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Cloning and heterologous expression of a novel insecticidal gene (tccC1) from Xenorhabdus nematophilus strain
BBRC Biochemical and Biophysical Research Communications 319 (2004) 1110–1116 www.elsevier.com/locate/ybbrc
Cloning and heterologous expression of a novel insecticidal gene (tccC1) from Xenorhabdus nematophilus strain Pom Joo Lee,a Ji-Young Ahn,a Yang-Hoon Kim,a,b Seung Wook Kim,a,b Ji-Yeon Kim,c Jae-Sung Park,c and Jeewon Leea,* a
Department of Chemical and Biological Engineering, Korea University, Anam-Dong 5-1, Sungbuk-Ku, Seoul 136-701, South Korea b Applied Rheology Center, Korea University, Anam-Dong 5-1, Sungbuk-ku, Seoul 136-701, South Korea c Advanced Biocontrol System Co., Ltd., Shindang-Dong 1000, Daegu 704-701, South Korea Received 26 April 2004 Available online 1 June 2004
Abstract We have identified and cloned a novel toxin gene (tccC1/xptB1) from Xenorhabdus nematophilus strain isolated from Koreaspecific entomophagous nematode Steinernema glaseri MK. The DNA sequence of cloned toxin gene (3048 bp) has an open reading frame encoding 1016 amino acids with a predicted molecular mass of 111058 Da. The toxin sequence shares 50–96% identical amino acid residues with the previously reported tccC1 cloned from X. nematophilus (AJ308438), Photorhabdus luminescens W14 (AF346499) P. luminescens TTO1 (BX571873), and Yersinia pestis CO92 (NC_003143). The toxin gene was successfully expressed in Escherichia coli, and the recombinant toxin protein caused a rapid cessation in mortality of Galleria mellonella larvae (80% death of larvae within 2 days). Conclusively, the heterologous expression of the novel gene tccC1 cloned into E. coli plasmid vector produced recombinant toxin with high insecticidal activity. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Toxin gene (tccC1/XptB1); Xenorhabdus nematophilus; Heterologous expression; High insecticidal activity
Recent strategies for the control of insect pests in agriculture have relied on the development of transgenic crops expressing insecticidal protein toxins. Currently, the most successful protein toxin applied to develop transgenic corps is based on the bacterium Bacillus thuringiensis [1,2] that produces insecticidal crystalline toxins during sporulation [3]. The wide use of B. thuringiensis on a large scale and the development and use of transgenic plants expressing these toxin genes [4,5] may enhance the development of resistant insect populations. New protein toxins are therefore required to provide a greater diversity of genes for use in pest control [6–8]. Entomophagous nematodes have long been used as biological control agents, and the insecticidal bacteria that they carry have also been studied in detail [9]. However, relatively we have had little idea of the active ingredients that these bacteria such as Xenorhabdus ne* Corresponding author. Fax: +82-2-926-6102. E-mail address: [email protected] (J. Lee).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.04.203
matophilus and Photorhabdus luminescens use to attack insects and of the novel genes that encode them. Xenorhabdus and Photorhabdus are two genera of bacteria that symbiotically associate with specific nematodes. The nematode–bacterium pair is capable of invading and killing the larval stage of numerous insects [10]. Both Xenorhabdus and Photorhabdus spp. are motile gram-negative bacteria belonging to the family Enterobacteriaceae [11,12]. Ensign et al. [13] have identified protein toxins from the bacterium P. luminescens, but when the toxin genes from P. luminescens were cloned and expressed in Escherichia coli they did not display insecticidal activity [14]. Since the direct use of X. nematophilus and P. luminescens as a biopesticide is severely limited due to their incapability in surviving in water or soil for long, it is important that the insecticidal toxicity of these bacteria can be reproduced in other bacteria, microorganisms, or plants for their exploitation. To enable this and to study the toxins in detail, these toxin proteins should be able to be expressed in
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heterologous host and their insecticidal activity be maintained. Recently, Morgan et al. [15] discovered a toxin gene from X. nematophilus and isolated an E. coli clone expressing a toxin protein that showed a high insecticidal activity against larvae of Pieris brassicae. In the search for new toxin genes, we have identified and cloned a novel toxin gene (tccC1/XptB1) from X. nematophilus strain isolated from Korean insecticidal nematode Steinernema glaseri MK. The toxin gene was successfully expressed in E. coli, and the recombinant toxin protein caused a rapid cessation in mortality of Galleria mellonella larvae. We describe here the identification of thediscovered strain of X. nematophilus, the sequence of the toxin gene cloned into the E. coli expression vector, and the insecticidal activity of the toxin gene product in E. coli.
