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CURRENT MICROBIOLOGY Vol. 41 (2000), pp. 65– 69
DOI: 10.1007/s002840010093
Current
Microbiology
An International Journal
© Springer-Verlag New York Inc. 2000
Cloning of a New Bacillus thuringiensis cry1I-Type Crystal
Protein Gene
Soo-Keun Choi,1 Byung-Sik Shin,1 Eun-Mee Kong,1 Hyune Mo Rho,3 Seung-Hwan Park2
1
Laboratory of Microbial and Bioprocess Engineering, Korea Research Institute of Bioscience & Biotechnology (KRIBB), P.O. Box 115,
Yusong, Taejon 305-600, Korea
2
Genome Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), P.O. Box 115, Yusong, Taejon 305-600,
Republic of Korea
3
Department of Molecular Biology and Research Center for Cell Differentiation, Seoul National University, Seoul, 151-742, Republic of Korea
Received: 19 January 2000 / Accepted: 22 February 2000
Abstract. A new cry1I-type gene, cry1Id1, was cloned from a B. thuringiensis isolate, and its nucleotide
sequence was determined. The deduced amino acid sequence of Cry1Id1 is 89.7%, 87.2%, and 83.4%
identical to the Cry1Ia, Cry1Ib, and Cry1Ic proteins, respectively. The upstream sequence of the cry1Id1
structural gene was not functional as promoter in B. subtilis. The Cry1Id1 protein, purified from
recombinant E. coli cells, had a toxicity comparable to that of Cry1Ia against Plutella xylostella, but it
was significantly less active than Cry1Ia against Bombyx mori. Cry1Id1 was not active against the
coleopteran insect, Agelastica coerulea.
Commercial products of B. thuringiensis strains have
been used as alternatives to chemical insecticides in the
control of various insect pests. However, reports of the
occurrence of insect resistance to Cry proteins call for
the strategies of resistance management [11]. It has been
reported that the insect resistance was due to the reduction of the binding affinity of the active toxin to the
membrane receptor in insect midgut [18]. This fact has
led many researchers to screen new cry genes because
the alternating use of two biochemically unrelated toxins
is expected to significantly reduce the rate of resistance
development.
Many of the genes encoding the crystal proteins
have been cloned and sequenced. They have been classified cry1 to cry28 and cyt according to the degree of
amino acid sequence homology [1]. In this study, we
observed a B. thuringiensis isolate having a new cry1Itype gene utilizing DNA dot blot hybridization and PCR.
We have cloned the cry1I-type gene cry1Id1 and determined its nucleotide sequence. The toxicities of Cry1Id1
purified from recombinant E. coli cells against the larvae
of Plutella xylostella, Bombyx mori, and Agelastica coerulae were also described.
Correspondence to: S.-H. Park; e-mail: [email protected]
Materials and Methods
Bacterial strains and plasmids. B. thuringiensis isolate BR30 was
previously isolated from the phylloplane in Korea [12]. E. coli DH5␣
was used for DNA manipulation and toxin expression. Plasmids
pGEM威-7Zf(⫹) for cloning and pKK223-3 for construction of expression vector were purchased from Promega Co. and Amersham Pharmacia Biotech, respectively. Plasmid pDG1728 was obtained from P.
Stragier.
Nucleic acid hybridization. The cry1I-specific primers, cry5A and
cry5B, and a cry1I-specific probe were described previously [15].
Total DNA from B. thuringiensis cells was extracted by the method
of Kalman et al. [8]. DNA dot blot and Southern hybridizations were
performed according to the method of Shin et al. [15]. Colony
hybridization was performed with DIG Nucleic acids detection kit
(Boehringer Mannheim Biochemicals) as recommended by the manufacturer.
Sequencing. Nested deletion mutants for sequencing were obtained
by the unidirectional progressive deletion method with exonuclease
III [7]. Sequencing by the Sanger dideoxy-mediate chain termination method was performed with a Dig Taq DNA sequencing kit
(Boehringer Mannheim Biochemicals) as recommended by the manufacturer.
Insect toxicity assays. The preparation of the inclusion bodies from
recombinant E. coli cells and insect toxicity assays against larvae of
diamondback moth (Plutella xylostella), silkworm (Bombyx mori),
and alder leaf beetle (Agelastica coerulea) were followed as previously described by Shin et al. [15]. Bioassays were repeated three
times.
