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Characterization of a new highly mosquitocidal isolate of Bacillus thuringiensis – An alternative to Bti?
Journal of Invertebrate Pathology 109 (2012) 217–222
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Characterization of a new highly mosquitocidal isolate of Bacillus thuringiensis – An alternative to Bti? Wenfei Zhang a,b, Neil Crickmore c, Zenas George c, Liu Xie b, Yong-Qiang He a, Youzhi Li a, Ji-Liang Tang a, Liang Tian b, Xi Wang b, Xuanjun Fang a,b,⇑ a b c
College of Life Science and Technology, Guangxi University, Nanning, Guangxi, China Hainan Institute of Tropical Agricultural Resources (HITAR), Sanya, Hainan, China School of Life Sciences, University of Sussex, Falmer, Brighton, UK
a r t i c l e
i n f o
Article history: Received 12 September 2011 Accepted 5 November 2011 Available online 22 November 2011 Keywords: Bacillus thuringiensis Mosquitocidal activity Cry-type gene PCR-RFLP
a b s t r a c t The mosquito is a very important vector involved in the worldwide transmission of disease-causing viruses and parasites. Controlling the mosquito population remains one of the best means for preventing the serious infectious diseases of malaria, yellow fever, dengue, filariasis and so on and there has been an increasing interest in developing biopesticides as a useful substitute to chemical insecticides. As a result, Bacillus thuringiensis subsp. israelensis (Bti) has been extensively used due to its specificity and high toxicity to a variety of mosquito larvae. However it is prudent to seek alternatives to Bti with alternative spectra of mosquitocidal activity or that are able to overcome any resistance that might develop against Bti. The Bt S2160-1 strain was isolated from soil samples collected from Southern China and found to have a comparable mosquitocidal activity to Bti. However there were significant differences in terms of their plasmid profiles, crystal proteins produced and cry gene complement. A PCR-restriction fragment length polymorphism identification system was developed and used in order to identify novel cry-type genes and four such genes (cry30Ea, cry30Ga, cry50Ba and cry54Ba) were identified in Bt S2160-1. In conclusion, Bt S2160-1 has been identified as a potential alternative to Bti, which could be used for the control of mosquito populations in order to reduce the incidence of mosquito-borne diseases. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Mosquitoes act worldwide as vectors transmitting diseasecausing viruses and parasites such as malaria, yellow fever, dengue fever, filariasis, St. Louis encephalitis and the West Nile virus between humans and animals (Tolle, 2009). The World Health Organization (WHO) estimates that half of the world’s population is at risk of malaria and there have been about 250 million cases leading to nearly 1 million deaths. Of these 91% were in Africa and 85% were of children under 5 years of age (WHO, 2008). Yellow fever causes 200,000 illnesses and 30,000 deaths every year. Dengue fever has become a major international public health concern in recent years; two-fifths of the world’s population is now at risk from dengue. It is estimated that there may be 50 million cases of dengue infection worldwide every year (Tolle, 2009; WHO, 2009). In Southern China, there is a large-scale cultivation of rice using the continuous submergence method for irrigation, which provides such a favorable habitat for mosquito breeding that mosquito-borne diseases are flourishing (Pal, 1982). ⇑ Corresponding author at: Hainan Institute of Tropical Agricultural Resources (HITAR), Sanya, Hainan 572025, China. Fax: +86 0771 3232621. E-mail address: [email protected] (X. Fang). 0022-2011/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2011.11.003
At present, the effective prophylactic drugs and vaccines against the West Nile virus, dengue, malaria, and filariasis are either too expensive or not available to those most at risk. To reduce the incidence of these diseases more reliance has been put on the control of mosquito populations. Until recently mosquito control was dominated by the spraying of synthetic chemical insecticides such as malathion and dichloro-diphenyl-trichloroethane (DDT) which were very effective in the short term. The mosquito though is prone to developing resistance to these insecticides, in addition chemical insecticides leave harmful residues which impact on non-target species, including humans and livestock (Federici et al., 2003). Due to the development of resistance, and the growing public concerns associated with adverse environmental effects of the chemical insecticides, it has been necessary to initiate an alternative strategy for mosquito control. Biopesticides, especially Bacillus thuringiensis (Bt), have received much attention due their lack of significant effect on humans, wildlife or beneficial insects (Bravo et al., 2007; Federici, 2005; Regis et al., 2001). B. thuringiensis subsp. israelensis (Bti) has been extensively studied for its specific and high toxicity to mosquito and black fly larvae since its discovery in 1976. The parasporal inclusion body of Bti consists of at least four major insecticidal crystal proteins (Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa) whilst others may be present in
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lesser amounts (Berry et al., 2002; Stein et al., 2006). Although Bti and its toxins have been successfully commercialized for mosquito control, screening programs have continued worldwide to identify and characterize new mosquitocidal Bt isolates and toxin genes (Bravo et al., 1998). Mosquitocidal Bt strains such as subsp. morrisoni PG-14, subsp. jegathesan, subsp. kyushuensis, subsp. medellin and subsp. darmstadiensis 73-E10-2 have previously been characterized but, with the exception of PG-14 (which is effectively Bti with an additional toxin), are less toxic to mosquitoes than Bti (Drobniewski and Ellar, 1989; Federici et al., 2003; Kawalek et al., 1995; Knowles et al., 1992; Ragni et al., 1996). We have initiated a screening program to collect more than 3000 soil samples from the Tropical Rainforest Nature Reserve in Hainan Province (Jianfengling, Wuzhishan, Dianluoshan and Bawangling) and the Subtropical Forest Nature Reserve in Guangxi Province (Dawangling, Shiwandashan and Longgang). More than 30 Bt isolates with mosquitocidal activity have been characterized with one, the isolate Bt S2160-1, having a comparable mosquitocidal activity to Bti.
Japan) and viewed on a HITACHI S-3400N (HITACHI, Japan) at a voltage of 20 kV. 2.3. Parasporal protein preparation Bt isolates were grown to lysis in G-Tris broth at 30 °C with shaking at 220 rpm and the spore–crystal mixture was harvested by centrifugation at 4 °C at 12,000g for 10 min. The pellet was washed twice in an equal volume of ice-cold 1 M NaCl containing 0.01% Triton X-100 and then twice in an equal volume of distilled water. Subsequently the suspension was sonicated 15 times (30 s, 30 s off) on ice at full amplitude (model VC-250, Sonics and Materials Inc., USA). After centrifugation at 4 °C at 12,000g for 10 min, the resulting pellet was suspended in 1/10 of the original volume of 50 mM Na2CO3 (pH10.0) and the protein concentration was measured using BCA (bicinchoninic acid) protein assay kit (Tiangen, Beijing, China) following the manufacturer’s instruction (Iracheta et al., 2000). Then SDS–PAGE analysis was conducted on mini-protean cell slab vertical apparatus (Bio-Rad, USA). 2.4. Bioassay against Plutella xylostella and mosquitoes
2. Materials and methods 2.1. Bacterial strains, growth conditions and plasmids The bacterial strains and plasmids used in this study are listed in Table 1. The wild type Bt isolate S2160-1 was isolated from soil samples in the Dawangling Forest Nature Reserve (Guangxi, China). Bt strains were incubated at 30 °C in Luria–Bertani (LB) medium for the preparation of plasmid DNA or in G-Tris medium (Aronson and Thompson, 1971) as a sporulation medium for SEM analysis and crystal protein extraction. Escherichia coli JM110, used as a cloning host for all constructed plasmids, was grown at 37 °C in LB broth. The pGEM-3Zf (+) plasmid served as a standard cloning vector for the construction of the Bt S2160-1 DNA library. Ampicillin (100 lg/ml) was added to E. coli culture medium as required.
