APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2008, p. 6997–7001
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 22
New Strategy for Isolating Novel Nematicidal Crystal Protein Genes
from Bacillus thuringiensis Strain YBT-1518?
Suxia Guo,1Mei Liu,1,2Donghai Peng,1Sisi Ji,1Pengxia Wang,1Ziniu Yu,1and Ming Sun1*
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology,1and College of Veterinary Medicine,2
Huazhong Agricultural University, Wuhan 430070, Hubei, People’s Republic of China
Received 16 June 2008/Accepted 19 September 2008
We have developed a strategy for isolating cry genes from Bacillus thuringiensis. The key steps are the
construction of a DNA library in an acrystalliferous B. thuringiensis host strain and screening for the formation
of crystal through optical microscopy observation and sodium dodecyl sulfate-polyacrylamide gel electrophore-
sis (SDS-PAGE) analyses. By this method, three cry genes—cry55Aa1, cry6Aa2, and cry5Ba2—were cloned from
rice-shaped crystals, producing B. thuringiensis YBT-1518, which consists of 54- and 45-kDa crystal proteins.
cry55Aa1 encoded a 45-kDa protein, cry6Aa2 encoded a 54-kDa protein, and cry5Ba2 remained cryptic in strain
YBT-1518, as shown by SDS-PAGE or microscopic observation. Proteins encoded by these three genes are all
toxic to the root knot nematode Meloidogyne hapla. The two genes cry55Aa1 and cry6Aa2 were found to be
located on a plasmid with a rather small size of 17.7 kb, designated pBMB0228.
Bacillus thuringiensis is a gram-positive, spore-forming bac-
terium that produces crystal inclusions during the sporulation
phase. The crystals comprise one or more Cry proteins (?-
endotoxins) that are specifically toxic to insect orders such as
Lepidoptera, Diptera, and Coleoptera and also to some nema-
todes, mites, and protozoa (23). The insecticidal Cry proteins
are encoded by cry genes. Since the cloning of the first cry gene
by Schnepf and Whiteley (24), more than 300 cry genes have
been isolated from B. thuringiensis (refer to Crickmore et al. 
and the toxin nomenclature website http://www.lifesci.sussex.ac
.uk/home/Neil_Crickmore/Bt/toxins2.html). These Cry pro-
teins are classified into families Cry1 to Cry54 on the basis of
their amino acid sequence homology. Among these 54 families,
Cry5, Cry6, Cry12, Cry13, Cry14, and Cry21 have been shown
to display nematicidal activity.
Agriculturally important nematodes usually live under-
ground (29), which makes them difficult to control using tra-
ditional B. thuringiensis insecticides. One of the most effective
approaches for controlling plant-parasitical nematodes has
been constructing transgenic plants with nematicidal cry genes.
However, most of the nematicidal cry genes have been de-
scribed only in patents with sparse data (28); except for Cry6,
most of the nematicidal Cry proteins are large proteins (90 to
140 kDa) that are difficult to use in transgenic manipulations.
It is therefore imperative that more-detailed studies be carried
out on the cloning of nematicidal cry genes, especially those
whose products have small molecular weights.
Typically, cry genes have been cloned by constructing B.
thuringiensis DNA libraries for Escherichia coli and then
screening a great number of colonies by Western blotting (20,
24) or a Southern hybridization-based method (4, 13, 15, 19).
Unfortunately, these methods require large amounts of Cry
proteins in order to prepare the antibody or to facilitate amino
acid sequencing, thus making it impossible to isolate cryptic or
silent cry genes. More recently, cry genes have been screened
with PCR-based methods (5, 6, 9, 14, 27), which are very rapid
and effective in principle. However, these methods depend
upon there being a high similarity between different cry genes;
in using these methods, novel cry genes that have little or no
identity to known genes would be lost. Furthermore, genes
with little or no identity in the primer regions would not be
amplified using this strategy. New strategies for identifying
such novel cry genes have yet to be developed.
Most B. thuringiensis cry genes reside on large plasmids (23).
