pat. Nos. 
4,683,195, 4,683,202, and 4,800,159; Saiki et al., 1985). PCR is based on 
the enzymatic amplification of a DNA fragment of interest that is flanked 
by two oligonucleotide primers that hybridize to opposite strands of the 
target sequence. The primers are oriented with the 3' ends pointing 
towards each other. Repeated cycles of heat denaturation of the template, 
annealing of the primers to their complementary sequences, and extension 
of the annealed primers with a DNA polymerase result in the amplification 
of the segment defined by the 5' ends of the PCR primers. Since the 
extension product of each primer can serve as a template for the other 
primer, each cycle essentially doubles the amount of DNA fragment produced 
in the previous cycle. This results in the exponential accumulation of the 
specific target fragment, up to several million-fold in a few hours. By 
using a thermostable DNA polymerase such as Taq polymerase, which is 
isolated from the thermophilic bacterium Thermus aquaticus, the 
amplification process can be completely automated. 
The DNA sequences of the subject invention can be used as primers for PCR 
amplification. In performing PCR amplification, a certain degree of 
mismatch can be tolerated between primer and template. Therefore, 
mutations, deletions, and insertions (especially additions of nucleotides 
to the 5' end) of the exemplified primers fall within the scope of the 
subject invention. Mutations, insertions and deletions can be produced in 
a given primer by methods known to an ordinarily skilled artisan. It is 
important to note that the mutational, insertional, and deletional 
variants generated from a given primer sequence may be more or less 
efficient than the original sequences. Notwithstanding such differences in 
efficiency, these variants are within the scope of the present invention. 
In addition, PCR-amplified DNA may serve as a hybridization probe. In order 
to analyze B.t. DNA using the nucleotide sequences of the subject 
invention as probes, the DNA can first be obtained in its native, 
double-stranded form. A number of procedures are currently used to isolate 
DNA and are well known to those skilled in this art. 
One approach for the use of the subject invention as probes entails first 
identifying by Southern blot analysis of a gene bank of the B.t. isolate 
all DNA segments homologous with the disclosed nucleotide sequences. Thus, 
it is possible, without the aid of biological analysis, to know in advance 
the probable activity of many new B.t. isolates, and of the individual 
endotoxin gene products expressed by a given B.t. isolate. Such a probe 
analysis provides a rapid method for identifying potentially commercially 
valuable insecticidal endotoxin genes within the multifarious subspecies 
of B.t. 
One hybridization procedure useful according to the subject invention 
typically includes the initial steps of isolating the DNA sample of 
interest and purifying it chemically. Either lysed bacteria or total 
fractionated nucleic acid isolated from bacteria can be used. Cells can be 
treated using known techniques to liberate their DNA (and/or RNA). The DNA 
sample can be cut into pieces with an appropriate restriction enzyme. The 
pieces can be separated by size through electrophoresis in a gel, usually 
agarose or acrylamide. The pieces of interest can be transferred to an 
immobilizing membrane in a manner that retains the geometry of the pieces. 
The membrane can then be dried and prehybfidized to equilibrate it for 
later immersion in a hybridization solution. The manner in which the 
nucleic acid is affixed to a solid support may vary. This fixing of the 
DNA for later processing has great value for the use of this technique in 
field studies, remote from laboratory facilities. 
The particular hybridization technique is not essential to the subject 
invention. As improvements are made in hybridization techniques, they can 
be readily applied. 
As is well known in the art, if the probe molecule and nucleic acid sample 
hybridize by forming a strong non-covalent bond between the two molecules, 
it can be reasonably assumed that the probe and sample are essentially 
identical. The probe's detectable label provides a means for determining 
in a known manner whether hybridization has occurred. 
The nucleotide segments of the subject invention which are used as probes 
can be synthesized by use of DNA synthesizers using standard procedures. 
In the use of the nucleotide segments as probes, the particular probe is 
labeled with any suitable label known to those skilled in the art, 
including radioactive and non-radioactive labels. Typical radioactive 
labels include .sup.32 P, .sup.35 S, or the like. A probe labeled with a 
radioactive isotope can be constructed from a nucleotide sequence 
complementary to the DNA sample by a conventional nick translation 
reaction, using a DNase and DNA polymerase. The probe and sample can then 
be combined in a hybridization buffer solution and held at an appropriate 
temperature until annealing occurs. Thereafter, the membrane is washed 
free of extraneous materials, leaving the sample and bound probe molecules 
typically detected and quantified by autoradiography and/or liquid 
scintillation counting. For synthetic probes, it may be most desirable to 
use enzymes such as polynucleotide kinase or terminal transferase to 
end-label the DNA for use as probes. 
Non-radioactive labels include, for example, ligands such as biotin or 
thyroxine, as well as enzymes such as hydrolases or perixodases, or the 
various chemiluminescers such as luciferin, or fluorescent compounds like 
fluorescein and its derivatives. The probes may be made inherently 
fluorescent as described in International Application No. WO93/16094. The 
probe may also be labeled at both ends with different types of labels for 
ease of separation, as, for example, by using an isotopic label at the end 
mentioned above and a biotin label at the other end. 
The amount of labeled probe which is present in the hybridization solution 
will vary widely, depending upon the nature of the label, the amount of 
the labeled probe which can reasonably bind to the filter, and the 
stringency of the hybridization. Generally, substantial excesses of the 
probe will be employed to enhance the rate of binding of the probe to the 
fixed DNA. 
Various degrees of stringency of hybridization can be employed. The more 
severe the conditions, the greater the complementarity that is required 
for duplex formation. Severity can be controlled by temperature, probe 
concentration, probe length, ionic strength, time, and the like. 
Preferably, hybridization is conducted under stringent conditions by 
techniques well known in the art, as described, for example, in Keller and 
Manak, 1987. 
Duplex formation and stability depend on substantial complementarity 
between the two strands of a hybrid, and, as noted above, a certain degree 
of mismatch can be tolerated. Therefore, the nucleotide sequences of the 
subject invention include mutations (both single and multiple), deletions, 
insertions of the described sequences, and combinations thereof, wherein 
said mutations, insertions and deletions permit formation of stable 
hybrids with the target polynucleotide of interest. Mutations, insertions, 
and deletions can be produced in a given polynucleotide sequence in many 
ways, and these methods are known to an ordinarily skilled artisan. Other 
methods may become known in the future. 
