- Email: [email protected]
Characterization of a novel mosquitocidal toxin of Cry50Ba and its potential synergism with other mosquitocidal toxins
Toxicon 138 (2017) 165e168
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Short communication
Characterization of a novel mosquitocidal toxin of Cry50Ba and its potential synergism with other mosquitocidal toxins Wenfei Zhang, Silan Yu, Silun Peng, Jianru Gong, Jiangzhao Qian, Jianqiao He, Wenyu Dai, Ruiping Wang* Ministry of Education Key Laboratory for Tropical Animal and Plant Ecology, College of Life Sciences, Hainan Normal University, Haikou, Hainan, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 April 2017 Received in revised form 25 August 2017 Accepted 30 August 2017 Available online 7 September 2017
A putative toxin gene of cry50Ba was successfully expressed in E. coli cells and confirmed that the purified Cry50Ba toxin had very high toxic activity against Culex quinquefasciatus larvae. Furthermore, the potential synergism of Cry50Ba toxin with Cry2Aa, Cry4Aa and Cry11Aa at a ratio of 1:1 was investigated. Although no significant synergism with other toxins was observed, the Cry50Ba as a novel toxin could be used to delay rapid onset of resistance in mosquito. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Bacillus thuringiensis Cry50Ba Synergism Culex quinquefasciatus
1. Introduction Bacillus thuringiensis (Bt), is recommended by the World Health Organization (WHO) for the control of mosquito pests owing to its efficiency and safety (Boyce et al., 2013; Setha et al., 2016). Bt strain S2160-1 was identified as a good candidate strain for the control of mosquito. The insecticidal crystal protein genes of cry30Ea (GenBank ID: EU503140), cry30Ga (GenBank ID: HQ638217), cry50Ba (GenBank ID: GU446675), cry54Ba (GenBank ID: GU446677) and cry4Cb (GenBank ID: KF753696) were successfully identified by using the approach of PCR-restriction fragment length polymorphism (RFLP) and mass spectrometry (Zhang et al., 2012, 2014). In this study, the cry50Ba gene was successfully cloned and expressed in Escherichia coli (E. coli) cells and the purified Cry50Ba toxin was detected to have mosquitocidal activity to the important disease vector of Culex quinquefasciatus larvae in laboratory conditions. Meanwhile, the potential synergism of Cry50Ba toxins with other mosquitocidal toxins of Cry2Aa (GenBank ID: AAA22335),
Cry4Aa (GenBank ID: CAA68485), Cry11Aa (GenBank ID: AAA22352) was investigated further. Unfortunately, there was no significant synergism between them, but the cry50Ba gene still could be a prospective candidate for delaying the rapid onset of resistance in mosquito. 2. Materials and methods 2.1. Bacterial strains, growth conditions and plasmids The wild-type Bt isolate S2160-1 and the reference strains of Bacillus thuringiensis subsp. Israelensis (Bti) were from Laboratory stock. Bt strains were incubated at 28 C in LuriaeBertani (LB) medium or in G-Tris medium as a sporulation medium for crystal protein extraction. E. coli strains were grown at 37 C in LB broth. The pET-30a plasmid served as a standard vector for the expression of cry50Ba genes in E. coli BL21 (DE3) cells (Zhang et al., 2012). 2.2. Sequence analysis
* Corresponding author. College of Life Sciences, Hainan Normal University, Haikou, Hainan 571158, China. E-mail addresses: [email protected], [email protected] (W. Zhang), [email protected] (S. Yu), [email protected] (S. Peng), [email protected] (J. Gong), [email protected] (J. Qian), [email protected] (J. He), 342364690@ qq.com (W. Dai), [email protected], [email protected] (R. Wang). http://dx.doi.org/10.1016/j.toxicon.2017.08.025 0041-0101/© 2017 Elsevier Ltd. All rights reserved.
The conserved blocks analysis was performed by multiple sequence alignment with CLUSTALW software as reported by Schnepf et al. (1998). The theoretical model of Cry50Ba was predicted by homology modelling on the structure of the Cry2Aa (PDB
166
W. Zhang et al. / Toxicon 138 (2017) 165e168
Fig. 1. Multiple sequence alignment and the three dimensional model of Cry50Ba. (A) Sequence alignment of Cry50Ba with homologous Cry proteins in five conserved blocks. (B) The first-approximation three-dimensional model of Cry50Ba toxin was generated by using the homology-modelling algorithm, SWISS-MODEL. The N-terminal Domain I (Red) consists ofa-helical bundle, is involved in membrane insertion and pore formation. Domain II (Blue) and domain III (Green) are involved in receptor recognition and binding. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
entries 1I5P). The amino acid sequence file of Cry50Ba toxin was submitted to Swiss-Model in the expasy server (http://www. expasy.ch/spdbv/) and a preliminary model for Cry50Ba was retrieved (Schwede et al., 2003). And conserved domains were predicted by the NCBI Conserved Domain Search server (https:// www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).