2.2. Scanning electron microscopy Bt strains were grown in G-Tris medium at 30 °C for over 72 h until sporulation was complete as determined by light microscopy. The spores and crystals were pelleted by centrifugation at 4 °C at 12,000g for 10 min then washed three times with ice-cold 1 M NaCl and then three times with sterile distilled water. The spore– crystal suspension was placed on aluminum mounts and fixed in 1% OsO4 after the samples had been air-dried overnight. Then the samples were coated with gold in an IB-5 ion coater (HITACHI,
A susceptible population of P. xylostella collected from Sanya, Hainan province, China and reared on pesticide-free greenhousegrown Chinese cabbage in an environment-controlled room. Disks of 8 cm diameter were cut from Chinese cabbage leaves and were immersed in serial dilutions of toxin crystals (in 0.02% Triton X-100) for 10 s, and allowed to air dry at ambient temperature for 2 h. The control leaf disks were immersed in distilled water with 0.02% Triton X-100. Each leaf disk was placed in an individual petri dish (10 cm diameter) lined with moistened filter paper. Five third-instar larvae of P. xylostella were introduced into each petri dish (Sayyed et al., 2001). Bioassays against mosquitoes were performed following the standard procedures recommended by WHO (Navon and Ascher, 2000; WHO, 1996). The parasporal proteins were diluted in plastic cups with 30 ml dechlorinated water for larvicidal activity test and cups with only 30 ml dechlorinated water were used as a negative control. Third instar larvae of Culex quinquefasciatus and Aedes albopictus, supplied by Guangxi Center for Diseases Prevention and Control (Guangxi, China), were exposed to serial dilutions of toxins. 30 mosquito larvae were introduced into each cup with a pipette and the mortality was recorded after 48 h. All the bioassays were conducted at 26 °C with 60–70% relative humidity and a photoperiod of 14 h light: 10 h dark. At least 400 third-instar larvae were tested with each protein preparation and 100 larvae for the controls. Protein samples were diluted into twofold serial dilutions with eight concentrations such that at least
Table 1 Bacterial strains and plasmids. Strains and plasmids
Description
Source
B. thuringiensis Bt S2160-1 Bt subsp. israelensis HD522 Bt subsp. israelensis AND508 Bt subsp. kurstaki HD1 Bt subsp. kurstaki HD73
Wild type strain of Bt Model strain of Bt haboring cry4Aa, cry4Ba, cry10Aa, cry11Aa, cyt1Aa, and cyt2Ba genes A variant of Bti AND406 cured of pTX14-2 and pTX14-3 Model strain of Bt haboring cry1Aa, cry1Ab, cry1Ac, cry1I, cry2Aa genes Model strain of Bt haboring cry1Ac gene
Lab Lab Lab Lab Lab
Escherichia coli JM110
Dam, dcm, supE44, hsdR17, thi,leu, rpsL1, lacY galK, galT, aratonA thr, tsx, D(lac-proAB) (F0, traD36, proAB, lacI qZDM15)
Novagen
Plasmids pMD18-T pGEM-3Zf(+) pMD30 pMD40 pGEMS4
AmpR, AmpR, AmpR, AmpR, AmpR,
Takara Promega This study This study This study
T-A cloning vector E. coli cloning vector pMD18-T harboring cry30Ea1 gene pMD18-T harboring cry50Ba1 gene pGEM-3Zf(+) harboring cry54Ba1 gene
stock stock stock stock stock
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five of the concentrations gave a range of mortalities between 10% and 90%, moreover the control mortality was less than 5%. The LC50 values were estimated by probit analysis using SPSS 13.0 software for windows (SPSS Inc., Chicago, USA). 2.5. Plasmid DNA isolation and plasmid profile Plasmid DNA was isolated from Bt strains grown to an OD600 value of 2 at 30 °C in LB broth with shaking at 220 rpm according the method of Song et al. (2003). CHEF MapperÒ XA Pulsed Field Electrophoresis System (Bio-Rad, USA) was used for the detection of the plasmid patterns of Bt. The parameters were set up for separating a DNA sample with a size range from 15 kb to 500 kb (voltage: 6 V/cm, run time: 22 h, included angle: 120°, initial switch interval time: 1.19 s, final switch interval time: 44.69 s). The plasmid DNA was subjected to electrophoresis in 1% low-melting-point agarose (Amresco, USA) at 14 °C with 0.5 TBE buffer. The plasmid DNA was stained in ethidium bromide (5 lg/ml) after electrophoresis and destained in double-distilled water (at 4 °C) for 24 h and then visualized under ultraviolet light. 2.6. Identification of cry-type genes by PCR–RFLP Pairs of universal oligonucleotide primers designed based on the conserved blocks from known cry-type genes, were using to screen Bt S2160-1 (Xie et al., 2009). The universal primer pairs of S5un4/S3un4, Up30-5/Up30-3 and Up39.40-5/Up39.40-3 (Table 2) were designed to detect cry4, cry10, cry30, cry39 and cry40 genes. PCR amplification was performed in a PTC200 Thermo Cycler (MJ Research, USA) using the following programme: an initial denaturation of 5 min at 94 °C, followed by 30 cycles (each cycle was comprised of 94 °C for 1 min, 53 °C for 1 min, 72 °C for 3 min), a final extension for 10 min at 70 °C. The resulting amplicons were purified with Tiangen Midi Purification Kit (Tiangen, Beijing, China). The appropriate restriction endonucleases (Fermentas) were selected to digest the PCR fragment and the resulting restriction fragments were separated according to their length by agarose gel electrophoresis (Kuo and Chak, 1996). The expected size of PCR products and restriction fragments of cry-type genes were determined in silico and the results are listed in Table 3. Purified PCR fragments were cloned into the pMD18-T vector for sequencing (BGI, Shenzen, China) and the resulting sequence was subjected to a blastx homology search against the nonredundant protein database of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/Blast.cgi).
Table 2 Oligonucleotides used in this study. Name of primer
Sequence of primer
Reference
S5un4 S3un4
50 -GTGTCAAGAGAACCAACAGTATG-30 50 -ACTAAGTCTCCTCCTGTATGACCAG30 50 -TTGGCTCAATATGTGTCAAAC-30 50 -GCTTTAACAGCAGGAATTTG-30 50 -TAAGAGGGTTTGTGGGAAGTAG-30 50 -ACTTTCTGGGAATACCTCTACTG-30 50 -ATGAGGGAGTGAAAAAGATGAAT-30 50 -TCTAAGCTTTAGTTCACTGTACAAG30 50 -ATACGAGGAGTGAAATAGATG-30 50 -CACTTGTAAACATCGGACT-30 50 -CAGCGACTGAATTAATGTCA-30 50 -GTCTATAGTGGTACAAAGTGGAGTA30 50 -TGGTAATGCGTCCACAACTCAAC-30 50 -TGAATGGCCCACAAAATGCATC-30
Song et al. (1998)
Up30-5 Up30-3 Up39.40-5 Up39.40-3 F30-5 F30-3 E40-5 E40-3 S30F1 S30F2 S30R1 S30R2
This study This study This study
This study This study
This study
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2.7. Cloning of the novel cry-type genes The primer pairs of F30-5/F30-3 and E40-5/E40-3 (Table 2) were designed from the sequences of the published cry30-type and cry40-type genes and designed to clone the full sequence of cry30A-like and cry40B-like genes respectively. Expand PCR was performed using Ex Taq polymerase (Takara, Dalian, China) and the PCR products purified by gel isolation were ligated into pMD18-T vector for sequencing. For the cloning of the full sequence of cry30G-like gene, the strategy of single oligonucleotide nested (SON)-PCR was used. The four specific primers were designed according the known sequence of the PCR fragment, the primers of S30R1 and S30R2 were for amplification of 50 region of the cry30G-like gene and the primers S30F1 and S30F2 were for 30 region (Antal et al., 2004). In order to clone the cry54A-like gene, a library from Bt S2160-1 plasmid DNA was constructed. The plasmid DNA of Bt S2160-1 was partially digested with BclI and fractionated by 0.7% agarose gel electrophoresis. Restriction fragments with sizes of 6–20 kb were purified from the gel and inserted into BamHI digested and dephosphorylated pGEM-3Zf (+) vector and then introduced into competent cells of E. coli JM110 to make the library. The method of limited growth PCR was utilized to screen the library for the presence of the cry54-like gene with the primer pairs of S5un4/S3un4 (Cheong and Gill, 1997; Ross and Gill, 1996). The positive recombinant plasmid was sequenced in both directions by primer walking (BGI, Shenzen, China). 