The known methods include, but are not limited to: 
(1) synthesizing chemically or otherwise an artificial sequence which is a 
mutation, insertion or deletion of the known sequence; 
(2) using a nucleotide sequence of the present invention as a probe to 
obtain via hybridization a new sequence or a mutation, insertion or 
deletion of the probe sequence; and 
(3) mutating, inserting or deleting a test sequence in vitro or in vivo. 
It is important to note that the mutational, insertional, and deletional 
variants generated from a given probe may be more or less efficient than 
the original probe. Notwithstanding such differences in efficiency, these 
variants are within the scope of the present invention. 
Thus, mutational, insertional, and deletional variants of the disclosed 
nucleotide sequences can be readily prepared by methods which are well 
known to those skilled in the art. These variants can be used in the same 
manner as the instant probe sequences so long as the variants have 
substantial sequence homology with the probes. As used herein, substantial 
sequence homology, refers to homology which is sufficient to enable the 
variant to function in the same capacity as the original probe. 
Preferably, this homology, is greater than 50%; more preferably, this 
homology is greater than 75%; and most preferably, this homology is 
greater than 90%. The degree of homology needed for the variant to 
function in its intended capacity will depend upon the intended use of the 
sequence. It is well within the skill of a person trained in this art to 
make mutational, insertional, and deletional mutations which are designed 
to improve the function of the sequence or otherwise provide a 
methodological advantage. 
It is well known in the art that the amino acid sequence of a protein is 
determined by the nucleotide sequence of the DNA. Because of the 
redundancy of the genetic code, i.e., more than one coding nucleotide 
triplet (codon) can be used for most of the amino acids used to make 
proteins, different nucleotide sequences can code for a particular amino 
acid. Thus, the genetic code can be depicted as follows: 
______________________________________ 
Phenylalanine (Phe) 
TTK Histidine (His) 
CAK 
Leucine (Leu) XTY Glutamine (Gln) 
CAJ 
Isoleucine (Ile) 
ATM Asparagine (Asn) 
AAK 
Methionine (Met) 
ATG Lysine (Lys) AAJ 
Valine (Val) GTL Aspartic acid (Asp) 
GAK 
Serine (Ser) QRS Glutamic acid (Glu) 
GAJ 
Preline (Pro) CCL Cysteine (Cys) 
TGK 
Threonine (Thr) 
ACL Tryptophan (Trp) 
TGG 
Alanine (Ala) GCL Arginine (Arg) 
WGZ 
Tyrosine (Tyr) 
TAK Glycine (Gly) GGL 
Termination signal 
TAJ Termination signal 
TGA 
______________________________________ 
Key: Each 3letter deoxynucleotide triplet corresponds to a trinucleotide 
of mMRNA, having a 5end on the left and a 3end on the right. All DNA 
sequences given herein are those of the strand whose sequence correspond 
to the mRNA sequence, with thymine substituted for uracil. The letters 
stand for the purine or pyrimidine bases forming the deoxynucleotide 
sequence. 
A = adenine 
G = guanine 
C = cytosine 
T = thymine 
X = T or C if Y is A or G 
X = C if Y is C or T 
Y = A, G, C or T if X is C 
Y = A or G if X is T 
W = C or A if Z is A or G 
W C if Z is C or T 
Z = A, G, C or T if W is C 
Z = A or G if W is A 
QR = TC if S is A, G, C or T; alternatively QR = AG if S is T or C 
J = A or G 
K = T or C 
L = A, T, C or G 
M = A, C or T 
The above shows that the amino acid sequence of B.t. toxins can be encoded 
by equivalent nucleotide sequences encoding the same amino acid sequence 
of the protein. Accordingly, the subject invention includes probes which 
would hybridize with various polynucleotide sequences which would all code 
for a given protein or variations of a given protein. In addition, it has 
been shown that proteins of identified structure and function may be 
constructed by changing the amino acid sequence if such changes do not 
alter the protein secondary structure (Kaiser, E. T., Kezdy, F. J. 1984! 
Science 223:249-255). 
The sequences and lengths of five cryV-specific primers useful according to 
the subject invention are shown in Table 1. 
TABLE 1 
__________________________________________________________________________ 
Primer name 
Sequence length 
__________________________________________________________________________ 
V3 GATCGTMTWGARTTTRTTCC (SEQ ID NO. 1) 
20-mer 
V5 AAAGTNGATGCMTTATCWGATGA (SEQ ID NO. 2) 
23-mer 
V7 ACACGTTATAHDGTTTCTGG (SEQ ID NO. 3) 
20-mer 
.DELTA.V5' 
TCATCWGATAAKGCATCNAC (SEQ ID NO. 4) 
20-mer 
.DELTA.V8' 
TGGACGDTCTTCAMKAATTTCYAAA (SEQ ID NO. 5) 
25-mer 
__________________________________________________________________________ 
Following is a table which provides characteristics of certain isolates 
useful according to the subject invention. 
