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Applied and Environmental Microbiology, August 1999, p. 3714-3716, Vol. 65, No. 8
Department of Life Sciences, Ben-Gurion
University of the Negev, Be'er-Sheva 84105, Israel1; Institute of Zoology, Uzbek
Academy of Science, Tashkent 700095, Uzbekistan2; Institute of Zoology,
Kazakhstan Academy of Science, Almaty 480032, Kazakhstan3; and Institute of Systematic
and Animal Ecology, Siberian Branch of Academic Science,
Novosibirsk 6300091, Russia4
Received 3 March 1999/Accepted 18 May 1999
An extended PCR method was established to rapidly identify and
classify Bacillus thuringiensis strains containing
cry (crystal protein) genes toxic to lepidopteran,
coleopteran, and dipteran pests (Ben-Dov et al., Appl. Environ.
Microbiol. 63:4883-4890, 1997). To optimize identification of all
reported cry genes, this methodology needs a complete PCR
set of primers. In the study reported here, a set of universal (Un9)
and specific primers for multiplex rapid screening for all four known
genes from the cry9 group was designed. PCR analyses were
performed for cry9 genes on 16 standard strains and 215 field isolates of B. thuringiensis. Among the standard
strains, only B. thuringiensis subsp. aizawai HD-133, which harbors cry1 and cry2 genes, was
positive with Un9 but negative to all four specific primers for
cry9 genes. DNA of 22 field-collected isolates was also
found to be positive with Un9. These isolates were classified into
three cry9 profiles using specific primers; all of them
harbor cry1 and cry2. This newly designed set
of primers complements the existing PCR methodology for most currently
known cry genes.
The soil bacterium Bacillus
thuringiensis fulfills the requisites of a microbiological control
agent against agricultural pests and vectors of diseases that lead to
its widespread commercial application. It is a gram-positive, aerobic,
endospore-forming saprophyte (1, 18). All known subspecies
of B. thuringiensis produce large quantities of insecticidal
crystal proteins (ICPs) which are segregated in parasporal bodies (also
known as Identifying novel B. thuringiensis isolates by bioassays is
a long and exhaustive process which is impeded by repeated isolation of
the same strains (18). Prediction of insecticidal activity of an unknown strain by serotyping seems impossible because it does not
necessarily reflect the specific cry gene class(es) the strain(s) contains (1, 12). Alternatively, PCR requires
minute amounts of DNA and allows quick, simultaneous screening of many B. thuringiensis samples, identification and classification
of cry genes, and subsequent prediction of their
insecticidal activities (3-5, 7-10, 13, 15, 16, 19, 21).
Extended PCR methodology has recently been exploited to rapidly
identify and classify cry genes of many groups (3,
5). A complete set of primers is required to optimize
identification of all reported cry genes (18).
cry9 genes are promising tools for effective control
(14, 26) and resistance management (22) of many
agronomically important lepidopteran species of insect pests. For
example, expression of Cry9Ca in transgenic corn protected the plant
against the European corn borer (Ostrina nubilalis)
(14). Cry9Ca is significantly more toxic to budworm
(Choristoneura fumiferana) than the currently used Cry1A-F
toxins (26) and displays high toxicity against Plutella xylostella (susceptible as well as resistant
larvae), Spodoptera exigua, Spodoptera
littoralis, Heliothis virescens, Agrotis
segetum, and silkworm (Bombyx mori) (20,
26). Another toxin belonging to the Cry9 group is Cry9Aa, the
major crystal component of B. thuringiensis subsp.
galleriae, which exhibits unique toxicity toward
Galleria mellonella larvae (25). The cryptic gene
cry9Ba was found to be localized upstream of
cry9Aa (23). The fourth protein in this group,
Cry9Da, toxic to scarabaeid larvae of the order Coleoptera, was found
in B. thuringiensis subsp. japonensis (2,
27).
In this study, we developed a new set of universal and specific primers
for multiplex rapid screening of B. thuringiensis strains
that harbor any of the four currently known cry9 genes.
B. thuringiensis strains were isolated as described
previously (3) and selected for appearance of parasporal
inclusions by phase-contrast microscopy. One pair of universal
oligonucleotide primers (Un9) was selected from a highly conserved
region present in the four cry9 genes (extracted from the
GenBank database in accordance with the method outlined in reference
11) to amplify a specific fragment from
cry9 genes by using the program Amplify 1.0 (Bill Engels,
University of Wisconsin) (Fig. 1, lanes 2 to 4). Primer sequences, match and mismatch positions on each gene of
the group, and the expected sizes of their amplicons are presented in
Table 1. Sequences and match positions of
the four specific primers, selected from highly variable regions in the
known cry9 genes, are presented in Table
2. A mixture of the four specific primers
with the universal reverse primer [Un9(r)] was used for multiplex PCR
screening to identify cry9 genes by different sizes of their
PCR products (Table 2; Fig. 1, lanes 5 to 7).
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Copyright © 1999, American Society for Microbiology. All rights reserved.
Multiplex PCR Screening To Detect cry9
Genes in Bacillus thuringiensis Strains
ABSTRACT
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- endotoxins) (6). The genes encoding ICPs
normally occur on large plasmids and direct the synthesis of a family
of related proteins classified as cry1-28 and
cyt1-2 groups according to their degree of amino acid
homology (2a, 11).
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FIG. 1.
Agarose gel (1%) electrophoresis of PCR products
obtained with universal and specific primers for cry9
genes. Lanes 1 and 8, molecular weight markers ( 
DNA
cleaved by HindIII), with sizes (in kilobases) indicated
on left; lanes 2 to 4, respectively, DNA of field-collected isolates
U-27, K-74, and B. thuringiensis subsp. aizawai
HD-133 amplified with Un9; lanes 5 to 7, respectively, DNA of
field-collected isolates U-27, K-74, and B. thuringiensis
subsp. aizawai HD-133 amplified with a mixture of four
specific primers and one reverse Un9(r).