2.8. Nucleotide sequence accession numbers The sequences of three novel cry-type genes cloned in this paper have been deposited in the GenBank database and also submitted to the Bt delta-endotoxin nomenclature committee to assign names. GenBank accession numbers: EU503140 (cry30Ea1), HQ638217 (cry30Ga2), GU446675 (cry50Ba1), GU446677 (cry54Ba1). 3. Results and discussion 3.1. Parasporal inclusion morphology and profile The crystalline inclusions produced during sporulation are the most important characteristic of Bt, and distinguish it from closely related species of Bacillus cereus and Bacillus anthracis. Moreover determination of the crystal morphology can identify certain toxin types such as the typical bipyramidal crystals of Cry1 or Cry9 proteins toxic to lepidopteran insects (Kaur and Singh, 2000). Bt S2160-1 was grown until crystals were observed through a light microscope, subsequently the crystals were examined by SEM. The electron micrograph of the parasporal crystals from Bt S2160-1 showed freely released crystals which were spherical in shape (Fig. 1A). SDS–PAGE analysis was employed to compare the polypeptide composition of parasporal proteins from Bt S2160-1 and Bti (Fig. 1B). Post sporulation samples from Bt S2160-1 contained four major polypeptides of around 140 kDa, 130 kDa, 75 kDa and 30 kDa which although similar to those produced by Bti did appear different on the gel. With 1 lg/ml trypsin treatment, the crystal proteins of Bt S2160-1 and Bti were processed primarily into 50 kDa fragments. 3.2. Plasmid profiles Bt commonly harbors a number of large plasmids of variable size, the vast majority of the Cry toxin genes are located on these plasmids (Carlson et al., 1996) and the diversity of plasmid sizes in Bt strains is frequently used as a tool to characterize specific strains (Porcar et al., 1999; Vilas-Boas and Lemos, 2004). Pulsed
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Table 3 PCR-RFLP patterns of the Bt S2160-1 amplicons compared with the theoretical sizes for known cry genes. Genes
Primers
Size of amplified product (bp)
Enzymes used to digest PCR product
Size of restriction fragments (bp)
S2160-1 cry4Aa cry4Ba cry10Aa S2160-1 cry30Aa cry30Ba cry30Ca S2160-1 cry40Aa cry40Ba cry40Da
S5un4/S3un4 S5un4/S3un4 S5un4/S3un4 S5un4/S3un4 Up30-5/Up30-3 Up30-5/Up30-3 Up30-5/Up30-3 Up30-5/Up30-3 Up39.40-5/Up39.40-3 Up39.40-5/Up39.40-3 Up39.40-5/Up39.40-3 Up39.40-5/Up39.40-3
1548 1551 1449 1467 1388,1463 1464 1461 1461 1005 1001 886 890
BanI + HaeIII BanI + HaeIII BanI + HaeIII BanI + HaeIII BglII + PstI BglII + PstI BglII + PstI BglII + PstI HindIII + HaeIII HindIII + HaeIII HindIII + HaeIII HindIII + HaeIII
1025, 523 1551 878, 341, 157, 73 742, 499, 226 1388, 906, 557 662, 560, 220 996, 426 864, 374, 184 1005 615, 386 615, 290, 96 450, 390, 89, 76
Fig. 1. Scanning electron micrograph and SDS–PAGE analysis of parasporal inclusions from Bt S2160-1. (A) Scanning electron micrograph of spores (SP) and crystals (C) from Bt S2160-1. (B) SDS–PAGE analysis of Bt S2160-1 and Bti. PM: protein molecular weight marker; lanes 1 and 4: samples taken before sporulation from Bti and Bt S2160-1 respectively; lanes 2 and 5: samples taken after sporulation from Bti and Bt S2160-1 respectively; lanes 3 and 6: samples from sporulated cultures of Bti and Bt S2160-1 respectively treated with 1 lg/ml trypsin.
Field Gel Electrophoresis (PFGE) was employed to compare the plasmid profiles of Bt S2160-1 with Bti and various other reference strains. Fig. 2 shows that the plasmid profile of Bt S2160-1 was significantly different to that of Bti and other strains and in particular lacked the major toxin-encoding pBtoxis plasmid of Bti. 3.3. Insecticidal activities of Bt S2160-1 against mosquitoes and P. xylostella Bioassays against P. xylostella and two species of mosquito were conducted using parasporal inclusions of Bt S2160-1 and Bti. The insecticidal activity was assessed using LC50 values. Table 4 reveals that the parasporal proteins of Bt S2160-1 and Bti both exhibited high toxicities to larvae of C. quinquefasciatus and A. albopictus, but not to P. xylostella. 3.4. Identification of cry-type genes Due to the high toxicity of Bt S2160-1, combined with the observation that it was phenotypically distinct from Bti, a PCRRFLP identification system was used to identify cry genes within this strain. 37 pairs of universal oligonucleotide primers were used to detect cry genes, of which the primer pairs of S5un4/S3un4, Up30-5/Up30-3 and Up39.40-5/Up39.40-3 produced PCR products. The PCR amplicons were digested with pairs of restriction endonucleases to further characterize the type of cry gene present (Fig. 3). Table 3 compares the results obtained with known cry genes and
Fig. 2. PFGE patterns of the large plasmids from Bt S2160-1 and other strains. Lane 1: Bt subsp kurstaki HD-73; lane 2: Bt subsp kurstaki HD-1; lane 3: Bt subsp israelensis AND508 with the 350 kb pXO16 and 128 kb pBtoxis plasmids (Wilcks et al., 2008) highlighted; lane 4: Bt S2160-1.
these data indicate the presence of novel cry genes due to the differences in band sizes obtained in comparison to the reference genes. It should also be noted that in contrast to Bti no cry4 or cry10 genes were identified using the S5un4/S3un4 primers and also that no PCR products were obtained from Bt S2160-1 using primers that successfully identified the cry11, cyt1 and cyt2 genes in Bti (data not shown). The PCR fragments obtained were cloned into the pMD18-T vector and sequenced. This analysis revealed that the deduced amino acid sequence of the cry genes encoded on the PCR products generated by primer pairs S5un4 /S3un4, and Up39.40-5/Up39.40-3 had highest identities with Cry54Aa1 (51%), and Cry40Ba1 (88%) respectively. The PCR fragment produced by Up30-5/Up30-3 was mixture of two different sequences, one can be digested into the fragments of 906 bp and 557 bp by BglII and PstI, the other has no recognition sites for these enzymes. Their deduced amino acid sequences show the highest identities with Cry30Aa1 (72%) and Cry30Ga1 (99%) respectively. 3.5. The cloning of novel cry genes In order to obtain the full length cry54A-like gene, a Bt S2160-1 plasmid DNA library was screened using the primer set S5un4/
W. Zhang et al. / Journal of Invertebrate Pathology 109 (2012) 217–222
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Table 4 Larvicidal activity of spore/crystal mixtures from Bt S2160-1 and Bti against Plutella xylostella and mosquitoes. Tested insect
S2160-1 LC50 (ng/ml) (95% FL)
Plutella xylostella Culex quinquefasciatus Aedes albopictus a
Bti a
>200,000 5.668 (4.262–7.464) 21.113 (12.608–36.393)
Slope
LC50 (ng/ml) (95% FL)a
Slope
– 0.184 0.173
>200,000 1.718 (1.216–2.313) 45.585 (34.998–62.385)
– 0.23 0.237
95% FL are the 95% fiducial limits in parentheses, as determined by probit analysis.
Fig. 3. PCR–RFLP patterns of cry-type genes from Bt S2160-1. M. DNA molecular weight marker; lanes 1, 2 and 3 are PCR amplification products using primer pairs S5un4/S3un4, Up30-5/Up30-3 and Up39.40-5/Up39.40-3 respectively; lanes 4, 5 and 6 represent the above PCR products digested with BanI/HaeIII, BglII/PstI and HindIII/HaeIII respectively.
