Biological Control 47 (2008) 335–339
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Investigation of the cyt gene in Bacillus thuringiensis and the biological activities of Bt isolates from the soil of China Yan Wu, Meiying Gao *, Shunying Dai, Dan Yi, Hongyu Fan Wuhan Institute of Virology, Chinese Academy of Sciences, Xiaohongshan 44#, Wuhan City, Hubei Province 430071, PR China
a r t i c l e
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Article history: Received 29 May 2008 Accepted 26 August 2008 Available online 2 September 2008 Keywords: Bacillus thuringiensis cyt gene Cytolytic activity Hemolytic activity
a b s t r a c t The presence of cyt genes was investigated in 80 type strains of Bacillus thuringiensis and 143 isolates obtained from soil samples of China by PCR ampliﬁcation using two pairs of primers for the cyt1 and cyt2 genes. Three type strains of serotypes H11ac, H14 and H36, eight isolates belonging to H3, H14, H18 and H21, and one isolate of unknown serotype harbored cyt genes. We also tested the cytolytic activity for mammal cells, the hemolytic activity for sheep erythrocytes and insecticidal activity against mosquitoes of ﬁve isolates that contained cyt genes but did not belong to B. thuringiensis serovar israelensis. The protein proﬁles of the ﬁve isolates were different from those of the type strains of B. thuringiensis serovar israelensis, and among the ﬁve isolates, only Y-5 showed mosquitocidal activity against larvae of Culex quinquefasciatus. All ﬁve of the isolates exhibited hemolytic activity, but only three could cause the cell death of A549 cells. The cytopathological changes induced by NX-4 in some A549 cells were characterized with cell-ballooning. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Bacillus thuringiensis (Bt) is a Gram-positive bacterium that is characterized by the production of insecticidal crystal proteins during the sporulation. It has been used as a successful agent of biological control for insect pests because of its high toxicity to certain insect species among the orders Lepidoptera, Diptera and Coleoptera (Hofte and Whiteley, 1989; Schnepf et al., 1998). The insecticidal crystal proteins produced by Bt have been categorized into two families, Cry and Cyt proteins, which do not share signiﬁcant sequence homology (Guerchicoff et al., 2001). Although both types of toxins appear to work through pore formation, which leads to cell lysis and irreversible damage to the midgut of the insect, Cyt toxins differed from Cry proteins because of their cytolytic activities against a broad range of cells, including mammalian cells such as erythrocytes (Crickmore et al., 1998; Gill and Hornung, 1987). These toxins are believed to lyse cells by forming an oligomeric transmembrane pore based on a b-barrel structure (Li et al., 1996). On the basis of amino acid identity, two cytolytic classes of Cyt toxins, Cyt1 and Cyt2, have been identiﬁed (Crickmore et al., 1998; http://www.lifesci.sussex.ac.uk/ home/Neil_Crickmore/Bt/index.html). The presence of Cyt toxins, which are not very toxic by itself, was always considered as a feature of all mosquitocidal strains. There is accumulating evidence, however, that the mosquitocidal activity of * Corresponding author. Fax: +86 027 87199355. E-mail address: [email protected]
(M. Gao). 1049-9644/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2008.08.020
Bt strains is not only mediated by the additive effect of each Cry toxin but by a complex synergistic interaction among the Cry and Cyt proteins in the insect gut (Crickmore et al., 1995; Guerchicoff et al., 2001; Salehi Jouzani et al., 2008). The mechanism by which Cyt proteins act synergistically with Cry proteins has also been researched. It has been reported that Cyt1Aa synergizes with or suppresses resistance to the Cry11Aa toxin by functioning as a membranebound receptor in B. thuringiensis subsp. israelensis (Perez et al., 2005). In addition, the positive correlation found between the coexistence of Cyt1 and Cyt2 proteins and high mosquitocidal activity, as well as the high conservation found among the cyt gene sequences determined so far, also point to an important biological role for Cyt proteins in the toxicity of the crystal proteins (Ibarra et al., 2003). Cyt proteins, combined with other mosquitocidal toxins, could be used