TABLE 2 
__________________________________________________________________________ 
Description of B.t. strains toxic to nematodes 
Culture 
Crystal Description Approx. MW (kDa) 
Serotype NRRL Deposit 
Deposit 
__________________________________________________________________________ 
Date 
PS32B attached amorphic, fibrous flat square 
86, 52, 26 non-motile 
B-21531 
3-14-96 
PS49C small dark sphere 133, 62 3 B-21532 
3-14-96 
PS52E3 
amorphic round 130, 127, 63 3 B-21533 
3-14-96 
PS54G2 
small bipyramid 140, 128, 112, 95, 62, 45, 43, 
non-motile 
B-21543 
3-20-96 
PS101CC3 
attached long 142, 135, 129 N.D. B-21534 
3-14-96 
PS178D4 
attached amorphic, round-ovoid, non-refractile 
95, 85, 65 non-motile 
B-21544 
3-20-96 
PS185L2 
attached multiple round amorphic, BP with ort, 
150, 98, 45, 39 
6, entomocidus 
B-21535 
3-14-96 
long thin 
PS197P3 
amorphic, long thin dark, smaller round 
35 non-motile 
B-21536 
3-14-96 
PS242B6 
multiple amorphic (many unlysed canoes) 
130, 65 N.D. B-21537 
3-14-96 
PS242G4 
multiple amorphic 130 N.D. B-21538 
3-14-96 
PS242H10 
spherical and large attached amorphic 
30 N.D. B-21439 
3-14-96 
PS242K17 
large attached multiple light amorphic 
55 N.D. B-21540 
3-14-96 
PS244A2 
amorphic, spore-sized 
130, 60 N.D. B-21541 
3-14-96 
PS244D1 
single large spherical, spore-sized with "nub" 
130 N.D. B-21542 
3-14-96 
PS74G1 
amorphic 145, 1215, 100, 90, 60 
10, darmstadiensis 
B-18397 
8-16-88 
PS75J1 
attached amorphic 86, 80, 74, 62 
non-motile 
B-18781 
3-7-91 
PS83E5 
multiple attached 42, 37 non-motile 
B-18782 
3-7-91 
PS86Q3 
attached long 155, 135, 98, 62, 58 
novel B-18765 
2-6-91 
PS98A3 
attached long 140, 130, 125 10, darmstadiensis 
B-18401 
8-16-88 
PS101Z2 
attached long 142, 135, 128 10, darmstadiensis 
B-18890 
10-1-91 
PS158C2 
amorphic, flat irregular 
130, 47, 38, 33 
4 B-18872 
8-27-91 
PS201T6 
amorphic, elliptical and bipyramid 
133, 31 24, neoleoensis 
B-18750 
9-1-91 
PS204C3 
attached multiple amorphic and ellipse 
100, 92, 47, 35 
non-motile 
B-21008 
6-10-92 
PS17 amorphic, attached long 
140, 90, 60 10, darmstadiensis 
B-18243 
7-28-87 
PS33F2 
long bipyramid 140, 115, 90, 60 
wuhanensis 
B-18244 
7-28-87 
PS63B amoprhic 84, 82, 78 wuhanensis 
B-18246 
7-28-87 
PS52A1 
multiple attached 58, 45 wuhanensis 
B-18245 
7-28-87 
PS69D1 
elongated 34, 38, 145 non-motile 
B-18247 
7-28-87 
PS80JJ1 
multiple attached 130, 90, 47, 37 
4a4b, sotto 
B-18679 
7-17-90 
PS158D5 
attached amorphic 80 novel B-18680 
7-17-90 
PS167P 
attached amorphic 120 novel B-18681 
7-17-90 
PS169E 
attached amorphic 150, 128, 33 non-motile 
B-18682 
7-17-90 
PS177F1 
multiple attached 140, 116, 103, 62 
non-motile 
B-18683 
7-17-90 
PS177G 
multiple attached 135, 125, 107, 98, 62 
non-motile 
B-18684 
7-17-90 
PS204G4 
multiple attached 105, 98, 90, 60, 44, 37 
non-motile 
B-18685 
7-17-90 
PS204G6 
long amorphic 23, 21 wuhanensis 
B-18686 
7-17-90 
__________________________________________________________________________ 
N.D. = not determined 
As noted in Table 2, certain B.t. isolates useful according to the subject 
invention are available from the permanent collection of the Agricultural 
Research Service Patent Culture Collection (NRRL), Northern Regional 
Research Center, 1815 North University Street, Peoria, Ill. 61604, USA. 
Cultures which have been deposited for the purposes of this patent 
application were deposited under conditions that assure that access to the 
cultures is available during the pendency of this patent application to 
one determined by the Commissioner of Patents and Trademarks to be 
entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposits will be 
available as required by foreign patent laws in countries wherein 
counterparts of the subject application, or its progeny, are filed. 
However, it should be understood that the availability of a deposit does 
not constitute a license to practice the subject invention in derogation 
of patent rights granted by governmental action. 
Further, the subject culture deposits will be stored and made available to 
the public in accord with the provisions of the Budapest Treaty for the 
Deposit of Microorganisms, i.e., they will be stored with all the care 
necessary to keep them viable and uncontaminated for a period of at least 
five years after the most recent request for the furnishing of a sample of 
the deposit, and in any case, for a period of at least thirty (30) years 
after the date of deposit or for the enforceable life of any patent which 
may issue disclosing the culture(s). The depositor acknowledges the duty 
to replace the deposit(s) should the depository be unable to furnish a 
sample when requested, due to the condition of a deposit. All restrictions 
on the availability to the public of the subject culture deposits will be 
irrevocably removed upon the granting of a patent disclosing them. 
Control of nematodes, or coleopterans, using the isolates, toxins, and 
genes of the subject invention can be accomplished by a variety of methods 
known to those skilled in the art. These methods include, for example, the 
application of B.t. isolates to the pests (or their location), the 
application of recombinant microbes to the pests (or their locations), and 
the transformation of plants with genes which encode the pesticidal toxins 
of the subject invention. Recombinant microbes may be, for example, a 
B.t., E. coli, or Pseudomonas. Transformations can be made by those 
skilled in the art using standard techniques. Materials necessary for 
these transformations are disclosed herein or are otherwise readily 
available to the skilled artisan. For example, the gene encoding the 167P 
toxin is provided herein as SEQ ID NO. 8. The deduced amino acid sequence 
for the 167P toxin is, provided in SEQ ID NO. 9. A description of the 
PS167P isolate can be found in WO94/16079, which also provides a 
description of the cloning of the 80JJ1 gene. The nematicidal toxin known 
as 86Q3(a) found in PS8603 is encoded by a gene described in WO92/20802. 
Similarly, U.S. Pat. No. 5,488,432 describes the 201T6 gene which can be 
used to encode nematicidal toxins according to the subject invention. 
Also, B.t. isolates harboring genes encoding pesticidal toxins have been 
deposited as described herein. 
Following are examples which illustrate procedures for practicing the 
invention. These examples should not be construed as limiting. All 
percentages are by weight and all solvent mixture proportions are by 
volume unless otherwise noted. 
Example 1-Culturing of B.t. Isolates Useful According to the Invention 
A subculture of B.t. isolates, or routants thereof, can be used to 
inoculate the following peptone, glucose, salts medium: 
______________________________________ 
Bacto Peptone 7.5 g/l 
Glucose 1.0 g/l 
KH.sub.2 PO.sub.4 3.4 g/l 
K.sub.2 HPO.sub.4 4.35 g/l 
Salt Solution 5.0 ml/l 
CaCl.sub.2 Solution 
5.0 ml/l 
pH 7.2 
Salts Solution (100 ml) 
MgSO.sub.4.7H.sub.2 O 
2.46 g 
MnSO.sub.4.H.sub.2 O 
0.04 g 
ZnSO.sub.4.7H.sub.2 O 
0.28 g 
FeSO.sub.4.7H.sub.2 O 
0.40 g 
CaCl.sub.2 Solution (100 ml) 
3.66 g 
CaCl.sub.2.2H.sub.2 O 
______________________________________ 
The salts solution and CaCl.sub.2 solution are filter-sterilized and added 
to the autoclaved and cooked broth at the time of inoculation. Flasks are 
incubated at 30.degree. C. on a rotary shaker at 200 rpm for 64 hr. 