2.3. Expression and purification of cry50Ba genes in E. coli cells To express the cloned cry50Ba genes in E. coli cells, the primer pairs of 50Ba-5(50 -CGggatccATGAATTCATATCAAAATACAAATG30 )/50Ba-3(50 -ACGCgtcgacTTAATTTGTAAATAGATTATCCACC-30 ) were designed to amplify the complete coding sequences (CDS) of cry50Ba gene. The BamHI and SalI restriction sites were introduced into 50 end of the forward and reverse primers respectively, which facilitated subcloning the cry50Ba genes into the expression vector pET-30a. The construct was sequenced to confirm that no frame shifts, stop codons or rearrangements had occurred and then it was transformed into E. coli BL21(DE3) cells. The transformants of pETcry50Ba containing cry50Ba genes and the control with no insert were incubated at 37 C in LB broth supplemented with 12.5 mg/ml Kanamycin with shaking at 220 rpm for about 1.5hr until an OD600 value of the culture reached 0.6. IPTG was added to the culture at a final concentration of 0.2 mM and incubation continued at 37 C for 8 h to induce the cry50Ba gene expression (Misra et al., 2002). The cells were collected by centrifugation at 4 C at 12,000 g for 5min and the pellets were washed with cooled 1M NaCl, sonicated and analyzed by SDSPAGE (Bio-Rad, USA). Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography (Sangon Biotech, Shanghai, China) was used to purify the expressed Cry50Ba proteins with 6 histidine (His) tag at its N-terminus (Gayen et al., 2012). The target recombinant protein was eluted with column buffer containing 250 mM imidazole and 5 mM.1, 4-dithiothreitol.
2.4. Preparation of cry toxin from Bt isolates The acrystalliferous B. thuringiensis subsp. israelensis strain IPS78/11 harboring plasmid pSVP27-cry2Aa (Bt2Aa), pSVP27cry4Aa (Bt4Aa), pSVP27-cry11Aa (Bt11Aa) kindly supplied by Dr Neil Crickmore (School of Biological Sciences, University of Sussex, UK) were used to isolate pure Cry toxins (Crickmore and Ellar, 1992; Crickmore et al., 1990). Cry proteins were produced in G-tris medium containing 5 mg/ml chloramphenicol after 3 days of incubation at 30 C. Spore/crystal mixtures were harvested and washed
Fig. 2. SDS-PAGE analysis of cry50Ba, cry2Aa, cry4Aa and cry11Aa genes expressed in E. coli BL21 (DE3) cells and acrystalliferous Bt strain IPS78/11 respectively. (A) SDSPAGE gel electrophoresis analysis of cry50Ba gene expressed in BL21 (DE3) cells. PM1: protein molecular weight marker; lane 1: E. coli BL21 (DE3) cells harboring pET30a incubated with IPTG; lane 2: E. coli BL21 (DE3) cell harboring pETcry50Ba incubated without IPTG; lane 3: E. coli BL21 (DE3) cells harboring pETcry50Ba incubated with IPTG; lane 4: purified Cry50Ba protein by Ni-NTA. (B) Expression of cry gene in the acrystalliferous Bt IPS78/11. PM2: protein molecular weight marker; lane 5, 6, 7: Purified crystal protein of Cry2Aa, Cry4Aa and Cry11Aa by centrifugation on discontinuous sucrose density gradients.
W. Zhang et al. / Toxicon 138 (2017) 165e168
167
Table 1 Bacterial strains and plasmids. Strains and plasmids B. thuringiensis Bt S2160-1 Bt subsp. israelensis HD522 Bt subsp. Israelensis IPS78/11 Bt2Aa Bt4Aa Bt11Aa Escherichia coli JM110 BL(DE3) Plasmids pET-30a pET50Ba
Description
Source
Wild strain of Bt haboring cry4Cb, cry30Ea, cry30Ga, cry50Ba and cry54Ba genes. Model strain of Bt haboring cry4Aa, cry4Ba, cry10Aa, cry11Aa, cyt1Aa, and cyt2Ba genes Acrystalliferous mutant of B. thuringiensis subsp. israelensis strain Bt subsp. Israelensis IPS78/11 transformed with the plasmid of pSVP27-cry2Aa Bt subsp. Israelensis IPS78/11 transformed with the plasmid of pSVP27-cry4Aa Bt subsp. Israelensis IPS78/11 transformed with the plasmid of pSVP27-cry11Aa
Lab stock Lab stock Dr Neil Crickmore Dr Neil Crickmore Dr Neil Crickmore Dr Neil Crickmore
Dam, dcm, supE44, hsdR17, thi,leu, rpsL1, lacY galK, galT, aratonA thr, tsx, D(lac-proAB) (F0, traD36, proAB, lacI qZDM15) F e ompT hsdSB(rBe mBe) gal dcm (DE3)
Novagen Novagen
KanR, HisTag/STag hsdSgal (lcTs857 indl Sam7nin5 lacUV5-T7gene) AmpR, pET-30a harboring cry50Ba1 gene
Novagen This study
three times with 1M cooled NaCl. Cry toxins were purified by centrifugation on discontinuous sucrose density gradients and solubilized by using 50 mM Na2CO3 for 1 h at pH11.0 (SilvaWerneck and Ellar, 2008).
five conserved sequence blocks common to a large majority of the Cry proteins. According to the conserved domain search and homology modelling analysis, three conserved domains were found out which indicated that the Cry50Ba was a typical three-domain insecticidal crystal protein (Fig. 1).