Materials and methods Strain isolation and identification. Killed larva of G. mellonella by the Korea-specific insecticidal nematode, S. glaseri MK, was sterilized and washed 3–4 times with 95% ethanol and sterile distilled water, respectively. After dissecting the sterilized dead larva, the hemolymph was spread out on NBT-agar (nutrient agar 20 g/L, bromothymol blue 25 mg/L, and triphenyltetrazolium 40 mg/L) plate and incubated for 3– 5 days at 28 °C. From the formed colonies, a pure and single colony was finally isolated through several stages of culturing in BUGM media (Biolog Universal Growth agar 57 g/L, maltose 2.5 g/L). Stock cultures were subcultured several times to increase their levels of metabolic activity. Also, a plastic disposable loop was used to collect colonies carefully so that there would be minimum carryover of nutrients from the agar when the growth was suspended in 0.85% saline. The turbidimeter was blanked with a tube of uninoculated saline. The suspension was then adjusted to fall within the low- and high-limit GN MicroPlate (Biolog, Hayward, CA) turbidity standards supplied by the manufacturer. The inoculum for Biolog system (Biolog, Hayward, CA) which was always used within 10 min of preparation was poured into a disposable plastic reservoir just prior to use. MicroPlates were inoculated with an eight-channel multipipette, with 150 ll of the inoculum being dispensed per well; plates were then generally incubated at 28 °C for 24 h. The metabolic profile of the bacterial strain was then compared automatically, by using the MicroLog software, with the MicroLog GN database (release 3.01A), and similarity index was calculated for strain identification. PCR conditions and PCR based cloning of toxin gene (tccC1). Polymerase chain reaction (PCR) was carried out in a total volume of 100 ll with the following reagents: 20 mM Tris–HCl, pH 8.3, 50 mM KCl, 2 mM MgSO4 , 10 mM (NH4 )2 SO4 , 0.1% Triton X-100, 0.25 mM dNTPs, 50 pmol of each primer, and Taq DNA polymerase (Takara, Japan). PCR cycling was as follows: 30 cycles of 94 or 95 °C (30 s or 1 min), 52 or 55 °C (30 s or 1 min), and 72 °C (1 min), where applicable. In order to amplify the core region and four fragments of genomic DNA encoding tccC1, we designed the following primer: (1) for core region amplification, 50 -GATTACAAAACCGTGCGT-30 (sense oligonucleotide), 50 -GGGGTTATTCCTGCACAT-30 (antisense oligonucleotide); (2) for amplification of fragment 1 (674 bp), 50 -TGCATATG A-AGAATTTCGTTC-30 (sense oligonucleotide), 50 -ACAGAAGTG AAGCTTTCCGGC-30 (antisense oligonucleotide); (3) for amplification of fragment 2 (1062 bp), 50 -GCCGGAAAGCTTCACTTCTGT-30 (sense oligonucleotide), 50 -TGATCATTGCTTGAT-ATCCG-30 (antisense oligonucleotide); (4) for amplification of fragment 3 (875 bp), 50 -C