The above procedure can be readily scaled up to large fermentors by 
procedures well known in the art. 
The B.t. spores and/or crystals, obtained in the above fermentation, can be 
isolated by procedures well known in the art. A frequently-used procedure 
is to subject the harvested fermentation broth to separation techniques, 
e.g., centrifugation. 
Example 2-Nematode Bioassay 
C. elegans eggs were purified from gravid adult hermaphrodites, followed by 
hatching, feeding B.t.-based materials to a population of L1 or L2 larvae, 
followed by a 3-day bioassay time. Hatched L1 larvae remain viable at 
15.degree. C. for more than two weeks and neither grow nor die in the 
absence of a bacterial food source. C. elegans adapt rapidly to a diet of 
non-toxic B.t. spores (and crystals), although they grow more slowly than 
on their usual bacterial food source, E. coli. 
This bioassay was validated by feeding approximately 100 L1 or L2 C. 
elegans larvae in 300 82 l bioassay volume in 24-well microliter trays 
with 4 doses (1, 5, 10, or 25 .mu.l of washed biomass) of the following 
bacterial cells: 
______________________________________ 
B.t. PS17 known nematicidal isolate 
B.t. PS80JJ1 known nematicidal isolate 
B.t. PS167P known nematicidal isolate 
B.t. HD-73 negative control strain 
E. coli strain MC1061 
normal C. elegans food source 
No bacteria 
______________________________________ 
All nematodes were dead after 3 days at room temperature when incubated 
with the three nematicidal isolates, but proceeded to their full-grown 
adult stage with HD-73 or MC1061. After 5 days, L2 progeny were observed 
along with fully-grown adults on high doses of MC1061; HD-73 yielded only 
fully grown adults during this time. 
Thus, all 3 known nematicidal isolates killed all nematodes, even at the 
lowest rate tested, while the non-toxic B.t. strain permitted full 
population growth without progeny interference even at the highest rate 
tested. 
Example 3-Isolation and Preparation of Cellular DNA for PCR 
DNA can be prepared from cells grown on Spizizen's agar, or other minimal 
agar known to those skilled in the art, for approximately 16 hours. 
Spizizen's casamino acid agar comprises 23.2 g/l Spizizen's minimal salts 
(NH.sub.4).sub.2 SO.sub.4, 120 g; K.sub.2 HPO.sub.4, 840 g; KH.sub.2 
PO.sub.4, 360 g; sodium citrate, 60 g; MgSO.sub.4. 7H.sub.2 O, 12 g. 
Total: 1392 g!; 1.0 g/l vitamin-free casamino acids; 15.0 g/l Difco agar. 
In preparing the agar, the mixture was autoclaved for 30 minutes, then a 
sterile, 50% glucose solution can be added to a final concentration of 
0.5% (1/100 vol). Once the cells are grown for about 16 hours, an 
approximately 1 cm.sup.2 patch of cells can be scraped from the agar into 
300/.mu.l of 10 mM Tris-HC1 (pH 8.0)-1 mM EDTA. Proteinase K was added to 
50 .mu.g/ml and incubated at 55.degree. C. for 15 minutes. Other suitable 
proteases lacking nuclease activity can be used. The samples were then 
placed in a boiling water bath for 15 minutes to inactivate the proteinase 
and denature the DNA. This also precipitates unwanted components. The 
samples are then centrifuged at 14,000 .times.g in an Eppendorf microfuge 
at room temperature for 5 minutes to remove cellular debris. The 
supernatants containing crude DNA were transferred to fresh tubes and 
frozen at -20.degree. C. until used in PCR reactions. 
Example 4-PCR Amplification 
Conditions for multiplex PCR amplification were: 
TABLE 3 
______________________________________ 
PCR amplification conditions 
Reagent Final reaction concentration 
______________________________________ 
Taq buffer 1x 
MgCl.sub.2 2.0 mM 
dNTPs 0.1 mM 
rRNA primers (forward & reverse) 
0.05 pmol/.mu.l each 
cryV-specific primers (forward & 
0.20 pmol/.mu.l each 
reverse) 
crude total B.t. DNA.sup.1 
15 .mu.l 
______________________________________ 
.sup.1 Total reaction volume: 49.5 .mu.l. 
Samples were preheated to 94.degree. C. for 3 minutes, then quick chilled 
on ice. 0.5 .mu.l Taq polymerase (5 units/ml) was added and overlaid with 
50 .mu.light mineral oil. Cycle conditions were: {94.degree. C., 1 minute; 
42.degree. C., 2 minutes; 72.degree. C., 3 minutes +5 second/cycle}, 
repeated for 30 cycles, and held at 4.degree. C. or -20.degree. C. until 
gel analysis. 
Internal positive controls for each PCR reaction in the screen were 
included: forward and reverse 16S rRNA gene primers, yielding a 
PCR-amplified fragment of 182 bp corresponding to nucleotide positions 
1188 to 1370 in the sequence (Ash, C. et al. 1991! Lett. Appl. Microbiol. 
13:202-206). This size is smaller than fragments expected from any of the 
cryV-specific primer pairs. The two rRNA primers were: 
TABLE 4 
______________________________________ 
Primer name 
Sequence length 
______________________________________ 
rRNAfor CCGGAGGAAGGTGGGGATG (SEQ ID NO. 6) 
19-mer 
rRNArev CGATTACTAGCGATTCC (SEQ ID NO. 7) 
17-mer 
______________________________________ 
TABLE 5 
__________________________________________________________________________ 
PCR amplification of nematode-active B.t. strains 
Expected size (bp) using primer pair 
Strain 
Gene Gene name 
V3-.DELTA.V5' 
V3-.DELTA.V8' 
V7-.DELTA.V8' 
V5-.DELTA.V8' 
__________________________________________________________________________ 
PS17 17a cryVAa 
817 1379 317 582 
PS17 17b cryVAb 
526 1088 317 582 
PS17 86Q3c-like 
cryVAc 
337 899 317 582 
PS86Q3 
86Q3a cryVD 562 1124 317 582 
PS86Q3 
86Q3c cryVAc 
337 899 317 582 
PS33F2 
33F2 cryVB 547 1112 320 585 
PS80JJ1 
80JJ1 cryVE 289 860 323 591 
PS167P 
167P 167P 196 800 332 599 
__________________________________________________________________________ 
Example 5-Cloning of Novel Pesticidal Genes Using Oligonucleotide Primers 
Nematicidal toxin genes of new B.t. strains can be obtained from their DNA 
by performing the standard polymerase chain reaction procedure as in 
Example 4 using the oligonucleotides of SEQ ID NO. 4 or SEQ ID NO. 5 as 
reverse primers and SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3 as forward 
primers. The expected PCR fragments are approximately 200 to 1000 bp with 
reverse primer SEQ ID NO. 4 and forward primer SEQ ID NO. 1. Fragments of 
about 300 to about 1500 bp are expected using the reverse primer SEQ ID 
NO. 5 and the forward primer SEQ ID NO. 1. The expected PCR fragments are 
approximately 400 to 800 bp using SEQ ID NO. 5 as reverse a primer, with 
SEQ ID NO. 2 as a forward primer. Fragments of approximately 200 to 650 bp 
are expected using the reverse primer SEQ ID NO. 5 and the forward primer 
SEQ ID NO. 3. Amplified DNA fragments of the indicated sizes can be 
radiolabeled and used as probes to clone the entire endotoxin gene. 