2.5. Mass spectrometry analysis The expressed of Cry proteins samples were analyzed using SDSPAGE gel electrophoresis. The target protein bands were excised from SDS- PAGE gel and analyzed with a 4800 MALDIeTOF/TOF mass spectrometer (Applied Biosystems, USA) as described by Wenfei Zhang et al. (2014). 2.6. Larvicidal assays and analysis The concentrations of expressed Cry proteins were estimated by using the Quantity One analysis software program followed the Instruction Manual (Bio-Rad, USA) (Fang et al., 2007). Mosquito larvicidal assays against 2nd instar Culex quinquefasciatus were performed as described previously (Zhang et al., 2012). The Cry50Ba toxin was mixed with Cry2Aa, Cry4Aa and Cry11Aa separately (1:1 ratio) and the resulting mixtures were prepared in twofold serial dilutions in dechlorinated water (seven concentrations: 14.6e1400 ng/ml). Mortality was recorded after incubation at 26 C for 48 h. The 50% lethal concentration (LC50) was determined by Probit analysis with software package SPSS 13.0 for windows (SPSS inc., Chicago, USA). The theoretical LC50 and synergism factor were evaluated from LC50 value of individual toxin by using the Tabashnik equation (Tabashnik, 1992; Wirth et al., 2000). 3. Results and discussion 3.1. Molecular model of Cry50Ba toxin Alignment of the Cry toxins reveals that Cry50Ba toxin presents
3.2. Over-expression of cry50Ba gene in E. coli cells and protein purification In an attempt to express the identified cry50Ba genes, the coding region amplified by PCR was inserted into pET-30a and the recombinant plasmids were transferred into E. coli BL21 (DE3) cells. The addition of IPTG to the recombinant strains resulted in the overexpression of Cry50Ba, which was detected on Coomassie blue stained SDS-PAGE gel with molecular weight of approximately 75 kDa (Fig. 2A). The recombinant Cry50Ba protein was then purified with Ni-NTA affinity chromatography, due to the presence of 6 histidine tail at the N-terminal end. The purified Cry50Ba proteins were corroborated by using mass spectrometry (see Table 1). 3.3. Mosquito larvicidal activity and synergism Mosquito larvicidal test results (Table 2) showed that Cry50Ba exhibited high activity against Culex quinquefasciatus larvae with a LC50 of 73.87 ng/ml, which was about seven times more toxic than Cry2Aa(LC50: 528.26 ng/ml). But Cry50Ba is nontoxic to lepidopterous larvae of plutella xylostella and Helicoverpa armigera (data not shown). When mixing the toxins of Cry50Ba and with Cry2Aa, Cry4Aa, Cry11Aa were mixed with a ratio of 1:1 separately, unfortunately the synergism among the toxins of Cry50Ba, Cry4Aa and Cry11Aa was not observed in this study. The combined toxins of Cry2Aa and Cry50Ba yielded a LC50 of 46.91 ng/ml that exhibited more insecticidal activity against Culex quinquefasciatus larvae than the observed activities for Cry50Ba and Cry2Aa alone. The
Table 2 Mosquito larvicidal activity of Cry50Ba, Cry2Aa, Cry4Aa and Cry11Aa alone and combination against 2nd instar larvae of Culex quinquefasciatus. Toxins
Experimental LC50(ng/ml)(95%FL minemaxb)
Theoretical LC50(ng/ml)c
Synergy factorc
Cry50Ba Cry2Aa Cry 4Aa Cry11Aa Cry50Ba þ Cry2Aaa Cry50Ba þ Cry4Aaa Cry50Ba þ Cry11Aaa
73.87(51.40e102.75) 528.26(374.86e835.58) 51.88 (37.86e69.82) 13.78 (10.04e18.56) 46.91(34.31e61.60) 54.24 (40.72e76.04) 31.01 (24.72e39.65)
129.61 60.98 23.23
2.76 1.12 0.75
a b c
Toxin mixtures are combinations of Cry50Ba and another Cry protein with ratio 1:1. 95% FL is the 95% fiducial limits in parentheses, as determined by probit analysis. Theoretical LC50 and Synergism factor (SF) calculated by the method of Tabashnik (1992).