The only exception to date was reported by Loeza-Lara et al.
in 2005 (17): a cry-like gene, cry14-4, found in a small plasmid,
pBMBt1, from a B. thuringiensis strain. The predicted protein
sequence showed low identity with the proteins CryC53
(24.6%) and Cry15Aa (27.8%). Apart from the coding se-
quence, there is no detailed experimental evidence available
regarding the Cry14-4 protein; whether other small plasmids
from B. thuringiensis harbor cry genes or not remains uncertain.
This work presents a strategy for isolating new cry genes by
screening a plasmid library in an acrystalliferous B. thuringien-
sis host strain. This method enables the isolation of the cry
genes cry55Aa1, cry6Aa2, and cryptic cry5Ba2 from the YBT-
1518 strain, which produces rice-shaped crystals and displays
nematicidal activity. Of these three genes, the first two were
found to be located on a 17.7-kb plasmid.
MATERIALS AND METHODS
Bacterial strains, plasmids, and cultural conditions. The bacterial strains and
plasmids used in this study are listed in Table 1. The nematicidal B. thuringiensis
strain YBT-1518 was isolated in China (30). The E. coli mutant strain DH10B
and the acrystalliferous “B. thuringiensis subsp. kurstaki” strain BMB171 from Li
and Yu (16) were used as the intermediate and final hosts, respectively, in the
plasmid library construction. The cloning vector was the E. coli-Bacillus shuttle
vector pHT304 (4). B. thuringiensis was grown at 28°C and E. coli at 37°C.
Plasmid isolation and DNA manipulation. Plasmids were extracted from B.
thuringiensis according to the procedure of Andrup et al. (2) and from E. coli
* Corresponding author. Mailing address: State Key Laboratory of
Agricultural Microbiology, College of Life Science and Technology, Hua-
zhong Agricultural University, Wuhan 430070, Hubei, People’s Republic
of China. Phone: 86-27-87283455. Fax: 86-27-87280670. E-mail: m98sun
?Published ahead of print on 26 September 2008.
according to the methods of Sambrook and Russell (22). The extraction of DNA
from gel was performed using an AxyPrep DNA gel extraction kit (Axygen
Scientific, Inc.). The DNA restriction and ligation operations were performed
according to the methods of Sambrook and Russell (22). The preparation of the
cloning vector pHT304 and determination of the insert size were carried out as
described by Luo and Wing (18).
Southern hybridization. DNA samples were separated in 0.8% agarose gel and
transferred onto a nylon membrane, according to the procedure of Sambrook
and Russell (22). The PCR-amplified probe labeling, hybridization, and detec-
tion of the result were all performed according to the insert of the DIG High
Prime DNA labeling and detection starter kit I (Roche Applied Science, Ger-
Sequence analysis. Open reading frame prediction was performed using the
CLC Free Workbench 4 program (CLC bio, Denmark). DNA and protein
sequence homology searches were performed using the BLAST algorithm (1)
against the GenBank database.
Electroporation of E. coli and B. thuringiensis. E. coli DH10B electroporation-
competent cells were prepared according to the procedure of Sambrook and
Russell (22); the transformation procedure was performed according to the
method of Luo and Wing (18). B. thuringiensis BMB171 transformation was
according to the modified method of Silo-Suh et al. (26). The transformants were
selected on LB (22) agar plates with erythromycin (25 ?g/ml).
Microscopy observation. An optical microscope was used to observe crystal
formation. Bacteria were cultivated on CCY agar plates containing casein hy-
drolysate and yeast for more than 72 h at 28°C (9); the cells were then stained
with basic fuchsin by a simple staining procedure and observed with an oil
immersion lens. Initially, 10 colonies per group were observed together, and then
every colony in the crystal-producing groups was observed separately. For scan-
ning electron microscopy observation, the bacteria were treated by following the
methods of Shao et al. (25).
SDS-PAGE and crystal protein purification. Sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis (SDS-PAGE) was used to examine the Cry pro-
teins from the positive colonies. For crystal protein purification, bacteria were
cultivated in liquid ICPM medium (25) for 36 h at 28°C with erythromycin (25
?g/ml). The crude spore lysate pellets were then treated using the method of
Griffitts et al. (10). The purified proteins were treated and loaded as described by
Shao et al. (25).