to delay the development of resistance and increase the activity of microbial insecticides (Georghiou and Wirth, 1997; Victor Juárez-Pérez et al., 2002; Wirth et al., 2000). Although Cyt proteins play an important role in the mosquitocidal activity of Bt, they are not presented only in the antidipteran strains. It has also been reported that cyt genes are present in some Bt strains that are not toxin to diptera larvae but are toxic to lepidopteran and coleopteran larvae (Guerchicoff et al., 2001, 1997). For example, cyt2Ba6 was found in the anticoleopteran B. thuringiensis subsp. tenebrionis, which belongs to the morrisoni serovar. During more than 30 years of work, a large number of Bt strains have been isolated in our laboratory from samples of soil and dead insects from China. The Bt isolates show great diversity and speciﬁcity on the basis of H-serotype, crystal proteins, plasmid proﬁle,
Y. Wu et al. / Biological Control 47 (2008) 335–339
cry types of genes and pesticidal activity ( Gao et al., 2008). In our research, which involves screening Bt strains for agents that are toxic to cancer cells, we have found that some strains, especially those that have little or no activity, exhibit cytolytic activity against cancer cells. Therefore, we decided to characterize the distribution and bioactivities of the Cyt proteins in our Bt collection. In this study, we investigated the presence of cyt genes by PCR ampliﬁcation using two pairs of primers, for cyt1 and cyt2, respectively. We also investigated the banding patterns of the crystal proteins, the cytolytic activity against mammalian cells, hemolytic activity for sheep erythrocytes and insecticidal activity against mosquitoes of the isolates. 2. Materials and methods 2.1. Bt type strains and isolates Eighty B. thuringiensis type strains, which belonged to serotypes H1–H29, H31–H47, H49–H70 and the type strain of wuhaniesis 140, were used. The type strains B. thuringiensis subsp. jinghongiensis (serotype H42), guiyangiensis (H43), rongseni (H56), pingluonsis (H60), zhaodongensis (H62), sinensis (H70) and wuhaniesis 140 were identiﬁed and named by Li et al.,2000, 1999). The other type strains were kindly provided by the Institut Pasteur (France). Native Bt isolates used herein were isolated and stored in our laboratory. 2.2. Primers and PCR ampliﬁcation Two pairs of primers were synthesized from conserved regions of the cyt1 and cyt2 genes as published previously (Ibarra et al., 2003). Templates and PCR mixtures were prepared as described previously by Gao et al. (2008). Thus, 0.1 lM of each primer, 1 U Taq DNA polymerase (MBI Fermentas) and 0.5 mM dNTPs were used in a total of 25 ll of PCR mixture. Ampliﬁcation was performed in a thermal cycler (PTC-100_ programmable Thermal Controller, MJ Research, Inc., Waltham, MA, USA) using a 29-cycle program with each cycle consisting of a denaturation step of 94 °C for 30 s, a 90 s annealing step of 52 or 50 °C for cyt1 and cyt2 primer pairs, respectively, and an extension step of 72 °C for 2 min. A ﬁnal extension step at 72 °C for 7 min was also included. 2.3. Preparation and analysis of parasporal inclusions The crude parasporal inclusions were prepared as described by Gao et al. (2008). Each Bt isolate was grown at 30 °C for 3–5 days on nutrient agar ([w/v]: 1% polypeptone, 0.5% NaCl, 0.3% meat extract, 1.5% agar, pH 7.5) until autolysis was completed. Spore/ crystal cultures were harvested by centrifugation and washed three times with 0.85% of NaCl solution. Spores that ﬂoated to the surface were removed with Whatman No.1 ﬁlter paper, and then the spore/crystal mixtures were resuspended in sterile water. The parasporal inclusions were solubilized in 0.1 M NaOH at 37 °C for 30 min, and then digested by trypsin at a ﬁnal concentration of 10 mg/ml. The concentration of inclusion proteins was determined by the method of Bradford (1976) using bovine serum albumin as the standard. Following this, SDS–PAGE was carried out as described previously by Laemmli (1970). 2.4. Assay of hemolytic and cytolytic activity The hemolytic activity of the Bt isolates was assayed using sheep erythrocytes according to a method modiﬁed from those of Saitoh et al. (1998) and Uemori et al. (2007). Brieﬂy, sheep erythrocytes were washed three times with 0.85% NaCl buffer, and resuspended to 2% (v/v) in the same buffer. A 1 ml of the crystal