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GGATATCAGCAATGATCA-30 (sense oligonucleotide), 50 -GCTGA CATTCCCGGGCCT-GA-30 (antisense oligonucleotide); and (5) for amplification of fragment 4 (495 bp), 50 -CTCAGG-CCCGGGAATGT CAGC-30 (sense oligonucleotide), 50 -TCATCGATTTATGCTTCGG AT-30 (antisense oligonucleotide) (the underlined sequences denote the restriction sites). The fragment was gel purified, digested with corresponding restriction enzymes, and ligated first into pGEM-T Easy vector system1 (Promega, Mannheim, Germany). After transformation into E. coli DH5a, each fragment clone was sequenced by Macrogen (Seoul, Korea) using appropriate primers. Each pGEM-T Easy vector containing fragment insert was cut with the corresponding restriction enzyme, and the fragments 1 and 2 were together ligated into a pGEM-T Easy vector again. Similarly, the fragments 3 and 4 were together ligated into a pGEM-T Easy vector, too. After properly digested, the two halffragments 1–2 and 3–4 were ligated into pGEM-T Easy vector, resulting in the construction of whole gene clone of tccC1. The resulting pGEM-T Easy vector-tccC1 was cut with NdeI and ClaI, and the 3056 bp insert was cloned into pET3a, and the resulting clone was named pET3a-TOX. Vectors and bacterial strains used for cloning and expression. Restriction digestion of PCR products and vectors as well as ligation of DNA was performed according to standard protocols [16]. PCR products were first cloned into pGEM-T Easy vector system1 (Promega), and pET3a (Novagen, USA) was used as the expression vector. For cloning and for propagation of plasmids, E. coli DH5a was applied. For expression of the cloned tccC1, E. coli Rosetta(DE3) (F . R ompT hsdSB (r B mB ) gal dcm (DE3) pRARE (Cm )) (Novagen) was used, which supplies tRNAs for the rare codons AUA, AGG, AGA, CUA, CCC, and GGA on a compatible chloramphenicol-resistant plasmid pRARE. LB-agar plates [16] were supplemented with ampicillin (100 mg/L) and chloramphenicol (100 mg/L), where applicable. Heterologous expression of pET3a-TOX. Escherichia coli Rosetta(DE3) cells harboring pET3a-TOX were grown at 37 °C in jar fermenter (Kobiotech, Incheon, Korea) (containing 3 L of LB medium with ampicillin (100 mg/L) and chloramphenicol (100 mg/L), pH 6.75), to an OD600 of 0.5. Then isopropyl-b-D -thiogalactopyranoside (IPTG) was added to a concentration of 1 mM and the cells were cultivated further for 14 h. Bacteria were harvested by centrifugation (6000 rpm, 10 min), washed with 10 ml of 1% NaCl, and resuspended in 5 ml distilled water. After cell disruption by using Branson Sonifier (Branson Ultrasonics, Danbury, CT), the cell-free supernatant and insoluble protein aggregates were separated at 13,000 rpm for 10 min, and the cell-free extracts were subjected to reducing SDS–PAGE analysis. Estimation of insecticidal activity of recombinant toxin. Cell-free extracts prepared from the induced cultures of E. coli Rosetta(DE3)/ pT7-TOX were 5-times concentrated using Microcon (MWCO 10 kDa, Amicon, MA, USA). Using microsyringe, 3 ll of the concentrated extracts was injected into each of 10 larvae of G. mellonella. Negative control (cell-free extracts from E. coli Rosetta(DE3) cells transformed with pET3a without insert) was prepared and injected to larvae using the same procedure. The larvae used for the toxicity test had been incubated for 25 days at 25 °C with the solid feed consisting of rice and wheat powder (500 g of each), 200-ml mixture of glycerol and honey, and dried yeast powder (3 g). The larvae injected with the recombinant toxin were incubated further for 5 days at 25 °C. The larva viability was monitored everyday for 5 days.