TABLE 1.
Characteristics of universal primers for cry9
group genesa
TABLE 2.
Characteristics of specific primers for cry9
genes
Amplification was carried out in a DNA MiniCycler (MJ Research, Inc., Watertown, Mass.) for 30 reaction cycles each. Reactions were routinely carried out in 25 µl; 1 µl of template DNA was mixed with reaction buffer, a 150 µM concentration of each deoxynucleoside triphosphate, a 0.2 to 0.5 µM concentration of each primer, and 0.5 U of Taq DNA polymerase (Appligene). Template DNA was denatured (1 min at 94°C) and annealed to primers (45 s at 56°C), and extensions of PCR products were achieved at 72°C for 50 s and 90 s for Un9 and specific primers, respectively. Each experiment was accompanied by a negative control (i.e., without DNA template).
Multiplex PCR screening for cry9 genes was performed on 16 B. thuringiensis standard strains previously used by us (3), as well as on 215 B. thuringiensis field isolates. Among the standard strains, only B. thuringiensis subsp. aizawai HD-133 yielded an amplicon with Un9 (Fig. 1, lane 4), though it was negative to the specific primers (lane 7). This strain contains cry2Ab (in addition to the four cry1 genes -Aa, -Ab, -Ca, and -Da [3, 10, 15]) and yielded a strong amplification product with universal primers for cry7 and cry8 (3). Another group (21) that claimed to have found cry2Ab in this strain only confirmed our previous finding (3). The low quality of their "degenerated family" primers for cry7 and cry8 (21) resulted in nonspecific amplicons (compare with reference 3). Another gene, cry1I (cryV in the old nomenclature), has also been found in this (21) and other B. thuringiensis subsp. aizawai strains (13, 24), and the product of this gene is known to be secreted into the medium in the early stationary phase (17).
A new gene, cry9Ea, has very recently been discovered in B. thuringiensis subsp. aizawai SSK-10 (2a) and should be recognized by Un9. Un9(d) hybridizes to nucleotides 2448 to 2471 with no mismatches, while Un9(r) hybridizes to nucleotides 2775 to 2798 with a single mismatch at residue 18. The resulting amplicon would be 351 bp in length. A new specific primer for cry9Ea should be designed to allow identification of cry9Ea in the strains that were positive to Un9 (see the Addendum in Proof).
Of the field-collected isolates, 22 yielded positive results with Un9
(Table 3). These were screened further
for the presence of four cry9 genes. Three different
cry9 gene profiles were found which contained also several
combinations of cry1 and cry2 (Table 3). Fifteen
isolates contained cry9Aa and cry9Ba (Fig. 1,
lane 5) and two contained only cry9Da (lane 6), whereas five
did not yield an amplicon by PCR with any of the four specific
cry9 primers tested. None of our field-collected isolates
contained cry9Ca.
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It is interesting to note that the specific primer EB-9B(d) nonspecifically amplified a cry9Da fragment of 1,534 bp (Fig. 1, lane 6). Alignment analysis discovered that it anneals with low binding strength to bases 1158 to 1181 in the cry9Da coding sequence. Increasing the temperature to 60 to 62°C can prevent this nonspecific annealing.
The recent report by Bravo et al. (5) on an expanded set of general and specific primers includes a set for detecting three genes of the cry9 group (excluding cry9Da). At least one of these specific primers (spe-cry9C), corresponding to bases 1853 to 1868 (yielding an amplicon of 306 bp), is predicted to nonspecifically anneal also to bases 1961 to 1976 in cry9Ca (to amplify a fragment of 198 bp); it may thus interfere with amplification of the 306-bp fragment of cry9Ca. In addition, spe-cry9C is predicted to anneal nonspecifically both directly and in the reverse direction to cry9Ca and cry9Aa, thus giving rise to further nonspecific amplifications.
Bravo et al. (5) detected cry9 genes in 2.6% of their B. thuringiensis strain collection, whereas we found them in 10.2% of our collection (Table 3). This apparent difference in frequencies may reflect a real difference in prevalence of cry9 genes between the Latin American and Asian collections. It may however be due to the fact that in addition to a set of four specific primers we used a pair of universal primers (Un9) which amplifies all five cry9 genes (and also potentially other unknown genes of this family).
Our screening procedure identified five field-collected B. thuringiensis isolates positive to Un9 but not to any of our specific primers for four cry9 genes. This may indicate that these isolates contain new cry9 genes. They may be potential biological control agents against insect pests.
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ACKNOWLEDGMENTS |
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This investigation was supported by INTAS project no. 96-1490, U.S.-Israel Cooperative Development Research Program, U.S. Agency for International Development Grant no. TA-MOU-CA13-067, and by a post-doctoral fellowship (to E.B.-D.) from the Israel Ministry of Science.
Gideon Raziel is gratefully acknowledged for producing the picture.
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ADDENDUM IN PROOF |
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The new specific primer EB-9E(a), 5'-GCGGCTGGCTTTACTTTACCGAG-3', designed to identify cry9Ea by hybridization to nucleotides 1975 to 1977, amplified PCR product of 824 bp with Un9(r). B. thuringiensis subsp. aizawai HD-133 and four of the five field-collected isolates (positive to Un9) yielded an amplicon specific to cry9Ea.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Life Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Be'er-Sheva 84105, Israel. Phone: 972-7-6461.340 or 972-7-6472.642. Fax: 972-7-6472.890. E-mail: yoelm{at}bgumail.bgu.ac.il.
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Applied and Environmental Microbiology, August 1999, p. 3714-3716, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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