proteins solutions (0.35 mg/ml) was added to 1 ml of 2% erythrocytes suspension and incubated at 37 °C. Following this, 150 ll of the mixture was centrifuged at 800g for 5 min and the supernatant was examined at OD540 nm each hour during 6 h inoculation. The A549 cells used in this study are human lung cancer cells, and they were kindly provided by Dr. Wang at the Wuhan Institute of Virology, Chinese Academy of Sciences, CAS. They were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37 °C and 5% CO2. The level of cytolytic activity of the proteins against A549 cells was assessed using the MTT assay (Behl et al., 1992; Heiss et al., 1997). Each well of a 96-well microtiter plate received 90 ll of cell suspension containing 1 104 cells, and incubated at 37 °C for 16 h. Protease-treated protein solution (10 ll of 0.35 mg/ml) was then delivered. After 24 h incubation, each well received 10 ll of 5 mg/ml MTT and was further incubated for 4 h, then 150 ll DMSO was delivered to each well and the plates were examined for absorbency at 570 nm. The average absorbance at 570 nm obtained from mock-inoculated cell culture was used as a control value. The assay was done in triplicate. The cytopathic effect (CPE) induced by the proteins on A549 cells were monitored for 24 h at appropriate intervals with inverted microscopy (Olympus XDS-1B, Japan). 2.5. DNA sequencing and analysis The cyt-like genes were ampliﬁed and the PCR products were extracted using gel extraction kit (Omega, BioTek) and then sequenced by Invitrogen Biotechnology Co., Ltd. The sequences of cyt-like genes were analyzed by NCBI BLAST. 2.6. Insecticidal assay Culex quinquefasciatus were provided by the laboratory of Dr. Yuan (Wuhan Institute of Virology, CAS), and were reared at 26 °C, 87% relative humidity, and a photoperiod of 12:12 (L:D). The insecticidal activity of Bt isolates was tested on larvae of C. quinquefasciatus as described previously (Ibarra et al., 2003). Thirty early third-instar larvae were placed in 50 ml of dechlorinated water. Four concentrations (0.1, 0.2, 0.4, 0.8 mg/ml) of the spore–crystal complex of each strain were added (three repetitions). The positive control was B. thuringiensis subsp. israelensis, and the negative control was dechlorinated water. Larvae were incubated in the conditions described above and examined after 24 h. 3. Results 3.1. Detection of cyt genes The present of cyt genes was investigated in 80 Bt type strains and 143 isolates from soil samples of China by PCR ampliﬁcation using two pairs of primers, for cyt1 and cyt2 gene, respectively. Among the 80 Bt type strains, only three strains, of which the serotypes were H11ac, H14 and H36, harbored cyt genes. The strains of H14 and H36 contained both cyt1 and cyt2 genes. In contrast, the H11ac strain only showed the PCR proﬁle of cyt2. Thirty three Bt isolates of unknown serotypes and 110 native isolates, which were distributed in 30 H-serotypes and belonged to 31 serovars, including auto-agglutination strains and no ﬂagellation strains, were also investigated for the present of cyt genes. The results are shown in Table 1. A total of nine isolates (6.29%) showed PCR proﬁles of cyt genes: eight of these isolates belonged to serotypes H3, H14, H18, H21, and one was of unknown serotype. Seven of the native isolates belonged to B. thuringiensis serovar israelensis were tested, only four of them contained cyt genes, and the other
Y. Wu et al. / Biological Control 47 (2008) 335–339 Table 1 Detection of the cyt gene in native Bacillus thuringienisis isolates
3.2. DNA sequencing and analysis
Number of isolates tested
Number of cyt gene positive strains
1 3a 3abc 3ac 3ade 4a 4ab 5 6 7 8 10 11 13 14 15 16 17 18 19 20a 20ab 21 22 24ab 24ac 25 26 27 28 29 42 43 52 Auto-agg Nonﬂage Unknown Total
1 6 3 2 1 6 4 4 1 3 4 10 1 5 7 2 2 2 3 1 1 2 3 7 2 1 2 1 1 1 1 2 3 2 11 2 33 143
— 1 — — — — — — — — — — — — 4 — — — 2 — — — 1 — — — — — — — — — — — — — 1 9
The PCR products of the cyt1 and cyt2 genes of the nine native isolates were sequenced, and the sequences were analyzed using the BLASTN programs. The GenBank Accession Nos. for the nucleotide sequences, and the identity of the partial sequences with previously published cyt genes are summarized in Table 2.
Unknown, serotype unknown; ﬂagellation. a Sub-factors, not identiﬁed.