S3un4. A positive clone designated as pGEMS4 was identified and sequenced. Genomic DNA fragments containing the full length sequences of cry30A-like and cry40B-like genes were obtained by expand PCR with the primer pairs of F30-5/F30-3 and E40-5/E40-3. These were then ligated into the pMD18-T vector to generate the recombinant plasmids of pMD30 and pMD40 for sequencing. The recombinant plasmid pMD30 was shown to harbor the full sequence of a cry30-like gene, whose deduced amino acid sequence indicated a protein with estimated molecular weight of 76 kDa with 75% sequence similarity to Cry30Aa1. With pMD40 and pGEMS4 putative toxins were identified that had 63% and 50% similarity with Cry50Aa1 and Cry54Aa1 respectively. The upstream and downstream flanking sequences of cry30G-like were obtained through SON–PCR strategy. The assembled sequence contains a 1,995 bp open reading frame, which encoded a 664 amino acid protein showed 99% identity with Cry30Ga1. The four novel cry-type genes were designated as cry30Ea1, cry30Ga2, cry50Ba1 and cry54Ba1 according to the nomenclature system proposed by Crickmore et al. (1998) and encoded proteins with predicted molecular weights of 77.6 kDa, 74.4 kDa, 76.3 kDa and 79.6 kDa respectively. 4. Conclusions In this paper, we have identified a new mosquiticidal Bt isolate S2160-1 with comparable toxicity to that of the reference strain of Bti against two species of mosquito. In all other respects the strains are dissimilar with differing plasmid profiles, cry genes and crystal proteins. Thus, it is believed that Bt S2160-1 could be applied to mosquito control as a potential alternative to Bti. Methods based on PCR amplification have been developed to detect cry genes in Bt strains and PCR–PFLP is becoming a popular and powerful approach, which is used not only to confirm the presence of the known genes, but also to discover novel ones (Bravo et al., 1998; Ibarra et al., 2003; Kuo and Chak, 1996; Noguera and Ibarra, 2010; Song et al., 2003). This approach was successfully used in this study to identify the cry30Ea, cry30Ga, cry50Ba and cry54Ba
genes in Bt S2160-1 but did not identify the genes encoding at least three of the main parasporal proteins (140 kDa, 130 kDa and 30 kDa). The SDS PAGE profile of parasporal inclusions from Bt S2160-1 were also different from those of subsp. jegathesan (77, 74, 72, 68, 55, 38, 35, 27, and 23 kDa) (Kawalek et al., 1995), subsp. kyushuensis (140, 85, 80, 70, 66, 50, 25 kDa) (Knowles et al., 1992), subsp. medellin (95, 67, 30 kDa) (Ragni et al., 1996) and subsp. darmstadiensis 73E10-2 (125, 50, 47, and 28 kDa) (Drobniewski and Ellar, 1989) suggesting that Bt S2160-1 is different to previously characterized mosquitocidal strains. Comparison of the plasmid profile of Bt S2160-1 with those published for subsp. jegathesan 163, subsp. kyushuensis, subsp. medellin and subsp. darmstadiensis 73-E10-2 (Reyes-Ramirez and Ibarra, 2008) also suggests that this strain is not one of the above subspecies. A report by Zhu et al. (2010) describes the cloning of cry30Ga, cry4Cb and a cry54-like toxin gene from a strain of Bt known as HS18-1. This strain bears some similarities to Bt S2160-1 although there also appear to be differences in the protein profile. No data are given on the mosquitocidal activity of HS18-1 although weak activity against Aedes aegypti was found for the recombinant Cry4Cb1 and Cry30Ga toxins. Although we have yet to fully characterize all the toxins present in the Bt S2160-1 strain, or to establish whether there may be synergistic interactions between them as observed for Bti (Chilcott and Ellar, 1988; Otieno-Ayayo et al., 2008), we suggest that this strain has potential as an alternative to Bti for mosquito control. Acknowledgments This work was initiated by the Project of China National Bt Collection Initiative and partly funded by the National Research and Development Program of China (Grant No: 2006AA02Z189). We would like to thank the fund from the State Administrative of Foreign Experts and the BBSRC for allowing Mr. Wenfei Zhang to be a visiting scholar at the University of Sussex. We also wish to thank Sarfraz Ali Shad from Sussex University for his technical assistance. We would like to thank Guangxi Center for Diseases Prevention and Control for help with the mosquito bioassay. References Antal, Z. et al., 2004. Single oligonucleotide nested PCR: a rapid method for the isolation of genes and their flanking regions from expressed sequence tags. Curr. Genet. 46, 240–246. Aronson, J.N., Thompson, F.M., 1971. Bacillus thuringiensis sporulation at suboptimal temperature. J. Bacteriol. 105, 445–448. Berry, C. et al., 2002. Complete sequence and organization of pBtoxis, the toxincoding plasmid of Bacillus thuringiensis subsp. Israelensis. Appl. Environ. Microbiol. 68, 5082–5095. Bravo, A. et al., 1998. Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection. Appl. Environ. Microbiol. 64, 4965–4972. Bravo, A. et al., 2007. Mode of action of Bacillus thuringiensis cry and cyt toxins and their potential for insect control. Toxicon 49, 423–435. Carlson, C.R. et al., 1996. The chromosome map of Bacillus thuringiensis subsp. canadensis HD224 is highly similar to that of the Bacillus cereus type strain ATCC 14579. FEMS Microbiol. Lett. 141, 163–167. Cheong, H., Gill, S.S., 1997. Cloning and characterization of a cytolytic and mosquitocidal delta-endotoxin from Bacillus thuringiensis subsp. Jegathesan. Appl. Environ. Microbiol. 63, 3254–3260.
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Chilcott, C.N., Ellar, D.J., 1988. Comparative toxicity of Bacillus thuringiensis var. Israelensis crystal proteins in vivo and in vitro. J. Gen. Microbiol. 134, 2551– 2558. Crickmore, N. et al., 1998. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 807–813. Drobniewski, F.A., Ellar, D.J., 1989. Purification and properties of a 28-kilodalton hemolytic and mosquitocidal protein toxin of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2. J. Bacteriol. 171, 3060–3067. Federici, B.A., 2005. Insecticidal bacteria: an overwhelming success for invertebrate pathology. J. Invertebr. Pathol. 89, 30–38. Federici, B.A. et al., 2003. Recombinant bacteria for mosquito control. J. Exp. Biol. 206, 3877–3885. Ibarra, J.E. et al., 2003. Diversity of Bacillus thuringiensis strains from Latin America with insecticidal activity against different mosquito species. Appl. Environ. Microbiol. 69, 5269–5274. Iracheta, M.M. et al., 2000. Screening for Bacillus thuringiensis crystal proteins active against the cabbage looper, Trichoplusia ni. J. Invertebr. Pathol. 76, 70–75. Kaur, A., Singh, J., 2000. Natural occurrence of Bacillus thuringiensis in leguminous phylloplanes in the new region of India. World J. Microbiol. Biotechnol. 16, 679– 682. Kawalek, M.D. et al., 1995. Isolation and identification of novel toxins from a new mosquitocidal isolate from Malaysia, Bacillus thuringiensis subsp. Jegathesan. Appl. Environ. Microbiol. 61, 2965–2969. Knowles, B.H. et al., 1992. A broad-spectrum cytolytic toxin from Bacillus thuringiensis var. Kyushuensis. Proc. Biol. Sci. 248, 1–7. Kuo, W.S., Chak, K.F., 1996. Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA. Appl. Environ. Microbiol. 62, 1369–1377. Navon, A., Ascher, K.R.S., 2000. Bioassys of Entomopathogenic Microbes and Nematodes. CABI Publishing, Wallingford, pp. 1–49. Noguera, P.A., Ibarra, J.E., 2010. Detection of new cry genes of Bacillus thuringiensis by use of a novel PCR primer system. Appl. Environ. Microbiol. 76, 6150–6155. Otieno-Ayayo, Z.N. et al., 2008. Variations in the mosquito larvicidal activities of toxins from Bacillus thuringiensis ssp. Israelensis. Environ. Microbiol. 10, 2191– 2199. Pal, R., 1982. Disease vector control in the People’s Republic of China. Mosq. News 42, 149–158. Porcar, M. et al., 1999. Identification and characterization of the new Bacillus thuringiensis serovars pirenaica (serotype H57) and iberica (serotype H59). J. Appl. Microbiol. 87, 640–648.