66
Results and Discussion
During screening for new cry genes from B. thuringiensis strains isolated from plant leaves in Korea by DNA
dot blot hybridization and PCR, we found a B. thuringiensis isolate, BR30, suggested to contain a new cry1Itype gene. The total DNA of this isolate responded
positively to a cry1I-specific probe by DNA dot blot
hybridization, but did not generate cry1I gene fragments
by PCR. Generally, the results of DNA dot blot analysis
reflect a degree of homology throughout the region hybridized to the specific probe, whereas the results of PCR
reflect only a homology at the primer binding site. Thus,
this result indicated that B. thuringiensis isolate BR30
carried cry1I-type genes differing from known cry1I
genes, at least in the binding sites of the primers cry5A
and/or cry5B. From the strain BR30, we have cloned a
new cry1I-type gene, cry1Id1.
When total DNA of B. thuringiensis BR30, digested
with various restriction enzymes, was analyzed by
Southern hybridization with a cry1I-specific probe, a
4-kb EcoRI fragment was detected (data not shown). The
4-kb EcoRI fragment was cloned into the plasmid
pGEM威-7Zf(⫹) by colony hybridization. Partial sequence analysis of the 4-kb EcoRI fragment revealed that
it contained the N-terminus half of the cry1I-type gene.
Further Southern hybridization with the same probe
showed that a 2.3-kb HindIII fragment contains the Cterminal part of the gene, and it overlapped 1 kb with the
4-kb EcoRI fragment. The 2.3-kb HindIII fragment was
also cloned into the plasmid pGEM威-7Zf(⫹), and the
full cry1I-type gene was obtained by annealing two fragments by using SpeI site located in the overlapping
region. The resulting plasmid was designated to pSK27.
The nucleotide sequence of the cloned fragment contains
an open reading frame, designated cry1Id1, encoding a
719-amino acid protein having a predicted molecular
weight of 81.4 kDa (Fig. 1). Alignment of the deduced
amino acid sequence of the Cry1Id1 with known crystal
proteins revealed that the Cry1Id1 protein is 89.7% identical to the Cry1Ia, 87.2% to the Cry1Ib, and 83.4% to
the Cry1Ic protein. The Cry1Id1 protein contains five
conserved blocks and truncated block 6 identified by
Schnepf et al. [13]. The potential ribosome binding site is
located 7 base pairs upstream of the translation start site
of the cry1Id1 gene, and the inverted repeat structure was
found downstream of the cry1Id1 structural gene. The
cry1I-type genes have been found to be located 0.5 kb
downstream of the cry1 genes, and this cry1– cry1I linkage seems to be very common in B. thuringiensis strains
[15]. But the cry1Id1 gene is not located closely downstream of the cry1 gene. The plasmid pSK27 contains the
2.9-kb upstream region from the cry1Id1 structural gene.
CURRENT MICROBIOLOGY Vol. 41 (2000)
Partial sequence analysis of the upstream region of
cry1Id1 gene showed that a cry1-type gene is located in
the 2.5-kb upstream of the cry1Id1 gene (Fig. 2).
Although it has been discussed that cry1I-type genes
are silent in B. thuringiensis [4, 15], a recent report
showed that Cry1I-type protein (CGCryV) is expressed
early in the stationary phase and the protein is secreted
into the media [9]. For the identification of upstream
promoter activities of the cry1Id1 gene, we constructed a
cry1Id1⬘-lacZ transcriptional fusion using the plasmid
pDG1728 [6] and measured ␤-galactosidase activities in
B. subtilis JH642 cultured at 37°C in DS medium [14].
The fused region of cry1Id1 was prepared by PCR with
primers
nr5P3
(5⬘-CGAATTCATGTAAGAGACCCCCTT-3⬘) and nr5P4 (5⬘-CCCAAGCTTACTAACAAACGGCTCTACAC-3⬘), and covered nucleotide
⫺542 to ⫹167 with respect to the translational start site
of cry1Id1 (nucleotide position ⫹1) (Fig. 2). The PCR
product was confirmed by sequencing and cloned into
the EcoRI and HindIII sites of the plasmid pDG1728 to
construct plasmid pSK35. The plasmid pSK35 was introduced into B. subtilis JH642. Time course analysis of
lacZ gene expression showed that the enzyme activities
were background levels, below 4 Miller units, at vegetative and through the sporulation stage (data not shown).
This result suggests that the cry1Id1 gene is silent or very
weakly expressed in B. subtilis.
For the overexpression of the cry1Id1 gene in E.
coli, we cloned the gene into the plasmid pKK223-3 to
allow that the cry1Id1 gene was under the control of tac
promoter. A 309-bp fragment containing the N-terminus
of cry1Id1 gene was amplified by PCR with primers
nr5a(5⬘-TCCCCCGGGATGAAATCAAAGAATCAAA
A-3⬘) and nr5b (5⬘-CCTTTGGGCCAAAGCTCACC3⬘), and substituted for the N-terminus of cry1Id1 gene
by using SmaI and NheI sites generating plasmid pSK28.