Nematode toxicity bioassay. The purified Cry proteins were used for bioassay.
Meloidogyne hapla eggs were harvested from the root knot of infected tomato,
and the second-stage juveniles were reared at 18 to 25°C and used to test the
toxicity of the Cry toxin. The bioassay procedure and 50% lethal concentration
(LC50) evaluation were undertaken according to the method of Yu et al. (30).
Nucleotide sequence accession numbers. The nucleotide sequences published
in this paper have been submitted to GenBank and assigned accession numbers
EU121521 (cry55Aa1), AF499736 (cry6Aa2), and EU121522 (cry5Ba2).
Construction of a library of plasmid DNA from strain YBT-
1518 in BMB171. B. thuringiensis strain YBT-1518 produces
rice-shaped crystals (Fig. 1A) and has at least three plasmids
(Fig. 1B). The plasmid DNA from YBT-1518 was isolated and
partially digested with HindIII. The resulting 5- to 12-kb frag-
ments were extracted and ligated into pHT304. The ligation
mix was transformed into E. coli DH10B. Plasmids present in
the recombinant E. coli pool were then extracted and trans-
ferred into the acrystalliferous B. thuringiensis strain BMB171
by electroporation, resulting in a library of strain YBT-1518
The average size of the inserts in the library was about 8 kb.
Approximately 1,000 recombinants were stored in the library,
of which 300 were first randomly selected for the screening
process described below.
Screening recombinants from the library harboring cry
genes. Each of the 300 recombinants selected in the previous
step was observed under an optical microscope to determine
whether it formed crystals. Seven recombinants were found to
produce the same rice-shaped crystal as that in strain YBT-
1518. SDS-PAGE analysis revealed that three of them could
produce a 45-kDa crystal protein (for example BMB0222 and
BMB0223) and that four of them could produce a 54-kDa
protein (such as BMB0249). There was one recombinant,
BMB0215, that produced a bipyramidal crystal (Fig. 2) com-
prising a 140-kDa protein (Fig. 3).
TABLE 1. Bacterial strains and plasmids used in this study
Strain Organism Plasmid Characteristic Cry protein production Source
Stored in our lab
Li and Yu (16)
Stored in our lab
FIG. 1. Crystal morphology and plasmid profile of YBT-1518.
(A) Crystal morphology of YBT-1518. Abbreviations: C, crystal; S,
Spore. Spores and crystals from strain YBT-1518 were harvested and
washed three times with 1 M NaCl and then three times with water.
The spore and crystal mix was resuspended in water and spread on a
microscope slide to be ion coated; it was then observed under a scan-
ning electron microscope (JEOL JSM-6390LV). The bar is 1 ?m.
(B) Plasmid profile of YBT-1518 in 0.8% agarose gel. Lane M, DNA
molecular mass ? DNA/HindIII markers; lane 1, plasmids from B.
thuringiensis HD-2 as size references; lane 2, plasmids from YBT-1518.
6998 GUO ET AL.APPL. ENVIRON. MICROBIOL.
Identification of cry55Aa1, cry6Aa2, and cry5Ba2. The anal-
ysis of a 45-kDa protein-producing recombinant, BMB0223,
showed that the recombinant plasmid it contained, pBMB0223,
possessed a 6.7-kb HindIII insertion fragment. The fragment was
predicted to bear six potential open reading frames (ORFs),
among which orf2 encoded a hypothetical protein with a molec-
ular mass of 40 kDa and a pI of 5.15. Significantly, the first 15
N-terminal sequence of this putative protein was identical to the
45-kDa crystal protein from strain YBT-1518, which was previ-
ously determined (data not shown). A 2.1-kb HindIII-HpaI frag-
ment containing only orf2 was then subcloned into pHT304, re-
sulting in pBMB0224, and transferred to BMB171, resulting in
BMB0224. The strain BMB0224 formed rice-shaped crystals (Fig.