Ragni, A. et al., 1996. Characterization of six highly mosquitocidal Bacillus thuringiensis strains that do not belong to H-14 serotype. Curr. Microbiol. 32, 48–54. Regis, L. et al., 2001. Bacteriological larvicides of dipteran disease vectors. Trends Parasitol. 17, 377–380. Reyes-Ramirez, A., Ibarra, J.E., 2008. Plasmid patterns of Bacillus thuringiensis type strains. Appl. Environ. Microbiol. 74, 125–129. Ross, L.S., Gill, S.S., 1996. Limited growth PCR screening of a plasmid library. Biotechniques 21 (382–4), 386. Sayyed, A.H. et al., 2001. Susceptibility of a field-derived, Bacillus thuringiensisresistant strain of diamondback moth to in vitro-activated Cry1Ac toxin. Appl. Environ. Microbiol. 67, 4372–4373. Song, F.P. et al., 1998. Establishment of PCR–RFLP identification system of cry genes from Bacillus thuringiensis. Sci. Agric. Sin. 31, 19–24. Song, F. et al., 2003. Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene. Appl. Environ. Microbiol. 69, 5207–5211. Stein, C. et al., 2006. Transcriptional analysis of the toxin-coding plasmid pBtoxis from Bacillus thuringiensis subsp. Israelensis. Appl. Environ. Microbiol. 72, 1771– 1776. Tolle, M.A., 2009. Mosquito-borne diseases. Curr. Probl. Pediatr. Adolesc. Health Care 39, 97–140. Vilas-Boas, G.T., Lemos, M.V., 2004. Diversity of cry genes and genetic characterization of Bacillus thuringiensis isolated from Brazil. Can. J. Microbiol. 50, 605–613. WHO, 1996. Report of the WHO informal consultation on the evaluation on the testing of insecticides. CTD_WHOPES, Geneva, pp. 31–48. WHO, 2008. World malaria report 2008. World Health Organization, Geneva. WHO, 2009. Dengue: guidelines for diagnosis, treatment, prevention and control – new edition. World Health Organization (WHO) and the Special Programme for Research and Training in Tropical Diseases, Geneva, pp. 3–86. Wilcks, A. et al., 2008. Germination and conjugation of Bacillus thuringiensis subsp. Israelensis in the intestine of gnotobiotic rats. J. Appl. Microbiol. 104, 1252– 1259. Xie, L. et al., 2009. Bacillus thuringiensis collection and isolates identification from Damingshan and Dawangling natural reserves in Guangxi province. Genomics Appl. Biol. 28, 62–68. Zhu, J. et al., 2010. Characterization and expression of cry4Cb1 and cry30Ga1 from Bacillus thuringiensis strain HS18-1. J. Invertebr. Pathol. 103, 200–202.
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Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Characterization of a new highly mosquitocidal isolate of Bacillus thuringiensis – An alternative to Bti? Wenfei Zhang a,b, Neil Crickmore c, Zenas George c, Liu Xie b, Yong-Qiang He a, Youzhi Li a, Ji-Liang Tang a, Liang Tian b, Xi Wang b, Xuanjun Fang a,b,⇑ a b c
College of Life Science and Technology, Guangxi University, Nanning, Guangxi, China Hainan Institute of Tropical Agricultural Resources (HITAR), Sanya, Hainan, China School of Life Sciences, University of Sussex, Falmer, Brighton, UK
a r t i c l e
i n f o
Article history: Received 12 September 2011 Accepted 5 November 2011 Available online 22 November 2011 Keywords: Bacillus thuringiensis Mosquitocidal activity Cry-type gene PCR-RFLP
a b s t r a c t The mosquito is a very important vector involved in the worldwide transmission of disease-causing viruses and parasites. Controlling the mosquito population remains one of the best means for preventing the serious infectious diseases of malaria, yellow fever, dengue, filariasis and so on and there has been an increasing interest in developing biopesticides as a useful substitute to chemical insecticides. As a result, Bacillus thuringiensis subsp. israelensis (Bti) has been extensively used due to its specificity and high toxicity to a variety of mosquito larvae. However it is prudent to seek alternatives to Bti with alternative spectra of mosquitocidal activity or that are able to overcome any resistance that might develop against Bti. The Bt S2160-1 strain was isolated from soil samples collected from Southern China and found to have a comparable mosquitocidal activity to Bti. However there were significant differences in terms of their plasmid profiles, crystal proteins produced and cry gene complement. A PCR-restriction fragment length polymorphism identification system was developed and used in order to identify novel cry-type genes and four such genes (cry30Ea, cry30Ga, cry50Ba and cry54Ba) were identified in Bt S2160-1. In conclusion, Bt S2160-1 has been identified as a potential alternative to Bti, which could be used for the control of mosquito populations in order to reduce the incidence of mosquito-borne diseases. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Mosquitoes act worldwide as vectors transmitting diseasecausing viruses and parasites such as malaria, yellow fever, dengue fever, filariasis, St. Louis encephalitis and the West Nile virus between humans and animals (Tolle, 2009). The World Health Organization (WHO) estimates that half of the world’s population is at risk of malaria and there have been about 250 million cases leading to nearly 1 million deaths. Of these 91% were in Africa and 85% were of children under 5 years of age (WHO, 2008). Yellow fever causes 200,000 illnesses and 30,000 deaths every year. Dengue fever has become a major international public health concern in recent years; two-fifths of the world’s population is now at risk from dengue. It is estimated that there may be 50 million cases of dengue infection worldwide every year (Tolle, 2009; WHO, 2009). In Southern China, there is a large-scale cultivation of rice using the continuous submergence method for irrigation, which provides such a favorable habitat for mosquito breeding that mosquito-borne diseases are flourishing (Pal, 1982). ⇑ Corresponding author at: Hainan Institute of Tropical Agricultural Resources (HITAR), Sanya, Hainan 572025, China. Fax: +86 0771 3232621. E-mail address: [email protected] (X. Fang). 0022-2011/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2011.11.003
At present, the effective prophylactic drugs and vaccines against the West Nile virus, dengue, malaria, and filariasis are either too expensive or not available to those most at risk. To reduce the incidence of these diseases more reliance has been put on the control of mosquito populations. Until recently mosquito control was dominated by the spraying of synthetic chemical insecticides such as malathion and dichloro-diphenyl-trichloroethane (DDT) which were very effective in the short term. The mosquito though is prone to developing resistance to these insecticides, in addition chemical insecticides leave harmful residues which impact on non-target species, including humans and livestock (Federici et al., 2003). Due to the development of resistance, and the growing public concerns associated with adverse environmental effects of the chemical insecticides, it has been necessary to initiate an alternative strategy for mosquito control. Biopesticides, especially Bacillus thuringiensis (Bt), have received much attention due their lack of significant effect on humans, wildlife or beneficial insects (Bravo et al., 2007; Federici, 2005; Regis et al., 2001). B. thuringiensis subsp. israelensis (Bti) has been extensively studied for its specific and high toxicity to mosquito and black fly larvae since its discovery in 1976. The parasporal inclusion body of Bti consists of at least four major insecticidal crystal proteins (Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa) whilst others may be present in
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lesser amounts (Berry et al., 2002; Stein et al., 2006). Although Bti and its toxins have been successfully commercialized for mosquito control, screening programs have continued worldwide to identify and characterize new mosquitocidal Bt isolates and toxin genes (Bravo et al., 1998). Mosquitocidal Bt strains such as subsp. morrisoni PG-14, subsp. jegathesan, subsp. kyushuensis, subsp. medellin and subsp. darmstadiensis 73-E10-2 have previously been characterized but, with the exception of PG-14 (which is effectively Bti with an additional toxin), are less toxic to mosquitoes than Bti (Drobniewski and Ellar, 1989; Federici et al., 2003; Kawalek et al., 1995; Knowles et al., 1992; Ragni et al., 1996). We have initiated a screening program to collect more than 3000 soil samples from the Tropical Rainforest Nature Reserve in Hainan Province (Jianfengling, Wuzhishan, Dianluoshan and Bawangling) and the Subtropical Forest Nature Reserve in Guangxi Province (Dawangling, Shiwandashan and Longgang). More than 30 Bt isolates with mosquitocidal activity have been characterized with one, the isolate Bt S2160-1, having a comparable mosquitocidal activity to Bti.