Example 6-Screening of B.t. Isolates for Genes Encoding Nematode- and 
Coleopteran-Active Toxins 
A large number of B.t. strains were screened by PCR as described above. 
Certain of these strains were identified as "cryV positive". In a 
preferred embodiment, "cryV positive" refers to strains for which PCR 
amplification using the primer pair V7-.DELTA.V8'(SEQ ID NOS. 3 and 5) 
yields a fragment of about 315 to about 325 bp. Most preferably, this 
fragment is about 320 bp. Approximate sizes of base pair fragments 
produced from those eleven strains were as follows: 
TABLE 6 
______________________________________ 
PCR amplification of DNA from miscellaneous B.t. strains 
Approximate size (bp) using primer pair 
Strain V3-.DELTA.V5' 
V3-.DELTA.V8' 
V7-.DELTA.V8'* 
V5-.DELTA.V8' 
______________________________________ 
PS54G2 470, 530 950, 590 320 (+) 
585 
PS62B1 600, 540, 480 
990, 590, 470 
320 (+) 
585 
PS72N 560 600, 540 850 (u) 
n.d. 
PS74G1 530 880, 590, 470 
320 (+) 
585 
PS75G2 560 n.d. 800 (u) 
n.d. 
PS86E 560 600, 540 800 (u) 
n.d. 
PS88F11 
560 n.d. 1000 (u) 
n.d. 
PS98A3 530, 390 900 320 (+) 
585 
PS177F1 
860, 530, 390 
880, 590, 470 
320 (+) 
585 
PS177G 530 n.d. 320 (+) 
585 
PS212 620, 530, 470 
950, 590, 470 
320 (+) 
585 
______________________________________ 
n.d. = not determined 
*symbols in parentheses indicate whether the strain was "cryV positive" 
(+) or "cryV unusual" (u). 
Example 7-Bioassay Results 
Bioassays for nematode activity were performed as described in Example 3. 
PCR using the V7-.DELTA.V8' primer pair was performed as described herein 
for many of the isolates which were bioassayed. The isolates were 
considered cryV PCR-positive if PCR using the V7-.DELTA.V8' primer pair 
yielded an approximately 320-bp fragment. If PCR with this primer pair 
yielded fragments of other sizes, then the isolate was designated 
"unusual". If no fragment was produced, then the isolate was designated as 
"negative". The results of these bioassays and the PCR experiment are 
shown in Table 7. Nematode activity was found to be very highly correlated 
with a "positive cryV" PCR profile. It should be noted that a negative or 
unusual cryV PCR profile does not preclude nematode activity. This is 
because there are nematicidal toxins other than cryV toxins which could 
still provide activity against nematodes. 
TABLE 7 
______________________________________ 
Summary of C. elegans bioassay results 
Strain/clone cryV PCR C.e. toxicity 
______________________________________ 
E. coli MC1061 N.D. - 
HD-73 negative - 
PS17 positive + 
PS33F2 positive + 
PS63B N.D. + 
PS69D1 N.D. + 
PS86A1 N.D. + 
PS158D5 negative + 
PS169E negative + 
PS177F1 positive + 
PS177G positive + 
PS204G4 N.D. + 
PS204G6 N.D. + 
PS80JJ1 positive + 
PS167P positive + 
PS54G2 positive + 
PS62B1 positive - 
PS72N unusual - 
PS74G1 positive + 
PS75G2 unusual - 
PS86E unusual - 
PS88F11 unusual - 
PS98A3 positive + 
PS212 positive - 
PS86Q3 positive + 
PS242B6 N.D. +/- 
PS242G4 N.D. +/- 
PS242H10 N.D. + 
PS242K17 N.D. + 
PS244A2 N.D. +/- 
PS244D1 N.D. +/- 
PS32B1 N.D. + 
PS49C neative + 
PS52E3 negative + 
PS75J negative + 
PS83E5 N.D. + 
PS101CC3 N.D. + 
PS101Z2 N.D. + 
PS158C2 N.D. +/- 
PS178D4 N.D. + 
PS185L2 negative + 
PS197P3 N.D. +/- 
PS201T6 N.D. +/- 
PS204C3 N.D. + 
______________________________________ 
+ = acute toxicity 
+/- = stunting of larvae 
- = little or no activity 
N.D. = not determined 
Recombinant hosts expressing specific toxins were constructed and tested in 
bioassays, as described above, to evaluate nematicidal activity. The 
results from these assays are shown in Table 8. 
TABLE 8 
______________________________________ 
Description of clones toxic to nematodes 
Toxin 
Toxin mol. wt. 
C. elegans 
Clone Host* expressed Parent strain 
(kDa) activity 
______________________________________ 
MR515 B.t. CryVD/Cry5B 
PS86Q3 140 + 
MR871 P.f. CryVD/Cry5B 
PS86Q3 140 + 
MR531 B.t. unnamed PS167P 132 + 
MR506 B.t. CryVIA/Cr6A 
PS86A1 54 + 
MR508 B.t. CryVB/Cry12A 
PS33F2 142 +/- 
cryB B.t. none -- -- -- 
MR839 P.f. none -- -- -- 
______________________________________ 
B.t. = Bacillus thuringiensis 
P.f. = Pseudomonas fluorescens 
Example 8-Insertion of Toxin Genes into Plants 
One aspect of the subject invention is the transformation of plants with 
genes encoding a toxin active against coleopteran and/or nematode pests. 