2) consisting of a 45-kDa protein (Fig. 3). A BLASTP homology
search revealed that the putative Cry protein encoded by orf2 did
not bear any homology to known crystal proteins in the GenBank
database. The proposed gene was designated cry55Aa1 by the B.
thuringiensis ?-endotoxin nomenclature committee.
The plasmid pBMB0249 harbored in the 54-kDa crystal pro-
tein-producing recombinant strain BMB0249 contained an in-
sertion with three HindIII fragments (4.2, 2.4, and 2.9 kb).
When the 2.9-kb HindIII fragment was subcloned into
pHT304, the resulting recombinant strain, BMB0250, formed
rice-shaped crystals (Fig. 2) consisting of 54-kDa proteins (Fig.
3). The cry gene in this fragment was identical to the cry6Aa2
crystal gene that we have described previously (30).
One recombinant strain, BMB0215, was found to produce a
bipyramidal crystal. The plasmid pBMB0215 harbored a 4.6-kb
HindIII fragment including an ORF that encoded a 140-kDa
protein displaying 99.99% identity to the known protein
Cry5Ba1. The gene was designated cry5Ba2. The presence of
the cry5Ba2 gene in wild-type strain YBT-1518 was confirmed
by PCR amplification (data not shown). Since the production
of the bipyramidal crystal and the 140-kDa crystal protein
could not be detected in YBT-1518, the cry5Ba2 gene may be
cryptic in this strain.
Localization of the 45- and 54-kDa protein genes. Alignment
of the relevant DNA sequences showed that there was a 4.2-kb
overlap between the insert in pBMB0222 carrying the cry55Aa1
gene and the insert in pBMB0249 carrying the cry6Aa2 gene,
suggesting that both cry genes may be located in close proxim-
ity within the genome. In support of this, an E. coli recombi-
nant strain isolated from a routine DNA library of strain YBT-
1518, EMB0228, was found to harbor a plasmid, designated
pEMB0228, with a 17.7-kb insert fragment that contained the
complete inserts from pBMB0222 and pBMB0249 within its
Sequence alignments revealed the presence in pBMB0249 of
features typical of rolling circle plasmids like pTX14-3 (3),
such as replication-associated protein, dso (double-stranded
origin), and sso (single-stranded origin) sequences, suggesting
that the plasmid harboring the two cry genes may not be very
large. To obtain the possible remaining sequence of this plas-
mid not represented in the insertion fragment in plasmid
pEMB0228, PCR was performed using primers designed ac-
cording to the end sequence of the insertion fragment borne by
pEMB0228. The sequence alignment of the PCR product
frag0228 (Fig. 4) showed that, in YBT-1518, there was no gap
between the two HindIII sites at the end of pEMB0228.
The above data suggested that the 17.7-kb insert in
pEMB0228 contained the entire plasmid bearing the cry55Aa1
and cry6Aa2 genes in strain YBT-1518. The native plasmid
harboring the two genes was designated pBMB0228. Southern
hybridization was performed against the plasmids from strain
YBT-1518 with a digoxigenin-labeled probe corresponding to
nucleotides 482 to 1032 of the cry55Aa1 gene to check whether
the plasmid pBMB0228 was indeed present and whether it
corresponded in size with that in the native strain. The result
showed that this probe hybridized to the smallest detectable
plasmid, with an estimated size of 18 kb, which is consistent
with the predicted size of 17.7 kb in strain YBT-1518 (Fig. 5).
Sequence alignment showed that, besides having the two cry
genes and pTX14-3-like rolling circle plasmid features,
pBMB0228 encoded two Mob proteins (Fig. 4) and a RepL-
FIG. 2. Optical microscope graphs of parasporal crystal proteins from the wild-type strain B. thuringiensis YBT-1518 and the recombinant
strains BMB0224, BMB0250, and BMB0215. (A) YBT-1518; (B) BMB0224 (harboring the 45-kDa protein gene cry55Aa1); (C) BMB0250
(harboring the 54-kDa protein gene cry6Aa2); (D) BMB0215 (harboring the 140-kDa protein gene cry5Ba2). Magnification, ?1,000.