168
W. Zhang et al. / Toxicon 138 (2017) 165e168
theoretical LC50 of the combined toxins calculated using the formula of Tabashnik was 129.61 ng/ml. As the result, the ratios of the experimental LC50 to theoretical LC50 or synergism factor (SF) was 2.76. The 95% fiducial limits of LC50 values for Cry50Ba and those of the mixture of Cry50Ba and Cry2Aa overlap so the data shows no significant synergism with the Cry2Aa toxin against Culex quinquefasciatus larvae. 4. Conclusions Bt strain S2160-1 is carrying insecticidal crystal protein genes of cry30Ea, cry30Ga, cry50Ba, cry54Ba and cry4Cb with a comparable mosquitocidal activity to Bti. It is believed that Bt S2160-1 could be applied to mosquito control as a potential alternative to Bti. But only the cry4Cb gene was successfully expressed in E. coli cells and confirmed the presence of a weak mosquitocidal activity. In the present paper, we not only successfully expressed cry50Ba gene in E. coli cells, but also found that expressed Cry50Ba protein exhibited very high toxic activity against Culex quinquefasciatus larvae. This would suggest that Cry50Ba plays a dominant role in the mosquitocidal toxicity of Bt S2160-1 although may be involved in synergistic interactions with other toxins, as yet unknown, as has been observed in Bti (Chen et al., 2007; Otieno-Ayayo et al., 2008). Thus, the novel Cry50Ba protein from Bt S2160-1 should be taken into consideration for preventing resistance development in mosquito. Acknowledgements This work was funded by the Foundation of Natural Science Foundation of China (31560527) and the Foundation of Education Bureau of Hainan Province (Hnky2017ZD-13). We would like to thank Dr Neil Crickmore kindly transferred Bt strain to the Lab of Hainan Normal University for the research use. References Boyce, R., Lenhart, A., Kroeger, A., Velayudhan, R., Roberts, B., Horstick, O., 2013.
Bacillus thuringiensis israelensis (Bti) for the control of dengue vectors: systematic literature review. Trop. Med. Int. Health 18, 564e577. Chen, J., Hua, G., Jurat-Fuentes, J.L., Abdullah, M.A., Adang, M.J., 2007. Synergism of Bacillus thuringiensis toxins by a fragment of a toxin-binding cadherin. Proc. Natl. Acad. Sci. U. S. A. 104, 13901e13906. Crickmore, N., Ellar, D.J., 1992. Involvement of a possible chaperonin in the efficient expression of a cloned CryIIA delta-endotoxin gene in Bacillus thuringiensis. Mol. Microbiol. 6, 1533e1537. Crickmore, N., Nicholls, C., Earp, D.J., Hodgman, T.C., Ellar, D.J., 1990. The construction of Bacillus thuringiensis strains expressing novel entomocidal deltaendotoxin combinations. Biochem. J. 270, 133e136. Fang, J., Xu, X., Wang, P., Zhao, J.Z., Shelton, A.M., Cheng, J., Feng, M.G., Shen, Z., 2007. Characterization of chimeric Bacillus thuringiensis Vip3 toxins. Appl. Environ. Microbiol. 73, 956e961. Gayen, S., Hossain, M.A., Sen, S.K., 2012. Identification of the bioactive core component of the insecticidal Vip3A toxin peptide of Bacillus thuringiensis. J. Plant Biochem. Biotechnol. 21, 128e135. Misra, H.S., Khairnar, N.P., Mathur, M., Vijayalakshmi, N., Hire, R.S., Dongre, T.K., Mahajan, S.K., 2002. Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae. J. Genet. 81, 5e11. Otieno-Ayayo, Z.N., Zaritsky, A., Wirth, M.C., Manasherob, R., Khasdan, V., Cahan, R., Ben-Dov, E., 2008. Variations in the mosquito larvicidal activities of toxins from Bacillus thuringiensis ssp. israelensis. Environ. Microbiol. 10, 2191e2199. Setha, T., Chantha, N., Benjamin, S., Socheat, D., 2016. Bacterial larvicide, Bacillus thuringiensis israelensis strain AM 65e52 water dispersible granule formulation impacts both dengue vector, Aedes aegypti (L.) population density and disease transmission in Cambodia. PLoS Negl. Trop. Dis. 10, e0004973. Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D.R., Dean, D.H., 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 775e806. Schwede, T., Kopp, J., Guex, N., Peitsch, M.C., 2003. SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res. 31, 3381e3385. Silva-Werneck, J.O., Ellar, D.J., 2008. Characterization of a novel Cry9Bb deltaendotoxin from Bacillus thuringiensis. J. Invertebr. Pathol. 98, 320e328. Tabashnik, B.E., 1992. Evaluation of synergism among Bacillus thuringiensis toxins. Appl. Environ. Microbiol. 58, 3343e3346. Wirth, M.C., Federici, B.A., Walton, W.E., 2000. Cyt1A from Bacillus thuringiensis synergizes activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae). Appl. Environ. Microbiol. 66, 1093e1097. Zhang, W., Crickmore, N., George, Z., Xie, L., He, Y.Q., Li, Y., Tang, J.L., Tian, L., Wang, X., Fang, X., 2012. Characterization of a new highly mosquitocidal isolate of Bacillus thuringiensisean alternative to Bti? J. Invertebr. Pathol. 109, 217e222. Zhang, W., Zhang, J., Crickmore, N., Wu, Z., Yang, Y., Qian, J., Wu, H., Wang, R., Fang, X., 2014. Identification of a mosquitocidal toxin from Bacillus thuringiensis using mass spectrometry. World J. Microbiol. Biotechnol. 30, 3273e3277.