Results and discussion Identification of insecticidal X. nematophilus strain A symbiotic bacterium was isolated from the hemolymph of killed larva of G. mellonella by the Koreaspecific insecticidal nematode, S. glaseri MK. For the
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identification of the symbiotic bacterium, the Biolog system (Biolog, Hayward, CA) was used, which reportedly performs best with oxidase-positive fermenters and biochemically active nonfermenters [17]. The Biolog system is based on tests for the oxidation of 95 substrates in a 96-well microtiter plate (MicroPlate). Each well contains a redox dye, tetrazolium violet, that permits colorimetric determination of the increased respiration that occurs when cells are oxidizing a carbon source. The inoculum of the symbiotic bacterium (in 0.85% saline) was dispensed per well of the MicroPlate where 95 different carbon sources (Table 1) were already coated on each well surface. After 4- or 24-h incubation, the metabolic profile of the symbiotic bacteria was then compared automatically, by using the MicroLog software, with the MicroLog GN database (release 3.01A) comprising 569 taxa. Since the similarity index of the genus/species was less than 0.750 as a result of similarity and distance calculation at 4 h, the MicroPlate was read
again after 24-h incubation. With metabolic profile obtained at 24 h (shown in Table 1), the similarity index of 0.728 was calculated by metabolic fingerprinting, resulting in a computer report that identified the symbiotic bacterium as X. nematophilus. Cloning and sequencing of insecticidal gene tccC1 from X. nematophilus strain The toxin complex gene of X. nematophilus consists of tcdA1, tcaC1, tccC1, and tcdA2 genes with an additional chitinase gene (tccB1), and among the three Tc elements, TccC is presumably the active delivered toxin while the others encode a delivery system [18]. According to phylogeny of the predicted TccC-like proteins, TccC1 (or XptB1: another homologous name) consists of three domains, i.e., core, extended core, and extension regions, and the ‘core’ represents the most highly conserved domain (>75%). Through database search in GenBank
Table 1 Metabolic responses to 95 different carbon sources dispensed per well of the MicroPlate for the identification of the symbiotic bacterium, using the Biolog system (Biolog, Hayward, CA) C-source
Response
C-source
Response
C-source
XR-MK
Water a-Cyclodestrin Dextrin Glycogen Tween 40 Tween 80 N-Acetyl-D -galactosamine N-Acetyl-D -glucosamine Adonitol L -Arabinose D -Arabitol Cellobiose i-Erythritol D -Fructose L -Frucose D -Galactose Gentiobiose a-D -Glucose m-Inositol a-D -Lactose Lactulose Maltose D -Mannitol D -Mannose D -Melibiose b-Methyl-D -glucoside D -Psicose D -Raffinose L -Rhamnose D -Sorbitol Sucrose D -Trehalose
) ) + w w w ) + ) w ) ) ) + ) ) ) + w ) ) + ) + ) ) ) ) ) ) ) +
Turanose Xylitol Methyl pyruvate Mono-methyl succinate Acetic acid cis-Acconitic acid Citric acid Formic acid D -Galactonic acid latone D -Galacturonic acid D -Gluconic acid D -Glucosaminic acid D -Glucuronic acid a-Hydroxybutyric acid b-Hydroxybutyric acid c-Hydroxybutyric acid p-Hydroxyphenylacetic acid Itaconic acid a-Keto butyric acid a-Keto glutaric acid a-Keto valeric acid D , L -Lactic acid Malonic acid Propionic acid Quinic acid D -Saccharic acid Sebacid acid Succinic acid Bromosuccinic acid Succinamic acid Glucuronamide Alaninamide
) ) + + w ) ) w ) ) ) ) ) ) ) + w ) ) ) ) w ) w ) ) ) w ) ) ) w
D -Alanine
acid acid Glycyl-L -aspartic acid Glycyl-L -glutamic acid L -Histidine Hydroxy L -proline L -Leucine L -Omithine L -Phenylalanine L -Proline L -Pyroglutamic acid D -Serine L -Serine L -Threonine D , L -Camitine c-Amino butyric acid Urocanic acid Inosine Uridine Thymidine Phenyl ethylamine Putrescine 2-Amino ethanol 2,3-Butanediol Glycerol D , L -Glycerol phosphate Glucose-1-phosphate Glucose-6-phosphate