Japan) and viewed on a HITACHI S-3400N (HITACHI, Japan) at a voltage of 20 kV. 2.3. Parasporal protein preparation Bt isolates were grown to lysis in G-Tris broth at 30 °C with shaking at 220 rpm and the spore–crystal mixture was harvested by centrifugation at 4 °C at 12,000g for 10 min. The pellet was washed twice in an equal volume of ice-cold 1 M NaCl containing 0.01% Triton X-100 and then twice in an equal volume of distilled water. Subsequently the suspension was sonicated 15 times (30 s, 30 s off) on ice at full amplitude (model VC-250, Sonics and Materials Inc., USA). After centrifugation at 4 °C at 12,000g for 10 min, the resulting pellet was suspended in 1/10 of the original volume of 50 mM Na2CO3 (pH10.0) and the protein concentration was measured using BCA (bicinchoninic acid) protein assay kit (Tiangen, Beijing, China) following the manufacturer’s instruction (Iracheta et al., 2000). Then SDS–PAGE analysis was conducted on mini-protean cell slab vertical apparatus (Bio-Rad, USA). 2.4. Bioassay against Plutella xylostella and mosquitoes
2. Materials and methods 2.1. Bacterial strains, growth conditions and plasmids The bacterial strains and plasmids used in this study are listed in Table 1. The wild type Bt isolate S2160-1 was isolated from soil samples in the Dawangling Forest Nature Reserve (Guangxi, China). Bt strains were incubated at 30 °C in Luria–Bertani (LB) medium for the preparation of plasmid DNA or in G-Tris medium (Aronson and Thompson, 1971) as a sporulation medium for SEM analysis and crystal protein extraction. Escherichia coli JM110, used as a cloning host for all constructed plasmids, was grown at 37 °C in LB broth. The pGEM-3Zf (+) plasmid served as a standard cloning vector for the construction of the Bt S2160-1 DNA library. Ampicillin (100 lg/ml) was added to E. coli culture medium as required.
2.2. Scanning electron microscopy Bt strains were grown in G-Tris medium at 30 °C for over 72 h until sporulation was complete as determined by light microscopy. The spores and crystals were pelleted by centrifugation at 4 °C at 12,000g for 10 min then washed three times with ice-cold 1 M NaCl and then three times with sterile distilled water. The spore– crystal suspension was placed on aluminum mounts and fixed in 1% OsO4 after the samples had been air-dried overnight. Then the samples were coated with gold in an IB-5 ion coater (HITACHI,
A susceptible population of P. xylostella collected from Sanya, Hainan province, China and reared on pesticide-free greenhousegrown Chinese cabbage in an environment-controlled room. Disks of 8 cm diameter were cut from Chinese cabbage leaves and were immersed in serial dilutions of toxin crystals (in 0.02% Triton X-100) for 10 s, and allowed to air dry at ambient temperature for 2 h. The control leaf disks were immersed in distilled water with 0.02% Triton X-100. Each leaf disk was placed in an individual petri dish (10 cm diameter) lined with moistened filter paper. Five third-instar larvae of P. xylostella were introduced into each petri dish (Sayyed et al., 2001). Bioassays against mosquitoes were performed following the standard procedures recommended by WHO (Navon and Ascher, 2000; WHO, 1996). The parasporal proteins were diluted in plastic cups with 30 ml dechlorinated water for larvicidal activity test and cups with only 30 ml dechlorinated water were used as a negative control. Third instar larvae of Culex quinquefasciatus and Aedes albopictus, supplied by Guangxi Center for Diseases Prevention and Control (Guangxi, China), were exposed to serial dilutions of toxins. 30 mosquito larvae were introduced into each cup with a pipette and the mortality was recorded after 48 h. All the bioassays were conducted at 26 °C with 60–70% relative humidity and a photoperiod of 14 h light: 10 h dark. At least 400 third-instar larvae were tested with each protein preparation and 100 larvae for the controls. Protein samples were diluted into twofold serial dilutions with eight concentrations such that at least
Table 1 Bacterial strains and plasmids. Strains and plasmids
Description
Source
B. thuringiensis Bt S2160-1 Bt subsp. israelensis HD522 Bt subsp. israelensis AND508 Bt subsp. kurstaki HD1 Bt subsp. kurstaki HD73
Wild type strain of Bt Model strain of Bt haboring cry4Aa, cry4Ba, cry10Aa, cry11Aa, cyt1Aa, and cyt2Ba genes A variant of Bti AND406 cured of pTX14-2 and pTX14-3 Model strain of Bt haboring cry1Aa, cry1Ab, cry1Ac, cry1I, cry2Aa genes Model strain of Bt haboring cry1Ac gene
Lab Lab Lab Lab Lab
Escherichia coli JM110
Dam, dcm, supE44, hsdR17, thi,leu, rpsL1, lacY galK, galT, aratonA thr, tsx, D(lac-proAB) (F0, traD36, proAB, lacI qZDM15)
Novagen
Plasmids pMD18-T pGEM-3Zf(+) pMD30 pMD40 pGEMS4
AmpR, AmpR, AmpR, AmpR, AmpR,
Takara Promega This study This study This study
T-A cloning vector E. coli cloning vector pMD18-T harboring cry30Ea1 gene pMD18-T harboring cry50Ba1 gene pGEM-3Zf(+) harboring cry54Ba1 gene
stock stock stock stock stock
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five of the concentrations gave a range of mortalities between 10% and 90%, moreover the control mortality was less than 5%. The LC50 values were estimated by probit analysis using SPSS 13.0 software for windows (SPSS Inc., Chicago, USA). 2.5. Plasmid DNA isolation and plasmid profile Plasmid DNA was isolated from Bt strains grown to an OD600 value of 2 at 30 °C in LB broth with shaking at 220 rpm according the method of Song et al. (2003). CHEF MapperÒ XA Pulsed Field Electrophoresis System (Bio-Rad, USA) was used for the detection of the plasmid patterns of Bt. The parameters were set up for separating a DNA sample with a size range from 15 kb to 500 kb (voltage: 6 V/cm, run time: 22 h, included angle: 120°, initial switch interval time: 1.19 s, final switch interval time: 44.69 s). The plasmid DNA was subjected to electrophoresis in 1% low-melting-point agarose (Amresco, USA) at 14 °C with 0.5 TBE buffer. The plasmid DNA was stained in ethidium bromide (5 lg/ml) after electrophoresis and destained in double-distilled water (at 4 °C) for 24 h and then visualized under ultraviolet light. 2.6. Identification of cry-type genes by PCR–RFLP Pairs of universal oligonucleotide primers designed based on the conserved blocks from known cry-type genes, were using to screen Bt S2160-1 (Xie et al., 2009). The universal primer pairs of S5un4/S3un4, Up30-5/Up30-3 and Up39.40-5/Up39.40-3 (Table 2) were designed to detect cry4, cry10, cry30, cry39 and cry40 genes. PCR amplification was performed in a PTC200 Thermo Cycler (MJ Research, USA) using the following programme: an initial denaturation of 5 min at 94 °C, followed by 30 cycles (each cycle was comprised of 94 °C for 1 min, 53 °C for 1 min, 72 °C for 3 min), a final extension for 10 min at 70 °C. The resulting amplicons were purified with Tiangen Midi Purification Kit (Tiangen, Beijing, China). The appropriate restriction endonucleases (Fermentas) were selected to digest the PCR fragment and the resulting restriction fragments were separated according to their length by agarose gel electrophoresis (Kuo and Chak, 1996). The expected size of PCR products and restriction fragments of cry-type genes were determined in silico and the results are listed in Table 3. Purified PCR fragments were cloned into the pMD18-T vector for sequencing (BGI, Shenzen, China) and the resulting sequence was subjected to a blastx homology search against the nonredundant protein database of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/Blast.cgi).