The transformed plants are resistant to attack by coleopterans and/or 
nematodes. 
Genes encoding pesticidal toxins, as disclosed herein, can be modified for 
optimum expression in plant, linked to a plant selectable marker gene, and 
inserted into a genome of plant cell using a variety of techniques which 
are well known to those skilled in the art. Any plant may be used in 
accordance with this invention, including angiosperms, gymnosperms, 
monocotyledons and dicotyledons. Preferred plants include soybean, 
sunflower, cotton, potato, alfalfa, maize, rice and wheat. The 
transformation method itself is not critical to the invention but may 
include transformation with T-DNA using Agrobacterium tumefaciens or A. 
rhizogenes as the transformation agent, liposome fusion, microinjection, 
microprojectile bombardment, chemical agent (PEG or calcium 
chloride)-assisted DNA uptake, or electroporation, as well as other 
possible methods. Reference may be made to the literature for full details 
of the known methods, especially Holsters et al., 1978; Fromm et al., 
1985; Horsch et al., 1985; Herrera-Estrella et al., 1983; Crossway et al., 
1986; Lin, 1966; and Steinkiss and Stabel, 1983. 
Use of a plant selectable marker in transformation allows for selection of 
transformed cells rather than cells that do not contain the inserted DNA. 
Various markers exist for use in plant cells and generally provide 
resistance to a biocide or antibiotic, including but not limited to, 
kanamycin, G418, hygromycin, and phosphinothricin. Visual markers 
including but not limited to b-glucuronidase, b-galactosidase, B-peru 
protein, green fluorescent protein, and luciferase may also be used. After 
transformation, those cells that have the DNA insert can be selected for 
by growth in a defined medium and assayed for marker expression, whether 
by resistance or visualization. Cells containing the DNA insert can be 
regenerated into plants. As long as stably transformed plants are 
obtained, the method used for regeneration will depend on the plant tissue 
and transformation method used and is not critical to the invention. 
However, for example, where cell suspensions have been used for 
transformation, transformed cells can be induced to produce calli and the 
calli subsequently induced to form shoots, which may then be transferred 
to an appropriate nutrient medium to regenerate plants. Alternatively, 
explants such as hypocotyl tissue or embryos may be transformed and 
regenerated by shoot induction in the appropriate media, followed by root 
and whole plant formation. Whatever regeneration method is used, the 
result will be stably transformed plants that can vegetatively and 
sexually transmit the transformed trait(s) to progeny, so that, if 
necessary, the transformed plant can be crossed with untransformed plants 
in order to transfer the trait to more appropriate germplasm for breeding 
purposes. 
It should be understood that the examples and embodiments described herein 
are for illustrative purposes only and that various modifications or 
changes in light thereof will be suggested to persons skilled in the art 
and are to be included within the spirit and purview of this application 
and the scope of the appended claims. 
______________________________________ 
References 
______________________________________ 
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U.S. Pat. No. 4,853,331. 
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______________________________________ 
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__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 9 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (synthetic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GATCGTMTWGARTTTRTTCC20 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (synthetic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
AAAGTNGATGCMTTATCWGATGA23 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (synthetic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
ACACGTTATAHDGTTTCTGG20 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (synthetic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
TCATCWGATAAKGCATCNAC20 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (synthetic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
TGGACGDTCTTCAMKAATTTCYAAA25 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (synthetic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