) + w + w + ) + w ) ) ) ) w ) w + ) ) ) ) + + w w w ) ) w + + +
Similarity Result of identification
0.728 X. nematophilus
+, positive response; ), negative response; and W, weak response
L -Alanine L -Alanyl-glycine L -Asparagine L -Aspartic
L -Glutamic
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(NCBI), we found only a single case of submitted tccC1 nucleotide sequence (3000 bp) (Accession No. AJ308438) from X. nematophilus [15]. To ascertain the existence of toxin gene in extracted genomic DNA, PCR was carried out for amplifying the core region using appropriate primers designed on the basis of the GenBank sequence of tccC1. As a result of agarose gel electrophoresis (Fig. 1A), a single PCR product was apparently visualized. The sequencing of the PCR product showed about 50% homology in amino acid sequence compared to the sequence AJ308438 and also very high homology (92%) with the amino acid sequence of TccC1Xn that Waterfield et al. [18] recently reported (Fig. 1B). These sequence homologies indicate that the extracted genomic DNA contains toxin gene tccC1 with highly conserved core region. Assuming the high homology with AJ308438, we first carried out six independent PCRs to get the toxin gene clone (tccC1) using appropriate primers designed on the basis of 50 - and 30 -UTR sequences of tccC1 (AJ308438). Through the sequence analysis, the six PCR products
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showed more than 90% homology with AJ308438, but the base sequences were all slightly different from each other when compared, which is due probably to the problems of erroneous long PCR (3000 bp) (data not shown). Fortunately, however, we found three restriction enzyme sites (HindIII, EcoRV, and SmaI) in the regions showing 100% sequencing reproducibility among six PCR products. To minimize probable errors in such a long PCR, we considered another PCR strategy of synthesizing the four different tccC1-derived fragments (50 -NdeI-fragment(1)-HindIII-30 , 50 -HindIIIfragment(2)-EcoRV-30 , 50 -EcoRV-fragment(3)-SmaI-30 , and 50 -SmaI-fragment(4)-ClaI-30 ), all of which can be combined together to give a whole tccC1 gene using the three restriction enzyme sites above. That is, the joining of fragments was possible because two fragments to be joined had the common nucleotide sequence including a restriction enzyme site at 50 - or 30 -end of each fragment. With 3–4 PCR products for each tccC1 fragment, the sequence analysis was repeated about 7-times, and correct nucleotide sequence was determined for each frag-
Fig. 1. (A) Result of PCR amplification followed by electrophoresis (1% agarose gel) for core region DNA fragment, using genomic DNA from symbiosis bacteria X. nematophilus MK as PCR template. (Arrow indicates that predicted core region size (180 bp). Lambda HindIII size marker bands indicate 23,130, 9416, 6557, 4361, 2322, 2027, and 564 bp from top to bottom.) (B) Alignment of amino acid sequences corresponding to TccC core region X. nematophilus toxin. (“PCR product”: amplified PCR product shown in (A) “TccXn”: core region sequences reported by Waterfield et al. [18], and “AJ308438”: core region sequences of toxin gene deposited in the GenBank (Accession No. AJ308438)).
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Fig. 2. Alignment of the protein sequences of the cloned toxin gene in this study and the previously reported protein sequences of X. nematophilus (AJ308438) [15] (“X. n XptB1”), P. luminescens W14 (AF346499) [18] (“P. 1 W14 TccC1”), P. luminescens TTO1 (BX571873) [19] (“P. 1 TTO1 TccC1”), and Y. pestis CO92 (NC_003143) [20] (“Y. p CO92 TccC1”).