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Short communication
Characterization of a novel mosquitocidal toxin of Cry50Ba and its potential synergism with other mosquitocidal toxins Wenfei Zhang, Silan Yu, Silun Peng, Jianru Gong, Jiangzhao Qian, Jianqiao He, Wenyu Dai, Ruiping Wang* Ministry of Education Key Laboratory for Tropical Animal and Plant Ecology, College of Life Sciences, Hainan Normal University, Haikou, Hainan, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 April 2017 Received in revised form 25 August 2017 Accepted 30 August 2017 Available online 7 September 2017
A putative toxin gene of cry50Ba was successfully expressed in E. coli cells and confirmed that the purified Cry50Ba toxin had very high toxic activity against Culex quinquefasciatus larvae. Furthermore, the potential synergism of Cry50Ba toxin with Cry2Aa, Cry4Aa and Cry11Aa at a ratio of 1:1 was investigated. Although no significant synergism with other toxins was observed, the Cry50Ba as a novel toxin could be used to delay rapid onset of resistance in mosquito. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Bacillus thuringiensis Cry50Ba Synergism Culex quinquefasciatus
1. Introduction Bacillus thuringiensis (Bt), is recommended by the World Health Organization (WHO) for the control of mosquito pests owing to its efficiency and safety (Boyce et al., 2013; Setha et al., 2016). Bt strain S2160-1 was identified as a good candidate strain for the control of mosquito. The insecticidal crystal protein genes of cry30Ea (GenBank ID: EU503140), cry30Ga (GenBank ID: HQ638217), cry50Ba (GenBank ID: GU446675), cry54Ba (GenBank ID: GU446677) and cry4Cb (GenBank ID: KF753696) were successfully identified by using the approach of PCR-restriction fragment length polymorphism (RFLP) and mass spectrometry (Zhang et al., 2012, 2014). In this study, the cry50Ba gene was successfully cloned and expressed in Escherichia coli (E. coli) cells and the purified Cry50Ba toxin was detected to have mosquitocidal activity to the important disease vector of Culex quinquefasciatus larvae in laboratory conditions. Meanwhile, the potential synergism of Cry50Ba toxins with other mosquitocidal toxins of Cry2Aa (GenBank ID: AAA22335),
Cry4Aa (GenBank ID: CAA68485), Cry11Aa (GenBank ID: AAA22352) was investigated further. Unfortunately, there was no significant synergism between them, but the cry50Ba gene still could be a prospective candidate for delaying the rapid onset of resistance in mosquito. 2. Materials and methods 2.1. Bacterial strains, growth conditions and plasmids The wild-type Bt isolate S2160-1 and the reference strains of Bacillus thuringiensis subsp. Israelensis (Bti) were from Laboratory stock. Bt strains were incubated at 28 C in LuriaeBertani (LB) medium or in G-Tris medium as a sporulation medium for crystal protein extraction. E. coli strains were grown at 37 C in LB broth. The pET-30a plasmid served as a standard vector for the expression of cry50Ba genes in E. coli BL21 (DE3) cells (Zhang et al., 2012). 2.2. Sequence analysis
* Corresponding author. College of Life Sciences, Hainan Normal University, Haikou, Hainan 571158, China. E-mail addresses: [email protected], [email protected] (W. Zhang), [email protected] (S. Yu), [email protected] (S. Peng), [email protected] (J. Gong), [email protected] (J. Qian), [email protected] (J. He), 342364690@ qq.com (W. Dai), [email protected], [email protected] (R. Wang). http://dx.doi.org/10.1016/j.toxicon.2017.08.025 0041-0101/© 2017 Elsevier Ltd. All rights reserved.
The conserved blocks analysis was performed by multiple sequence alignment with CLUSTALW software as reported by Schnepf et al. (1998). The theoretical model of Cry50Ba was predicted by homology modelling on the structure of the Cry2Aa (PDB
166
W. Zhang et al. / Toxicon 138 (2017) 165e168
Fig. 1. Multiple sequence alignment and the three dimensional model of Cry50Ba. (A) Sequence alignment of Cry50Ba with homologous Cry proteins in five conserved blocks. (B) The first-approximation three-dimensional model of Cry50Ba toxin was generated by using the homology-modelling algorithm, SWISS-MODEL. The N-terminal Domain I (Red) consists ofa-helical bundle, is involved in membrane insertion and pore formation. Domain II (Blue) and domain III (Green) are involved in receptor recognition and binding. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
entries 1I5P). The amino acid sequence file of Cry50Ba toxin was submitted to Swiss-Model in the expasy server (http://www. expasy.ch/spdbv/) and a preliminary model for Cry50Ba was retrieved (Schwede et al., 2003). And conserved domains were predicted by the NCBI Conserved Domain Search server (https:// www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).