FIG. 3. SDS-PAGE analysis of crystal proteins from the wild-type
strain and recombinant strains of B. thuringiensis. Lane M, molecular
mass standards; lane 1, YBT-1518; lane 2, BMB0224 (harboring the
45-kDa protein gene cry55Aa1); lane 3, BMB0250 (harboring the 54-
kDa protein gene cry6Aa2; lane 4, BMB0215 (harboring the 140-kDa
protein gene cry5Ba2).
VOL. 74, 2008 NEMATICIDAL CRYSTAL PROTEIN GENES FROM B. THURINGIENSIS 6999
like protein having the conserved domain of the RepL family
of plasmid replication proteins characterized for the Firmi-
cutes. This unusual collection of features suggested that
pBMB0228 was not an ordinary plasmid, by B. thuringiensis
Toxicity to root knot nematode Meloidogyne hapla. Since the
three cry genes identified in this study all originated from a
nematicidal strain of B. thuringiensis (30) and cry5B is a known
nematicidal protein gene (28), we examined the toxicity of
their protein products against the root knot nematode Meloi-
dogyne hapla. Three kinds of Cry proteins—Cry55Aa1 protein
from strain BMB0224, Cry6Aa2 from strain BMB0250, and
Cry5Ba2 from strain BMB0215—were prepared and assayed in
parallel against a second-stage juvenile of Meloidogyne hapla.
As a result, all three proteins were found by the bioassay to be
toxic to Meloidogyne hapla, with LC50s of 23.2 ?g/ml, 23.9
?g/ml, and 18.1 ?g/ml, respectively (Table 2).
In this study, we screened cry genes for the formation of a
crystal from a B. thuringiensis plasmid library. Compared to
previous methods for screening E. coli libraries (4, 8, 13, 15, 19,
20, 24) or PCR amplification (5, 6, 9, 27), this strategy is
advantageous in terms of isolating cry genes that have little
identity to known genes or to some cryptic genes with func-
tional promoters. In this process, the high electroporation ef-
ficiency of B. thuringiensis BMB171 played an important role.
By modifying the method of Silo-Suh et al. (26), the electro-
poration efficiency reached 8 ? 104CFU ?g?1ml?1, while
29.1-kb plasmids were transferred to BMB171; for the small
6.5-kb plasmids, the efficiency was 5 ? 109CFU ?g?1ml?1
(21). In addition, the shuttle vector pHT304 was used for
transfer to the intermediate host, E. coli DH10B, for convert-
ing the ligation mix to covalently closed circular plasmids,
making the recombinant plasmids much more likely to be
transferred (22); use of the method of Luo and Wing (18)
made it possible to produce a highly efficient vector. All these
crucial technical points of the protocol made the DNA library
construction in B. thuringiensis possible.
By applying the method described above, we cloned the cry
genes cry55Aa1 and cry6Aa2 and the cryptic cry5Ba2 gene from
the B. thuringiensis rice-shaped crystal strain YBT-1518. Bio-
assay data showed that all three proteins displayed effective
toxicity to the nematode Meloidogyne hapla, which causes tre-
mendous crop damage throughout the world (29). In this con-
text, the 45-kDa protein gene cry55Aa1, which showed no iden-
tity to other known crystal protein genes, is particularly
interesting, because the small molecular weight of its protein
makes it a prime candidate for use in the creation of nema-
tode-resistant transgenic plants.
Unlike with the previous report (23), in our study, the two
typical cry genes, cry55Aa1 and cry6Aa2, were found to be
located on a plasmid of uncharacteristically modest size (17.7
kb) designated pBMB0228 (Fig. 4). The plasmid harbors two
Rep-like protein genes—rep14-3-like and repL-like, which play
critical roles in its replication (12)—and two Mob protein
genes, both of which have significant similarity with Mob14-4,
which is necessary for plasmid pBMbt1 mobilization (17). The
apparent functional redundancies of the replication and mo-
bilization elements could be explained as a result of a recom-
bination process that fuses two plasmids into one in the evo-
FIG. 4. Physical map of plasmid pBMB0228, harboring both
cry55Aa2 and cry6Aa2. Genes are indicated by arrows. The filled ar-
rows indicate the genes with known functions, and the open arrows
indicate genes having unknown functions. The filled box indicates the
position of the PCR fragment frag0228, while the open boxes indicate
the predicted positions of dso (double-stranded origin) and sso (single-
stranded origin). The rep14-3-like and repL-like genes are the putative
replication initiator protein genes, while two mob-like genes contribute
to the mobilization of this plasmid. R2 is a negative-regulator gene of
cry6Aa2, referred to as orf2 by Yu et al. (30).