The PCR-amplified fragment was confirmed by sequencing. The plasmid pSK28 was digested with SmaI and
NsiI, and the DNA fragment containing cry1Id1 gene
was cloned into the SmaI and PstI sites of plasmid
pKK223-3 to construct plasmid pSK29. Recombinant E.
coli DH5␣ cells harboring the plasmid pSK29 produced
inclusion bodies without the addition of isopropyl-␤-Dthiogalactopyranoside when they were grown overnight
at 37°C in LB medium containing ampicillin (50 ␮g
ml⫺1). We prepared the inclusion bodies from recombinant E. coli cells and performed insect toxicity assays
against larvae of the diamondback moth (Plutella xylostella), silkworm (Bombyx mori), and alder leaf beetle
(Agelastica coerulea). According to the analyses of crystal structures of Cry1Aa and Cry3Aa toxins [5, 10],
domain II is involved in recognition and binding to cell
surface receptors. In particular, the mutations in se-
S.-K. Choi et al.: Cloning of a New Bacillus thuringiensis cry1I-Type Gene
67
Fig. 1. Nucleotide sequence of the cry1Id1 gene (GenBank accession number AF047579) and deduced amino acid sequence of Cry1Id1 protein. The
box indicates the ribosome binding site. The inverted repeats are marked with long arrows below the sequence. Conserved amino acid blocks for
the known crystal proteins are in boldface. The Cry1Id1 amino acid sequence was aligned with those of Cry1Ia and Cry1Ib proteins; only the
differences in the open reading frame are shown.
68
CURRENT MICROBIOLOGY Vol. 41 (2000)
Fig. 2. Restriction map of cry1Id1
compared with other cry1Igenes.
Black boxes indicate open
reading frames of the genes.
The numbers indicate the
distance between cry1I genes
and other cry1 genes located
in the upstream region of the
cry1I genes. The dashed box
indicates the DNA region
used for lacZ transcriptional fusion.
Table 1. Toxicity of Cry1Ia3 and Cry1Id1 proteins produced by
E. coli recombinant cells
LC50a (␮g/ml) against
Proteins
Bombyx mori
Plutella xylostella
Agelastica
coerulea
Cry1Ia3
Cry1Id1
7.08 (4.11–10.77)
439.56 (214.28–709.12)
2.57 (0.88–5.32)
4.26 (2.44–7.47)
⬎1,900
⬎960
a
95% confidence intervals are shown in parentheses.
quences encoding loop regions of domain II had either a
negative or positive effect on receptor binding and toxicity [13]. The analysis of the deduced amino acid sequence of the Cry1Id1 protein showed that domain II
(residues 294 – 492) is 97% identical to that of the
Cry1Ia3 protein. Moreover, the amino acid sequences
corresponding to three loops (loop 1, residues 349 –355;
loop 2, residues 409 – 419; loop 3, residues 480 – 483)
proposed by Tabashnik et al. [16] in domain II of
Cry1Id1 are perfectly matched with those of Cry1Ia3.
This enabled us to predict that the insecticidal spectrum
of the Cry1Id1 protein might be similar to that of the
Cry1Ia3 protein. Bioassay results showed that the toxicity of the Cry1Id1 protein was comparably to that of
Cry1Ia3 against P. xylostella. However, counter to expectation, Cry1Id1 was significantly less active than
Cry1Ia3 against B. mori (Table 1). It has been reported
that the domain III of Cry protein might also participate
in receptor binding [2, 3]. The domain III (residues
501– 642) of Cry1Id1 shares 90.1% homology with that
of Cry1Ia3. Therefore, a possible explanation of the
bioassay result was that the difference in the toxicity
between Cry1Id1 and Cry1Ia3 against B. mori might
originate from domain III of each toxin.
Cry1Id1 did not show toxicity against a coleopteran
insect, Agelastica coerulea. Similarly, other groups reported that Cry1I-type proteins were active solely against
lepidopteran insects [4, 9, 15], even though the Cry1I
protein was discovered to possess a property of dual
insecticidal activity against lepidopterans and coleopterans [17].
ACKNOWLEDGMENTS
We are grateful to P. Stragier for the generous gift of plasmid
pDG1728. This study was supported by a grant (HS2720) from the
Ministry of Science and Technology of Korea.
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