3.3. Analysis of parasporal inclusions The ﬁve Bt isolates that contained cyt genes but did not belong to B. thuringiensis subsp. israelensis were further characterized by SDS–PAGE of their crystal protein products. The protein proﬁles of the ﬁve isolates were different from the proﬁle of B. thuringiensis subsp. israelensis (HD-567), which contains major proteins of 130, 70, and 28 kDa, and exhibited diversity (Fig. 1). Isolate NX-6 was found to contain two major crystal proteins with molecular masses of 95 and 27 kDa; SX-1 was also composed of two proteins with molecular masses of 95 and 25 kDa; SY1-1 was contained three major proteins of 95, 42 and 28 kDa; isolate NX-4 contained no proteins larger than 70 kDa, but had three major proteins of 65, 37 and 28 kDa; isolates Y-5 contained four major proteins with molecular masses of 80, 40, 27 and 26 kDa. Each of these ﬁve isolates contained a similar band to that of Cyt protein of HD-567 with molecular masses ranging from 25 to 28 kDa. 3.4. Hemolytic and cytolytic activity
three strains did not detected for harboring cyt genes. The nine isolates that harbored cyt genes are shown in Table 2. Seven contained both cyt1 and cyt2 genes, while strain NX-6 showed only the PCR proﬁle of cyt1 and SX-1 only contained the cyt2 gene.
The ﬁve isolates were assayed for hemolytic activity against sheep erythrocytes (Fig. 2). Spore/crystal proteins of NX-6 and Y5 were less hemolytic than that of HD-567 to sheep erythrocytes during a 6 h incubation. The hemolytic activity of SY1-1 was similar to that of distilled water for up to 4 h incubation but rose slightly later. In contrast, the hemolytic activity of SX-1 did not showed during the ﬁrst hour, but rose quickly and became higher than that of HD-567 after 6 h. The hemolytic activity of NX-4 was higher than that of HD-567 during the ﬁrst and last hours and was similar to it during the other 4 h. The cytolytic activities of the ﬁve isolates are shown in Table 3. The alkali-solubilized, trypsin-digested proteins of NX-6 and SY1-1 exhibited no cytolytic activity against A549 cells. The trypsin-digested proteins of SX-1 and Y-5 induced relatively high cell death rates of 75.27% and 58.38%, respectively, and caused rapid cell lysis that was similar to that caused by HD-567. The NX-4 protein not only induced a 66.18% cell death rate and the lysis of A549 cells,
Table 2 Bacillus thuringienisis isolates containing cyt genes Strain
cyt gene type
Identity (%) to known cyt gene
GenBank accession numbers
cyt1 cyt2 cyt1 cyt2 cyt1 cyt2 cyt1 cyt2 cyt1 cyt2 cyt1 cyt1 cyt2 cyt1 cyt2 cyt2
cyt1Ab1 (97%) cyt2Bc (98%) cyt1Aa1 (99%) cyt2B-like (99%) cyt1 (100%) NT cyt1 (100%) NT cyt1Aa1 (98%) cyt2Ba8 (99%) NT cyt1Ab1 (98%) NT cyt1Aa (76%) cyt2Ba (98%) NT
EU917075 FJ009214 EU917076 EU917078 FJ009210 NT FJ009211 NT EU917077 FJ009212 NT EU917074 NT EU917073 FJ009213 NT
NT, not submit to GenBank and analyze with the BLASTN programs.
Y. Wu et al. / Biological Control 47 (2008) 335–339
NX-4, NX-6 and SX-1 showed no signiﬁcant mosquitocidal activity. Strain Y-5 was highly toxic to larvae of the insect, and application of 0.1 mg/ml of the spore/crystal cultures resulted in approximately 93.3% mortality of C. quinquefasciatus after 24 h of exposure. However, the mosquitocidal activity of Y-5 was still slightly lower than that of HD-567. The mosquitocidal activity of the tested Bt isolates is shown in Table 4. 4. Discussion
Fig. 1. Protein proﬁles of spore/crystal cultures of NX-6, SX-1, SY1-1, NX-4, Y-5 and HD-567. M. molecular marker; 1. NX-6; 2. SX-1; 3. SY1-1; 4. NX-4; 5. Y-5; 6. HD567.
Fig. 2. Hemolytic activity of six B. thuringiensis isolates against sheep erythrocytes.