2.3. Expression and purification of cry50Ba genes in E. coli cells To express the cloned cry50Ba genes in E. coli cells, the primer pairs of 50Ba-5(50 -CGggatccATGAATTCATATCAAAATACAAATG30 )/50Ba-3(50 -ACGCgtcgacTTAATTTGTAAATAGATTATCCACC-30 ) were designed to amplify the complete coding sequences (CDS) of cry50Ba gene. The BamHI and SalI restriction sites were introduced into 50 end of the forward and reverse primers respectively, which facilitated subcloning the cry50Ba genes into the expression vector pET-30a. The construct was sequenced to confirm that no frame shifts, stop codons or rearrangements had occurred and then it was transformed into E. coli BL21(DE3) cells. The transformants of pETcry50Ba containing cry50Ba genes and the control with no insert were incubated at 37 C in LB broth supplemented with 12.5 mg/ml Kanamycin with shaking at 220 rpm for about 1.5hr until an OD600 value of the culture reached 0.6. IPTG was added to the culture at a final concentration of 0.2 mM and incubation continued at 37 C for 8 h to induce the cry50Ba gene expression (Misra et al., 2002). The cells were collected by centrifugation at 4 C at 12,000 g for 5min and the pellets were washed with cooled 1M NaCl, sonicated and analyzed by SDSPAGE (Bio-Rad, USA). Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography (Sangon Biotech, Shanghai, China) was used to purify the expressed Cry50Ba proteins with 6 histidine (His) tag at its N-terminus (Gayen et al., 2012). The target recombinant protein was eluted with column buffer containing 250 mM imidazole and 5 mM.1, 4-dithiothreitol.
2.4. Preparation of cry toxin from Bt isolates The acrystalliferous B. thuringiensis subsp. israelensis strain IPS78/11 harboring plasmid pSVP27-cry2Aa (Bt2Aa), pSVP27cry4Aa (Bt4Aa), pSVP27-cry11Aa (Bt11Aa) kindly supplied by Dr Neil Crickmore (School of Biological Sciences, University of Sussex, UK) were used to isolate pure Cry toxins (Crickmore and Ellar, 1992; Crickmore et al., 1990). Cry proteins were produced in G-tris medium containing 5 mg/ml chloramphenicol after 3 days of incubation at 30 C. Spore/crystal mixtures were harvested and washed
Fig. 2. SDS-PAGE analysis of cry50Ba, cry2Aa, cry4Aa and cry11Aa genes expressed in E. coli BL21 (DE3) cells and acrystalliferous Bt strain IPS78/11 respectively. (A) SDSPAGE gel electrophoresis analysis of cry50Ba gene expressed in BL21 (DE3) cells. PM1: protein molecular weight marker; lane 1: E. coli BL21 (DE3) cells harboring pET30a incubated with IPTG; lane 2: E. coli BL21 (DE3) cell harboring pETcry50Ba incubated without IPTG; lane 3: E. coli BL21 (DE3) cells harboring pETcry50Ba incubated with IPTG; lane 4: purified Cry50Ba protein by Ni-NTA. (B) Expression of cry gene in the acrystalliferous Bt IPS78/11. PM2: protein molecular weight marker; lane 5, 6, 7: Purified crystal protein of Cry2Aa, Cry4Aa and Cry11Aa by centrifugation on discontinuous sucrose density gradients.
W. Zhang et al. / Toxicon 138 (2017) 165e168
167
Table 1 Bacterial strains and plasmids. Strains and plasmids B. thuringiensis Bt S2160-1 Bt subsp. israelensis HD522 Bt subsp. Israelensis IPS78/11 Bt2Aa Bt4Aa Bt11Aa Escherichia coli JM110 BL(DE3) Plasmids pET-30a pET50Ba
Description
Source
Wild strain of Bt haboring cry4Cb, cry30Ea, cry30Ga, cry50Ba and cry54Ba genes. Model strain of Bt haboring cry4Aa, cry4Ba, cry10Aa, cry11Aa, cyt1Aa, and cyt2Ba genes Acrystalliferous mutant of B. thuringiensis subsp. israelensis strain Bt subsp. Israelensis IPS78/11 transformed with the plasmid of pSVP27-cry2Aa Bt subsp. Israelensis IPS78/11 transformed with the plasmid of pSVP27-cry4Aa Bt subsp. Israelensis IPS78/11 transformed with the plasmid of pSVP27-cry11Aa
Lab stock Lab stock Dr Neil Crickmore Dr Neil Crickmore Dr Neil Crickmore Dr Neil Crickmore
Dam, dcm, supE44, hsdR17, thi,leu, rpsL1, lacY galK, galT, aratonA thr, tsx, D(lac-proAB) (F0, traD36, proAB, lacI qZDM15) F e ompT hsdSB(rBe mBe) gal dcm (DE3)
Novagen Novagen
KanR, HisTag/STag hsdSgal (lcTs857 indl Sam7nin5 lacUV5-T7gene) AmpR, pET-30a harboring cry50Ba1 gene
Novagen This study
three times with 1M cooled NaCl. Cry toxins were purified by centrifugation on discontinuous sucrose density gradients and solubilized by using 50 mM Na2CO3 for 1 h at pH11.0 (SilvaWerneck and Ellar, 2008).
five conserved sequence blocks common to a large majority of the Cry proteins. According to the conserved domain search and homology modelling analysis, three conserved domains were found out which indicated that the Cry50Ba was a typical three-domain insecticidal crystal protein (Fig. 1).