FIG. 5. Detection of plasmid pBMB0228. (A) Plasmid profile by
0.8% agarose gel electrophoresis. (B) Southern hybridization, detect-
ing the existence of pBMB0228 in the native strain YBT-1518. The
PCR fragment corresponding to nucleotides 482 to 1032 of the
cry55Aa1 gene was used as a probe. Lane M, DNA molecular mass ?
DNA/HindIII markers; lane 1, total plasmids from YBT-1518; lane 2,
plasmid pBMB0223 digested with HindIII, as a positive control; lane 3,
total plasmids from YBT-1518 digested with HindIII; lane 4, cloning
vector pHT304 digested with HindIII as a negative control. The arrows
point to the position of plasmid pBMB0228.
TABLE 2. Activities of Cry55Aa1, Cry6Aa2, and Cry5Ba2 against
Crystal proteinRegression equation
y ? 2.6911x ? 1.4288
y ? 1.8736x ? 2.4415
y ? 1.6544x ? 2.9475
aThe second-stage juvenile of Meloidogyne hapla was tested, and LC50s were
determined on the fifth day.
bBovine serum albumin (BSA) was used as the negative control; it has no
toxicity to Meloidogyne hapla.
7000GUO ET AL.APPL. ENVIRON. MICROBIOL.
lutionary history of this plasmid. In any case, these features, Download full-text
combined with the fact that pBMB0228 harbors cry genes,
make this unusual plasmid a very interesting subject for further
It was interesting to observe that the cry5Ba2 gene was
expressed in the BMB171 host strain, as evidenced by the
formation of a bipyramidal crystal, but not in the original
strain, YBT-1518. According to previous reports, cryptic or
silent cry genes have been found to lack functional promoters,
resulting in their display of little or no expression (8, 11, 27). In
the case of cry5Ba2, with this strategy, it has been shown that
the cry5Ba2 gene possesses a functional promoter that allows it
to be expressed in the acrystalliferous B. thuringiensis strain
BMB171. The reason for the gene’s cryptic behavior in its
original strain therefore seems to be different from those men-
tioned above. What has been proved is that the high level of
expression of cry55Aa1 and cry6Aa2 should not affect the issue
of cry5Ba2 expression in the wild-type strain. The expression of
gene cry5Ba2 still cannot be detected (data not shown) after
pBMB0228 is cured by deletion of the two genes but not
cry5Ba2 from YBT-1518. In this strain, a negative regulator of
cry6Aa2 had been isolated downstream of the gene by Yu et al.
(30). There was likely another negative regulator for the
cry5Ba2 gene in the strain. The substantive mechanism respon-
sible for the lack of expression in strain YBT-1518 is still under
investigation in our laboratory.
In conclusion, in this study, we proposed a strategy for iso-
lating cry genes from B. thuringiensis. This process was applied
to a strain that produces rice-shaped crystals—YBT-1518—
resulting in the cloning of three nematicidal cry genes. One of
those genes was genuinely novel, one was cryptic, and one was
a traditional cry gene, and we also found that a plasmid smaller
than the usual Cry plasmids (17.7 kb) could encode typical cry
We thank Geraldine A. Van der Auwera (Laboratory of Food and
Environmental Microbiology in Belgium) for her critical review of the
This research was supported by grants from the Key Project of China
National Programs for Fundamental Research and Development
(grant 2003CB114201) and the National Programs for High Technol-
ogy Research and Development of China (grants 2006AA02Z174 and
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