but the cytopathological changes of some cells were characterized by cell-ballooning (Fig. 3). 3.5. Mosquitocidal activity The mosquitocidal activities of isolates Y-5, NX-4, NX-6, SX-1 and HD-567 were performed on larvae of C. quinquefasciatus. Strain
The Cyt proteins of a B. thuringiensis strain that is highly toxic to diptera have been previously thoroughly studied by different methodologies (Guerchicoff et al., 2001; Li et al., 1996). In this study, we focused on investigating the distribution of cyt genes in the type strains of Bt and in native isolates maintained in our laboratory, and the bioactivities of some isolates containing Cyt proteins were also studied. The investigation of cyt genes in native Bt isolates showed that the cyt gene is not a common in the Bt, but is distributed in some serotypes. In our research, we also found that some Bt isolates of serovar israelensis lacked Cyt proteins. Although strains belonging to serotype 14 (Bti) have been previously reported uniformly to produce Cyt protein and show mosquitocidal activity, Yasutake et al. (2007) has also reported that organisms belonging to serovar israelensis but lacking mosquitocidal activity are widely distributed throughout Japan. Our ﬁnding that some strains belonging to serovar israelensis did not contain Cyt proteins agree with the result of Yasutake et al., and indicates that strains lacking mosquitocidal activity and Cyt proteins are common in Bt strains belonging to serotype 14 (Bti). Our result may partially explain the phenomenon that some Bti strains do not exhibit mosquitocidal activity. This result also indicates that the distribution of Cyt proteins is not tightly co-related with Bt serovarieties. It has long been known that the Cyt proteins are found particularly, but not exclusively, among strains that display antidipteran activity. In this study, we detected cyt genes in some isolates from the Bt collection in our laboratory and found that three of these isolates showed no mosquitocidal activity against C. quinquefasciatus. In addition, the antidipteran activity of the strains did not correlate with their hemolytic activity; for example NX-4 and SX-1 showed high hemolytic activity but very low mosquitocidal activity. The main reason for this may be that hemolytic activity is attributable only to the presence of Cyt protein, while antidipteran activity re-
Table 3 Cytotoxic activities of six Bacillus thuringienisis strains against A549 cells Strain
MTT 570 nm Cell death rate (%)
Buffer control containing trypsin (10 mg/ml).
Fig. 3. Micrographs of A549 cells treated with proteolytic proteins of B. thuringiensis isolates for 24 h under light microscopy showing cytopathic effect of Bt isolates NX-4 (a), HD-567 (b), mock-inoculated A549 cells (c).
Y. Wu et al. / Biological Control 47 (2008) 335–339 Table 4 The mosquitocidal activity of Bt isolates against the larvae of C. quinquefasciatus after 24 h of exposure Strain
Y-5 NX-4 NX-6 SX-1 HD-567 Control
Average mortality 0.8 mg/ml
100% 0 3.3% 0 100% 0
100% 0 0 0 100%
96.7% 0 0 0 100%
93.3% 0 0 0 100%
Control, C. quinquefasciatus treated with dechlorinated water.
quires complex synergistic interaction among the Cry and Cyt proteins. Isolates that contain Cyt proteins but show no mosquitocidal activity may be useful for combating insecticide resistance and for increasing the activity of microbial insecticides when combined with other Bt strains (Victor Juárez-Pérez et al., 2002). Proteins of isolates SX-1 and Y-5 exhibited hemolytic activity against erythrocytes and cytolytic activity against A549 cells according to MTT assay, and the CPE was typically cytolysis, therefore the cell death of A549 cells were totally considered as cytolysis by Cyt proteins. It has been reported that strains belonging to serovar colmeri (H21) exhibited strong larvicidal activity against A. aegypti, giving 100% mortality in 24 h (Yasutake et al., 2007). In contrast, strain NX-4, which also belongs to serovar colmeri, showed no mosquitocidal activity against C. quinquefasciatus, but was highly hemolytic. In addition, the cytopathological changes induced by NX-4 in some A549 cells were characterized by cell-ballooning, which is very similar to the reported effect of parasporin in causing the death of cancer cells (Mizuki et al., 1999; Yamashita et al., 2000). Therefore, NX-4 could also be a candidate for harboring crystal proteins that are toxic to cancer cells. Our future research will be focused on separating the cytotoxic proteins from the crystal proteins of NX-4 and characterizing their genes. It has been reported previously that the cyt2Ba3 gene has been found in a Bt strain belonging to fuokukaensis (H3ade) (Guerchicoff et al., 1997). Similarly, our research also showed that the strain Y-5, which belongs to H3, had both cyt1 and cyt2 genes. In addition, Y-5 was almost as toxic to larvae of C. quinquefasciatus as B. thuringiensis serovar israelensis but produces different polypeptides. Bt strains of serotype H3 have been considered as insecticidal strains against lepidoptera pests; the insecticidal activity of Y-5 against mosquitoes was unique and will be characterized in our further work. Acknowledgments This study was supported by the National Natural Science Foundation of China (No.30370778; No.30170025) and ‘‘863” project (2006AA02Z174). References Behl, C., Davis, J., Cole, G.M., Schubert, D., 1992. Vitamin E protects nerve cells from amyloid beta protein toxicity. Biochemical Biophysical and Research Communication 186, 944–950. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.
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