Table 2 Oligonucleotides used in this study. Name of primer
Sequence of primer
Reference
S5un4 S3un4
50 -GTGTCAAGAGAACCAACAGTATG-30 50 -ACTAAGTCTCCTCCTGTATGACCAG30 50 -TTGGCTCAATATGTGTCAAAC-30 50 -GCTTTAACAGCAGGAATTTG-30 50 -TAAGAGGGTTTGTGGGAAGTAG-30 50 -ACTTTCTGGGAATACCTCTACTG-30 50 -ATGAGGGAGTGAAAAAGATGAAT-30 50 -TCTAAGCTTTAGTTCACTGTACAAG30 50 -ATACGAGGAGTGAAATAGATG-30 50 -CACTTGTAAACATCGGACT-30 50 -CAGCGACTGAATTAATGTCA-30 50 -GTCTATAGTGGTACAAAGTGGAGTA30 50 -TGGTAATGCGTCCACAACTCAAC-30 50 -TGAATGGCCCACAAAATGCATC-30
Song et al. (1998)
Up30-5 Up30-3 Up39.40-5 Up39.40-3 F30-5 F30-3 E40-5 E40-3 S30F1 S30F2 S30R1 S30R2
This study This study This study
This study This study
This study
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2.7. Cloning of the novel cry-type genes The primer pairs of F30-5/F30-3 and E40-5/E40-3 (Table 2) were designed from the sequences of the published cry30-type and cry40-type genes and designed to clone the full sequence of cry30A-like and cry40B-like genes respectively. Expand PCR was performed using Ex Taq polymerase (Takara, Dalian, China) and the PCR products purified by gel isolation were ligated into pMD18-T vector for sequencing. For the cloning of the full sequence of cry30G-like gene, the strategy of single oligonucleotide nested (SON)-PCR was used. The four specific primers were designed according the known sequence of the PCR fragment, the primers of S30R1 and S30R2 were for amplification of 50 region of the cry30G-like gene and the primers S30F1 and S30F2 were for 30 region (Antal et al., 2004). In order to clone the cry54A-like gene, a library from Bt S2160-1 plasmid DNA was constructed. The plasmid DNA of Bt S2160-1 was partially digested with BclI and fractionated by 0.7% agarose gel electrophoresis. Restriction fragments with sizes of 6–20 kb were purified from the gel and inserted into BamHI digested and dephosphorylated pGEM-3Zf (+) vector and then introduced into competent cells of E. coli JM110 to make the library. The method of limited growth PCR was utilized to screen the library for the presence of the cry54-like gene with the primer pairs of S5un4/S3un4 (Cheong and Gill, 1997; Ross and Gill, 1996). The positive recombinant plasmid was sequenced in both directions by primer walking (BGI, Shenzen, China). 2.8. Nucleotide sequence accession numbers The sequences of three novel cry-type genes cloned in this paper have been deposited in the GenBank database and also submitted to the Bt delta-endotoxin nomenclature committee to assign names. GenBank accession numbers: EU503140 (cry30Ea1), HQ638217 (cry30Ga2), GU446675 (cry50Ba1), GU446677 (cry54Ba1). 3. Results and discussion 3.1. Parasporal inclusion morphology and profile The crystalline inclusions produced during sporulation are the most important characteristic of Bt, and distinguish it from closely related species of Bacillus cereus and Bacillus anthracis. Moreover determination of the crystal morphology can identify certain toxin types such as the typical bipyramidal crystals of Cry1 or Cry9 proteins toxic to lepidopteran insects (Kaur and Singh, 2000). Bt S2160-1 was grown until crystals were observed through a light microscope, subsequently the crystals were examined by SEM. The electron micrograph of the parasporal crystals from Bt S2160-1 showed freely released crystals which were spherical in shape (Fig. 1A). SDS–PAGE analysis was employed to compare the polypeptide composition of parasporal proteins from Bt S2160-1 and Bti (Fig. 1B). Post sporulation samples from Bt S2160-1 contained four major polypeptides of around 140 kDa, 130 kDa, 75 kDa and 30 kDa which although similar to those produced by Bti did appear different on the gel. With 1 lg/ml trypsin treatment, the crystal proteins of Bt S2160-1 and Bti were processed primarily into 50 kDa fragments. 3.2. Plasmid profiles Bt commonly harbors a number of large plasmids of variable size, the vast majority of the Cry toxin genes are located on these plasmids (Carlson et al., 1996) and the diversity of plasmid sizes in Bt strains is frequently used as a tool to characterize specific strains (Porcar et al., 1999; Vilas-Boas and Lemos, 2004). Pulsed
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Table 3 PCR-RFLP patterns of the Bt S2160-1 amplicons compared with the theoretical sizes for known cry genes. Genes
Primers
Size of amplified product (bp)
Enzymes used to digest PCR product
Size of restriction fragments (bp)
S2160-1 cry4Aa cry4Ba cry10Aa S2160-1 cry30Aa cry30Ba cry30Ca S2160-1 cry40Aa cry40Ba cry40Da
S5un4/S3un4 S5un4/S3un4 S5un4/S3un4 S5un4/S3un4 Up30-5/Up30-3 Up30-5/Up30-3 Up30-5/Up30-3 Up30-5/Up30-3 Up39.40-5/Up39.40-3 Up39.40-5/Up39.40-3 Up39.40-5/Up39.40-3 Up39.40-5/Up39.40-3
1548 1551 1449 1467 1388,1463 1464 1461 1461 1005 1001 886 890
BanI + HaeIII BanI + HaeIII BanI + HaeIII BanI + HaeIII BglII + PstI BglII + PstI BglII + PstI BglII + PstI HindIII + HaeIII HindIII + HaeIII HindIII + HaeIII HindIII + HaeIII
1025, 523 1551 878, 341, 157, 73 742, 499, 226 1388, 906, 557 662, 560, 220 996, 426 864, 374, 184 1005 615, 386 615, 290, 96 450, 390, 89, 76
Fig. 1. Scanning electron micrograph and SDS–PAGE analysis of parasporal inclusions from Bt S2160-1. (A) Scanning electron micrograph of spores (SP) and crystals (C) from Bt S2160-1. (B) SDS–PAGE analysis of Bt S2160-1 and Bti. PM: protein molecular weight marker; lanes 1 and 4: samples taken before sporulation from Bti and Bt S2160-1 respectively; lanes 2 and 5: samples taken after sporulation from Bti and Bt S2160-1 respectively; lanes 3 and 6: samples from sporulated cultures of Bti and Bt S2160-1 respectively treated with 1 lg/ml trypsin.
Field Gel Electrophoresis (PFGE) was employed to compare the plasmid profiles of Bt S2160-1 with Bti and various other reference strains. Fig. 2 shows that the plasmid profile of Bt S2160-1 was significantly different to that of Bti and other strains and in particular lacked the major toxin-encoding pBtoxis plasmid of Bti. 3.3. Insecticidal activities of Bt S2160-1 against mosquitoes and P. xylostella Bioassays against P. xylostella and two species of mosquito were conducted using parasporal inclusions of Bt S2160-1 and Bti. The insecticidal activity was assessed using LC50 values. Table 4 reveals that the parasporal proteins of Bt S2160-1 and Bti both exhibited high toxicities to larvae of C. quinquefasciatus and A. albopictus, but not to P. xylostella. 3.4. Identification of cry-type genes Due to the high toxicity of Bt S2160-1, combined with the observation that it was phenotypically distinct from Bti, a PCRRFLP identification system was used to identify cry genes within this strain. 37 pairs of universal oligonucleotide primers were used to detect cry genes, of which the primer pairs of S5un4/S3un4, Up30-5/Up30-3 and Up39.40-5/Up39.40-3 produced PCR products. The PCR amplicons were digested with pairs of restriction endonucleases to further characterize the type of cry gene present (Fig. 3). Table 3 compares the results obtained with known cry genes and
Fig. 2. PFGE patterns of the large plasmids from Bt S2160-1 and other strains. Lane 1: Bt subsp kurstaki HD-73; lane 2: Bt subsp kurstaki HD-1; lane 3: Bt subsp israelensis AND508 with the 350 kb pXO16 and 128 kb pBtoxis plasmids (Wilcks et al., 2008) highlighted; lane 4: Bt S2160-1.