2.5. Mass spectrometry analysis The expressed of Cry proteins samples were analyzed using SDSPAGE gel electrophoresis. The target protein bands were excised from SDS- PAGE gel and analyzed with a 4800 MALDIeTOF/TOF mass spectrometer (Applied Biosystems, USA) as described by Wenfei Zhang et al. (2014). 2.6. Larvicidal assays and analysis The concentrations of expressed Cry proteins were estimated by using the Quantity One analysis software program followed the Instruction Manual (Bio-Rad, USA) (Fang et al., 2007). Mosquito larvicidal assays against 2nd instar Culex quinquefasciatus were performed as described previously (Zhang et al., 2012). The Cry50Ba toxin was mixed with Cry2Aa, Cry4Aa and Cry11Aa separately (1:1 ratio) and the resulting mixtures were prepared in twofold serial dilutions in dechlorinated water (seven concentrations: 14.6e1400 ng/ml). Mortality was recorded after incubation at 26 C for 48 h. The 50% lethal concentration (LC50) was determined by Probit analysis with software package SPSS 13.0 for windows (SPSS inc., Chicago, USA). The theoretical LC50 and synergism factor were evaluated from LC50 value of individual toxin by using the Tabashnik equation (Tabashnik, 1992; Wirth et al., 2000). 3. Results and discussion 3.1. Molecular model of Cry50Ba toxin Alignment of the Cry toxins reveals that Cry50Ba toxin presents
3.2. Over-expression of cry50Ba gene in E. coli cells and protein purification In an attempt to express the identified cry50Ba genes, the coding region amplified by PCR was inserted into pET-30a and the recombinant plasmids were transferred into E. coli BL21 (DE3) cells. The addition of IPTG to the recombinant strains resulted in the overexpression of Cry50Ba, which was detected on Coomassie blue stained SDS-PAGE gel with molecular weight of approximately 75 kDa (Fig. 2A). The recombinant Cry50Ba protein was then purified with Ni-NTA affinity chromatography, due to the presence of 6 histidine tail at the N-terminal end. The purified Cry50Ba proteins were corroborated by using mass spectrometry (see Table 1). 3.3. Mosquito larvicidal activity and synergism Mosquito larvicidal test results (Table 2) showed that Cry50Ba exhibited high activity against Culex quinquefasciatus larvae with a LC50 of 73.87 ng/ml, which was about seven times more toxic than Cry2Aa(LC50: 528.26 ng/ml). But Cry50Ba is nontoxic to lepidopterous larvae of plutella xylostella and Helicoverpa armigera (data not shown). When mixing the toxins of Cry50Ba and with Cry2Aa, Cry4Aa, Cry11Aa were mixed with a ratio of 1:1 separately, unfortunately the synergism among the toxins of Cry50Ba, Cry4Aa and Cry11Aa was not observed in this study. The combined toxins of Cry2Aa and Cry50Ba yielded a LC50 of 46.91 ng/ml that exhibited more insecticidal activity against Culex quinquefasciatus larvae than the observed activities for Cry50Ba and Cry2Aa alone. The
Table 2 Mosquito larvicidal activity of Cry50Ba, Cry2Aa, Cry4Aa and Cry11Aa alone and combination against 2nd instar larvae of Culex quinquefasciatus. Toxins
Experimental LC50(ng/ml)(95%FL minemaxb)
Theoretical LC50(ng/ml)c
Synergy factorc
Cry50Ba Cry2Aa Cry 4Aa Cry11Aa Cry50Ba þ Cry2Aaa Cry50Ba þ Cry4Aaa Cry50Ba þ Cry11Aaa
73.87(51.40e102.75) 528.26(374.86e835.58) 51.88 (37.86e69.82) 13.78 (10.04e18.56) 46.91(34.31e61.60) 54.24 (40.72e76.04) 31.01 (24.72e39.65)
129.61 60.98 23.23
2.76 1.12 0.75
a b c
Toxin mixtures are combinations of Cry50Ba and another Cry protein with ratio 1:1. 95% FL is the 95% fiducial limits in parentheses, as determined by probit analysis. Theoretical LC50 and Synergism factor (SF) calculated by the method of Tabashnik (1992).