CCGGAGGAAGGTGGGGATG19 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (synthetic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
CGATTACTAGCGATTCC17 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3504 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(C) INDIVIDUAL ISOLATE: 167P 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
ATGACAAATCCAACTATACTATATCCTAGTTACCATAATGTATTAGCTCATCCGATTAGA60 
TTAGATTCTTTTTTTGATCCATTTGTAGAGACATTTAAGGATTTAAAAGGGGCTTGGGAA120 
GAATTCGGAAAAACGGGATATATGGACCCCTTAAAACAACACCTTCAAATCGCATGGGAT180 
ACTAGTCAAAATGGAACAGTGGATTATTTAGCATTAACAAAAGCATCTATATCTCTCATA240 
GGTTTAATTCCTGGTGCAGACGCTGTAGTCCCTTTTATTAATATGTTTGTAGACTTTATT300 
TTTCCGAAATTATTTGGAAGAGGTTCTCAACAAAATGCTCAAGCTCAATTTTTCGAACTA360 
ATCATAGAAAAAGTTAAAGAACTTGTTGATGAAGATTTTAGAAACTTTACCCTTAATAAT420 
CTACTCAATTACCTTGATGGTATGCAAACAGCCTTATCACATTTCCAAAACGATGTACAA480 
ATTGCTATTTGTCAAGGAGAACAACCAGGACTTATGCTAGATCAAACACCAACGGCTTGT540 
ACTCCTACTACAGACCATTTAATTTCTGTAAGAGAATCTTTTAAAGATGCTCGAACTACA600 
ATTGAAACAGCTTTACCACATTTTAAAAATCCTATGCTATCCACAAATGATAACACTCCA660 
GATTTTAATAGCGACACTGTCTTATTAACATTACCAATGTATACAACAGCAGCGACTTTA720 
AATCTTATATTACATCAAGGGTATATTCAATTCGCAGAAAGATGGAAATCTGTAAATTAT780 
GATGAAAGTTTTATAAATCAAACAAAAGTTGATTTGCAACGTCGTATTCAGGACTATTCT840 
ACTACTGTATCTACCACTTTTGAAAAATTCAAACCTACTCTAAATCCATCAAATAAAGAA900 
TCTGTTAATAAGTATAATAGATATGTTCGTTCCATGACTCTTCAATCTTTAGACATTGCT960 
GCAACATGGCCTACTTTAGATAATGTTAATTACCCTTCCAATGTAGATATTCAATTGGAT1020 
CAAACTCGCTTAGTATTTTCAGATGTTGCAGGACCTTGGGAAGGTAATGATAATATAACT1080 
TCGAATATTATAGATGTATTAACACCAATAAATACAGGGATAGGATTTCAAGAAAGTTCA1140 
GATCTTAGAAAATTCACTTATCCACGAATAGAATTACAAAGCATGCAATTCCATGGACAA1200 
TATGTAAACTCAAAAAGTGTAGAACATTGTTATAGCGATGGTCTTAAATTAAATTATAAA1260 
AATAAAACTATAACTGCAGGTGTAAGTAATATTGATGAAAGTAATCAAAATAATAAACAT1320 
AACTATGGTCCTGTAATAAATAGTCCTATTACTGATATCAACGTAAATTCCCAAAATTCT1380 
CAATATTTAGATTTAAATTCAGTCATGGTAAATGGTGGTCAAAAAGTAGCCGGGTGTTCA1440 
CCACTTAGTTCAAATGGTAATTCTAATAATGCTGCTTTACCTAATCAAAAAATAAATGTT1500 
ATTTATTCAGTACAATCAAATGATAAACCAGAAAAACATGCAGACACTTATAGAAAATGG1560 
GGATATATGAGCAGTCATATTCCTTATGATCTTGTTCCAGAAAATGTAATTGGAGATATA1620 
GATCCGGATACTAAACAACCGTCATTGCTTCTTAAAGGGTTTCCGGCAGAAAAAGGATAT1680 
GGTGACTCAATTGCATATGTATCAGAACCTTTAAATGGTGCGAATGCAGTTAAACTTACT1740 
TCATATCAAGTTCTCAAAATGGAAGTTACAAATCAAACAACTCAAAAATATCGTATTCGC1800 
ATACGTTATGCTACAGGTGGAGATACAGCTGCTTCTATATGGTTTCATATTATTGGTCCA1860 
TCTGGAAATGATTTAACAAACGAAGGCCATAACTTCTCTAGTGTATCTTCTAGAAATAAA1920 
ATGTTTGTTCAGGGTAATAACGGAAAATATGTATTGAACATCCTTACAGATTCAATAGAA1980 
TTACCATCAGGACAACAAACTATTCTTATTCAAAATACTAATTCTCAAGATCTTTTTTTA2040 
GATCGTATTGAATTTATTTCTCTCCCTTCTACTTCTACTCCTACTTCTACTAATTTTGTA2100 
GAACCTGAATCATTAGAAAAGATCATAAACCAAGTTAATCAATTATTTAGCTCCTCATCT2160 
CAAACTGAATTGGCTCACACTGTAAGCGATTATAAAATTGATCAAGTAGTGCTAAAAGTA2220 
AATGCCTTATCCGACGATGTATTTGGTGTAGAGAAAAAAGCATTACGTAAACTTGTGAAT2280 
CAGGCCAAACAACTCAGTAAAGCACGAAATGTATTGGTCGGTGGAAACTTTGAAAAAGGT2340 
CATGAATGGGCACTAAGCCGTGAAGCAACAATGGTCGCAAATCATGAGTTATTCAAAGGG2400 
GATCATTTATTATTACCACCACCAACCCTATATCCATCGTATGCATATCAAAAAATTGAT2460 
GAATCGAAATTAAAATCCAATACACGTTATACTGTTTCCGGCTTTATTGCGCAAAGTGAA2520 
CATCTAGAAGTCGTTGTGTCTCGATACGGGAAAGAAGTACATGACATGTTAGATATCCCG2580 
TATGAAGAAGCCTTACCAATTTCTTCTGATGAGAGTCCAAATTGTTGCAAACCAGCTGCT2640 
TGTCAGTGTTCATCTTGTGATGGTAGTCAATCAGATTCTCATTTCTTTAGCTATAGTATC2700 
GATGTTGGTTCCCTACAATCAGATGTAAATCTCGGCATTGAATTCGGTCTTCGTATTGCG2760 
AAACCAAACGGATTTGCGAAAATCAGTAATCTAGAAATTAAAGAAGATCGTCCATTAACA2820 
GAAAAAGAAATCAAAAAAGTACAACGTAAAGAACAAAAATGGAAAAAAGCATTTAACCAA2880 
GAACAAGCCGAAGTAGCGACAACACTCCAACCAACGTTAGATCAAATCAATGCTTTGTAT2940 
CAAAATGAAGATTGGAACGGTTCCGTTCACCCGCATGTGACCTATCAACATCTGTCCGCT3000 
GTTGTTGTACCAACGTTACCAAAACAAAGACATTGGTTTATGGAGGATCGAGAAGGCGAA3060 
CATGTTGTTCTGACGCAACAATTCCAACAAGCATTGGATCGTGCGTTCCAACAAATCGAA3120 
GAACAAAACTTAATCCACAATGGTAATTTTGCGAATGGATTAACAGATTGGACTGTCACA3180 
GGAGATGCACAACTTACGATCTTTGACGAAGATCCAGTATTAGAACTAGCGCATTGGGAT3240 
GCAAGTATCTCTCAAACCATTGAAATTATGGATTTTGAAGAAGACACAGAATACAAACTG3300 
CGTGTACGTGGAAAAGGCAAAGGAACAGTTACCGTTCAACATGGAGAAGAAGAATTAGAA3360 
ACGATGACATTCAATACAACGAGTTTTACAACACAAGAACAAACCTTCTACTTCGAAGGA3420 
GATACAGTGGACGTACATGTTCAATCAGAGAATAACACATTCCTGATAGATAGTGTGGAA3480 
CTCATTGAAATCATAGAAGAGTAA3504 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1168 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vi) ORIGINAL SOURCE: 
(C) INDIVIDUAL ISOLATE: 167p 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
MetThrAsnProThrIleLeuTyrProSerTyrHisAsnValLeuAla 
151015 
HisProIleArgLeuAspSerPhePheAspProPheValGluThrPhe 