these data indicate the presence of novel cry genes due to the differences in band sizes obtained in comparison to the reference genes. It should also be noted that in contrast to Bti no cry4 or cry10 genes were identified using the S5un4/S3un4 primers and also that no PCR products were obtained from Bt S2160-1 using primers that successfully identified the cry11, cyt1 and cyt2 genes in Bti (data not shown). The PCR fragments obtained were cloned into the pMD18-T vector and sequenced. This analysis revealed that the deduced amino acid sequence of the cry genes encoded on the PCR products generated by primer pairs S5un4 /S3un4, and Up39.40-5/Up39.40-3 had highest identities with Cry54Aa1 (51%), and Cry40Ba1 (88%) respectively. The PCR fragment produced by Up30-5/Up30-3 was mixture of two different sequences, one can be digested into the fragments of 906 bp and 557 bp by BglII and PstI, the other has no recognition sites for these enzymes. Their deduced amino acid sequences show the highest identities with Cry30Aa1 (72%) and Cry30Ga1 (99%) respectively. 3.5. The cloning of novel cry genes In order to obtain the full length cry54A-like gene, a Bt S2160-1 plasmid DNA library was screened using the primer set S5un4/
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Table 4 Larvicidal activity of spore/crystal mixtures from Bt S2160-1 and Bti against Plutella xylostella and mosquitoes. Tested insect
S2160-1 LC50 (ng/ml) (95% FL)
Plutella xylostella Culex quinquefasciatus Aedes albopictus a
Bti a
>200,000 5.668 (4.262–7.464) 21.113 (12.608–36.393)
Slope
LC50 (ng/ml) (95% FL)a
Slope
– 0.184 0.173
>200,000 1.718 (1.216–2.313) 45.585 (34.998–62.385)
– 0.23 0.237
95% FL are the 95% fiducial limits in parentheses, as determined by probit analysis.
Fig. 3. PCR–RFLP patterns of cry-type genes from Bt S2160-1. M. DNA molecular weight marker; lanes 1, 2 and 3 are PCR amplification products using primer pairs S5un4/S3un4, Up30-5/Up30-3 and Up39.40-5/Up39.40-3 respectively; lanes 4, 5 and 6 represent the above PCR products digested with BanI/HaeIII, BglII/PstI and HindIII/HaeIII respectively.
S3un4. A positive clone designated as pGEMS4 was identified and sequenced. Genomic DNA fragments containing the full length sequences of cry30A-like and cry40B-like genes were obtained by expand PCR with the primer pairs of F30-5/F30-3 and E40-5/E40-3. These were then ligated into the pMD18-T vector to generate the recombinant plasmids of pMD30 and pMD40 for sequencing. The recombinant plasmid pMD30 was shown to harbor the full sequence of a cry30-like gene, whose deduced amino acid sequence indicated a protein with estimated molecular weight of 76 kDa with 75% sequence similarity to Cry30Aa1. With pMD40 and pGEMS4 putative toxins were identified that had 63% and 50% similarity with Cry50Aa1 and Cry54Aa1 respectively. The upstream and downstream flanking sequences of cry30G-like were obtained through SON–PCR strategy. The assembled sequence contains a 1,995 bp open reading frame, which encoded a 664 amino acid protein showed 99% identity with Cry30Ga1. The four novel cry-type genes were designated as cry30Ea1, cry30Ga2, cry50Ba1 and cry54Ba1 according to the nomenclature system proposed by Crickmore et al. (1998) and encoded proteins with predicted molecular weights of 77.6 kDa, 74.4 kDa, 76.3 kDa and 79.6 kDa respectively. 4. Conclusions In this paper, we have identified a new mosquiticidal Bt isolate S2160-1 with comparable toxicity to that of the reference strain of Bti against two species of mosquito. In all other respects the strains are dissimilar with differing plasmid profiles, cry genes and crystal proteins. Thus, it is believed that Bt S2160-1 could be applied to mosquito control as a potential alternative to Bti. Methods based on PCR amplification have been developed to detect cry genes in Bt strains and PCR–PFLP is becoming a popular and powerful approach, which is used not only to confirm the presence of the known genes, but also to discover novel ones (Bravo et al., 1998; Ibarra et al., 2003; Kuo and Chak, 1996; Noguera and Ibarra, 2010; Song et al., 2003). This approach was successfully used in this study to identify the cry30Ea, cry30Ga, cry50Ba and cry54Ba
genes in Bt S2160-1 but did not identify the genes encoding at least three of the main parasporal proteins (140 kDa, 130 kDa and 30 kDa). The SDS PAGE profile of parasporal inclusions from Bt S2160-1 were also different from those of subsp. jegathesan (77, 74, 72, 68, 55, 38, 35, 27, and 23 kDa) (Kawalek et al., 1995), subsp. kyushuensis (140, 85, 80, 70, 66, 50, 25 kDa) (Knowles et al., 1992), subsp. medellin (95, 67, 30 kDa) (Ragni et al., 1996) and subsp. darmstadiensis 73E10-2 (125, 50, 47, and 28 kDa) (Drobniewski and Ellar, 1989) suggesting that Bt S2160-1 is different to previously characterized mosquitocidal strains. Comparison of the plasmid profile of Bt S2160-1 with those published for subsp. jegathesan 163, subsp. kyushuensis, subsp. medellin and subsp. darmstadiensis 73-E10-2 (Reyes-Ramirez and Ibarra, 2008) also suggests that this strain is not one of the above subspecies. A report by Zhu et al. (2010) describes the cloning of cry30Ga, cry4Cb and a cry54-like toxin gene from a strain of Bt known as HS18-1. This strain bears some similarities to Bt S2160-1 although there also appear to be differences in the protein profile. No data are given on the mosquitocidal activity of HS18-1 although weak activity against Aedes aegypti was found for the recombinant Cry4Cb1 and Cry30Ga toxins. Although we have yet to fully characterize all the toxins present in the Bt S2160-1 strain, or to establish whether there may be synergistic interactions between them as observed for Bti (Chilcott and Ellar, 1988; Otieno-Ayayo et al., 2008), we suggest that this strain has potential as an alternative to Bti for mosquito control. Acknowledgments This work was initiated by the Project of China National Bt Collection Initiative and partly funded by the National Research and Development Program of China (Grant No: 2006AA02Z189). We would like to thank the fund from the State Administrative of Foreign Experts and the BBSRC for allowing Mr. Wenfei Zhang to be a visiting scholar at the University of Sussex. We also wish to thank Sarfraz Ali Shad from Sussex University for his technical assistance. We would like to thank Guangxi Center for Diseases Prevention and Control for help with the mosquito bioassay. References Antal, Z. et al., 2004. Single oligonucleotide nested PCR: a rapid method for the isolation of genes and their flanking regions from expressed sequence tags. Curr. Genet. 46, 240–246. Aronson, J.N., Thompson, F.M., 1971. Bacillus thuringiensis sporulation at suboptimal temperature. J. Bacteriol. 105, 445–448. Berry, C. et al., 2002. Complete sequence and organization of pBtoxis, the toxincoding plasmid of Bacillus thuringiensis subsp. Israelensis. Appl. Environ. Microbiol. 68, 5082–5095. Bravo, A. et al., 1998. Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection. Appl. Environ. Microbiol. 64, 4965–4972. Bravo, A. et al., 2007. Mode of action of Bacillus thuringiensis cry and cyt toxins and their potential for insect control. Toxicon 49, 423–435. Carlson, C.R. et al., 1996. The chromosome map of Bacillus thuringiensis subsp. canadensis HD224 is highly similar to that of the Bacillus cereus type strain ATCC 14579. FEMS Microbiol. Lett. 141, 163–167. Cheong, H., Gill, S.S., 1997. Cloning and characterization of a cytolytic and mosquitocidal delta-endotoxin from Bacillus thuringiensis subsp. Jegathesan. Appl. Environ. Microbiol. 63, 3254–3260.
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