168
W. Zhang et al. / Toxicon 138 (2017) 165e168
theoretical LC50 of the combined toxins calculated using the formula of Tabashnik was 129.61 ng/ml. As the result, the ratios of the experimental LC50 to theoretical LC50 or synergism factor (SF) was 2.76. The 95% fiducial limits of LC50 values for Cry50Ba and those of the mixture of Cry50Ba and Cry2Aa overlap so the data shows no significant synergism with the Cry2Aa toxin against Culex quinquefasciatus larvae. 4. Conclusions Bt strain S2160-1 is carrying insecticidal crystal protein genes of cry30Ea, cry30Ga, cry50Ba, cry54Ba and cry4Cb with a comparable mosquitocidal activity to Bti. It is believed that Bt S2160-1 could be applied to mosquito control as a potential alternative to Bti. But only the cry4Cb gene was successfully expressed in E. coli cells and confirmed the presence of a weak mosquitocidal activity. In the present paper, we not only successfully expressed cry50Ba gene in E. coli cells, but also found that expressed Cry50Ba protein exhibited very high toxic activity against Culex quinquefasciatus larvae. This would suggest that Cry50Ba plays a dominant role in the mosquitocidal toxicity of Bt S2160-1 although may be involved in synergistic interactions with other toxins, as yet unknown, as has been observed in Bti (Chen et al., 2007; Otieno-Ayayo et al., 2008). Thus, the novel Cry50Ba protein from Bt S2160-1 should be taken into consideration for preventing resistance development in mosquito. Acknowledgements This work was funded by the Foundation of Natural Science Foundation of China (31560527) and the Foundation of Education Bureau of Hainan Province (Hnky2017ZD-13). We would like to thank Dr Neil Crickmore kindly transferred Bt strain to the Lab of Hainan Normal University for the research use. References Boyce, R., Lenhart, A., Kroeger, A., Velayudhan, R., Roberts, B., Horstick, O., 2013.
Bacillus thuringiensis israelensis (Bti) for the control of dengue vectors: systematic literature review. Trop. Med. Int. Health 18, 564e577. Chen, J., Hua, G., Jurat-Fuentes, J.L., Abdullah, M.A., Adang, M.J., 2007. Synergism of Bacillus thuringiensis toxins by a fragment of a toxin-binding cadherin. Proc. Natl. Acad. Sci. U. S. A. 104, 13901e13906. Crickmore, N., Ellar, D.J., 1992. Involvement of a possible chaperonin in the efficient expression of a cloned CryIIA delta-endotoxin gene in Bacillus thuringiensis. Mol. Microbiol. 6, 1533e1537. Crickmore, N., Nicholls, C., Earp, D.J., Hodgman, T.C., Ellar, D.J., 1990. The construction of Bacillus thuringiensis strains expressing novel entomocidal deltaendotoxin combinations. Biochem. J. 270, 133e136. Fang, J., Xu, X., Wang, P., Zhao, J.Z., Shelton, A.M., Cheng, J., Feng, M.G., Shen, Z., 2007. Characterization of chimeric Bacillus thuringiensis Vip3 toxins. Appl. Environ. Microbiol. 73, 956e961. Gayen, S., Hossain, M.A., Sen, S.K., 2012. Identification of the bioactive core component of the insecticidal Vip3A toxin peptide of Bacillus thuringiensis. J. Plant Biochem. Biotechnol. 21, 128e135. Misra, H.S., Khairnar, N.P., Mathur, M., Vijayalakshmi, N., Hire, R.S., Dongre, T.K., Mahajan, S.K., 2002. Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae. J. Genet. 81, 5e11. Otieno-Ayayo, Z.N., Zaritsky, A., Wirth, M.C., Manasherob, R., Khasdan, V., Cahan, R., Ben-Dov, E., 2008. Variations in the mosquito larvicidal activities of toxins from Bacillus thuringiensis ssp. israelensis. Environ. Microbiol. 10, 2191e2199. Setha, T., Chantha, N., Benjamin, S., Socheat, D., 2016. Bacterial larvicide, Bacillus thuringiensis israelensis strain AM 65e52 water dispersible granule formulation impacts both dengue vector, Aedes aegypti (L.) population density and disease transmission in Cambodia. PLoS Negl. Trop. Dis. 10, e0004973. Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D.R., Dean, D.H., 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 775e806. Schwede, T., Kopp, J., Guex, N., Peitsch, M.C., 2003. SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res. 31, 3381e3385. Silva-Werneck, J.O., Ellar, D.J., 2008. Characterization of a novel Cry9Bb deltaendotoxin from Bacillus thuringiensis. J. Invertebr. Pathol. 98, 320e328. Tabashnik, B.E., 1992. Evaluation of synergism among Bacillus thuringiensis toxins. Appl. Environ. Microbiol. 58, 3343e3346. Wirth, M.C., Federici, B.A., Walton, W.E., 2000. Cyt1A from Bacillus thuringiensis synergizes activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae). Appl. Environ. Microbiol. 66, 1093e1097. Zhang, W., Crickmore, N., George, Z., Xie, L., He, Y.Q., Li, Y., Tang, J.L., Tian, L., Wang, X., Fang, X., 2012. Characterization of a new highly mosquitocidal isolate of Bacillus thuringiensisean alternative to Bti? J. Invertebr. Pathol. 109, 217e222. Zhang, W., Zhang, J., Crickmore, N., Wu, Z., Yang, Y., Qian, J., Wu, H., Wang, R., Fang, X., 2014. Identification of a mosquitocidal toxin from Bacillus thuringiensis using mass spectrometry. World J. Microbiol. Biotechnol. 30, 3273e3277.