202530 
LysAspLeuLysGlyAlaTrpGluGluPheGlyLysThrGlyTyrMet 
354045 
AspProLeuLysGlnHisLeuGlnIleAlaTrpAspThrSerGlnAsn 
505560 
GlyThrValAspTyrLeuAlaLeuThrLysAlaSerIleSerLeuIle 
65707580 
GlyLeuIleProGlyAlaAspAlaValValProPheIleAsnMetPhe 
859095 
ValAspPheIlePheProLysLeuPheGlyArgGlySerGlnGlnAsn 
100105110 
AlaGlnAlaGlnPhePheGluLeuIleIleGluLysValLysGluLeu 
115120125 
ValAspGluAspPheArgAsnPheThrLeuAsnAsnLeuLeuAsnTyr 
130135140 
LeuAspGlyMetGlnThrAlaLeuSerHisPheGlnAsnAspValGln 
145150155160 
IleAlaIleCysGlnGlyGluGlnProGlyLeuMetLeuAspGlnThr 
165170175 
ProThrAlaCysThrProThrThrAspHisLeuIleSerValArgGlu 
180185190 
SerPheLysAspAlaArgThrThrIleGluThrAlaLeuProHisPhe 
195200205 
LysAsnProMetLeuSerThrAsnAspAsnThrProAspPheAsnSer 
210215220 
AspThrValLeuLeuThrLeuProMetTyrThrThrAlaAlaThrLeu 
225230235240 
AsnLeuIleLeuHisGlnGlyTyrIleGlnPheAlaGluArgTrpLys 
245250255 
SerValAsnTyrAspGluSerPheIleAsnGlnThrLysValAspLeu 
260265270 
GlnArgArgIleGlnAspTyrSerThrThrValSerThrThrPheGlu 
275280285 
LysPheLysProThrLeuAsnProSerAsnLysGluSerValAsnLys 
290295300 
TyrAsnArgTyrValArgSerMetThrLeuGlnSerLeuAspIleAla 
305310315320 
AlaThrTrpProThrLeuAspAsnValAsnTyrProSerAsnValAsp 
325330335 
IleGlnLeuAspGlnThrArgLeuValPheSerAspValAlaGlyPro 
340345350 
TrpGluGlyAsnAspAsnIleThrSerAsnIleIleAspValLeuThr 
355360365 
ProIleAsnThrGlyIleGlyPheGlnGluSerSerAspLeuArgLys 
370375380 
PheThrTyrProArgIleGluLeuGlnSerMetGlnPheHisGlyGln 
385390395400 
TyrValAsnSerLysSerValGluHisCysTyrSerAspGlyLeuLys 
405410415 
LeuAsnTyrLysAsnLysThrIleThrAlaGlyValSerAsnIleAsp 
420425430 
GluSerAsnGlnAsnAsnLysHisAsnTyrGlyProValIleAsnSer 
435440445 
ProIleThrAspIleAsnValAsnSerGlnAsnSerGlnTyrLeuAsp 
450455460 
LeuAsnSerValMetValAsnGlyGlyGlnLysValAlaGlyCysSer 
465470475480 
ProLeuSerSerAsnGlyAsnSerAsnAsnAlaAlaLeuProAsnGln 
485490495 
LysIleAsnValIleTyrSerValGlnSerAsnAspLysProGluLys 
500505510 
HisAlaAspThrTyrArgLysTrpGlyTyrMetSerSerHisIlePro 
515520525 
TyrAspLeuValProGluAsnValIleGlyAspIleAspProAspThr 
530535540 
LysGlnProSerLeuLeuLeuLysGlyPheProAlaGluLysGlyTyr 
545550555560 
GlyAspSerIleAlaTyrValSerGluProLeuAsnGlyAlaAsnAla 
565570575 
ValLysLeuThrSerTyrGlnValLeuLysMetGluValThrAsnGln 
580585590 
ThrThrGlnLysTyrArgIleArgIleArgTyrAlaThrGlyGlyAsp 
595600605 
ThrAlaAlaSerIleTrpPheHisIleIleGlyProSerGlyAsnAsp 
610615620 
LeuThrAsnGluGlyHisAsnPheSerSerValSerSerArgAsnLys 
625630635640 
MetPheValGlnGlyAsnAsnGlyLysTyrValLeuAsnIleLeuThr 
645650655 
AspSerIleGluLeuProSerGlyGlnGlnThrIleLeuIleGlnAsn 
660665670 
ThrAsnSerGlnAspLeuPheLeuAspArgIleGluPheIleSerLeu 
675680685 
ProSerThrSerThrProThrSerThrAsnPheValGluProGluSer 
690695700 
LeuGluLysIleIleAsnGlnValAsnGlnLeuPheSerSerSerSer 
705710715720 
GlnThrGluLeuAlaHisThrValSerAspTyrLysIleAspGlnVal 
725730735 
ValLeuLysValAsnAlaLeuSerAspAspValPheGlyValGluLys 
740745750 
LysAlaLeuArgLysLeuValAsnGlnAlaLysGlnLeuSerLysAla 
755760765 
ArgAsnValLeuValGlyGlyAsnPheGluLysGlyHisGluTrpAla 
770775780 
LeuSerArgGluAlaThrMetValAlaAsnHisGluLeuPheLysGly 
785790795800 
AspHisLeuLeuLeuProProProThrLeuTyrProSerTyrAlaTyr 
805810815 
GlnLysIleAspGluSerLysLeuLysSerAsnThrArgTyrThrVal 
820825830 
SerGlyPheIleAlaGlnSerGluHisLeuGluValValValSerArg 
835840845 
TyrGlyLysGluValHisAspMetLeuAspIleProTyrGluGluAla 
850855860 
LeuProIleSerSerAspGluSerProAsnCysCysLysProAlaAla 
865870875880 
CysGlnCysSerSerCysAspGlySerGlnSerAspSerHisPhePhe 
885890895 
SerTyrSerIleAspValGlySerLeuGlnSerAspValAsnLeuGly 
900905910 
IleGluPheGlyLeuArgIleAlaLysProAsnGlyPheAlaLysIle 
915920925 
SerAsnLeuGluIleLysGluAspArgProLeuThrGluLysGluIle 
930935940 
LysLysValGlnArgLysGluGlnLysTrpLysLysAlaPheAsnGln 
945950955960 
GluGlnAlaGluValAlaThrThrLeuGlnProThrLeuAspGlnIle 
965970975 
AsnAlaLeuTyrGlnAsnGluAspTrpAsnGlySerValHisProHis 
980985990 
ValThrTyrGlnHisLeuSerAlaValValValProThrLeuProLys 
99510001005 
GlnArgHisTrpPheMetGluAspArgGluGlyGluHisValValLeu 
101010151020 
ThrGlnGlnPheGlnGlnAlaLeuAspArgAlaPheGlnGlnIleGlu 
1025103010351040 
GluGlnAsnLeuIleHisAsnGlyAsnPheAlaAsnGlyLeuThrAsp 
104510501055 
TrpThrValThrGlyAspAlaGlnLeuThrIlePheAspGluAspPro 
106010651070 
ValLeuGluLeuAlaHisTrpAspAlaSerIleSerGlnThrIleGlu 
107510801085 
IleMetAspPheGluGluAspThrGluTyrLysLeuArgValArgGly 
109010951100 
LysGlyLysGlyThrValThrValGlnHisGlyGluGluGluLeuGlu 
1105111011151120 
ThrMetThrPheAsnThrThrSerPheThrThrGlnGluGlnThrPhe 
112511301135 
TyrPheGluGlyAspThrValAspValHisValGlnSerGluAsnAsn 
114011451150 
ThrPheLeuIleAspSerValGluLeuIleGluIleIleGluGluMet 
115511601165 
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