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ment. The DNA sequence of cloned toxin gene (3048 bp) has an open reading frame encoding 1016 amino acids with a predicted molecular mass of 111,058 Da (Fig. 2). From the alignment of the protein sequences, the determined toxin sequence shares 96%, 51%, 50%, and 52% identical amino acid residues with the X. nematophilus (AJ308438) [15], P. luminescens W14 (AF346499) [18], P. luminescens TTO1 (BX571873) [19], and Yersinia pestis CO92 (NC_003143) [20], respectively (Fig. 2).
Fig. 3. Result of SDS–PAGE analysis of cell-free extracts from E. coli culture (Rosetta(DE3)/pT7-TOX) in 5-L jar fermenter, showing timecourse variations in soluble recombinant exotoxin. (M: size marker, lanes 1-8: cell-free extracts from recombinant E. coli cultures sampled at 0 h (lane 1), 2 h (lane 2), 4 h (lane 3), 6 h (lane 4), 8 h (lane 5), 10 h (lane 6), 12 h (lane 7), and 14 h (lane 8) after gene expression is induced by adding IPTG (1 mM).)
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Heterologous expression of tccC1 gene in E. coli and insecticidal activity of recombinant TccC1 The whole gene of tccC1 was cloned into expression vector, pET3a-TOX, that was used to transform the E. coli strain Rosetta(DE3) (Novagen, USA) that expresses rare tRNAs on a compatible chloramphenicolresistant plasmid pRARE (Materials and methods). Cultures of E. coli Rosetta(DE3)/pT7-TOX were induced with IPTG, the bacterial cells were harvested, and the cell-free extracts were separated by reducing SDS– PAGE. A prominent band of about 110 kDa was visible, which was not found in control cells (E. coli Rosetta(DE3)) transformed with pET3a without insert (Fig. 3). The size of the 110 kDa protein band, found by SDS–PAGE, is in accordance with the calculated size of 111,058 Da of the expressed protein. An equal volume (3 ll) of cell-free extracts prepared from the induced cultures of E. coli Rosetta(DE3)/ pT7-TOX was injected into 10 larvae of G. mellonella. Cell-free extracts from E. coli Rosetta(DE3) cells transformed with pET3a without insert were used as negative control. After the injection of recombinant toxin, the larva viability was monitored everyday for 5 days at 28 °C. Negative control never caused larva death until 5th day, whereas under the same assay conditions, 80% of the larvae that the cell extracts containing recombinant toxin TccC1 were applied to
Table 2 Test results of insecticidal activity of recombinant exotoxin applied to 10 larvae of G. mellonella Percentage mortality (%)a
Negative control (supernatant of host E. coli lysates) Supernatant of recombinant E. coli lysates containing recombinant exotoxin a
Day 1
Day 2
Day 3
Day 4
Day 5
0 60
0 80
0 80
0 80
0 80
(Number of dead larvae) (total number of larvae tested) 100.
Fig. 4. Test results of insecticidal activity of recombinant exotoxin at 5 days after applied to G. mellonella larvae. (A) Larvae injected with negative control (supernatant of host E. coli (Rosetta(DE3) lysates); (B) larvae injected with supernatant of recombinant E. coli (Rosetta(DE3)/pT7-TOX) lysates containing recombinant exotoxin).
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was killed within only 2 days (Table 2 and Fig. 4). We therefore conclude that the heterologous expression of the cloned gene tccC1 produced recombinant toxin with high insecticidal activity.
Acknowledgments This work was supported by Ministry of Commerce, Industry, and Energy in South Korea.
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Cloning and heterologous expression of a novel insecticidal gene (tccC1) from Xenorhabdus nematophilus strain Pom Joo Lee,a Ji-Young Ahn,a Yang-Hoon Kim,a,b Seung Wook Kim,a,b Ji-Yeon Kim,c Jae-Sung Park,c and Jeewon Leea,* a
Department of Chemical and Biological Engineering, Korea University, Anam-Dong 5-1, Sungbuk-Ku, Seoul 136-701, South Korea b Applied Rheology Center, Korea University, Anam-Dong 5-1, Sungbuk-ku, Seoul 136-701, South Korea c Advanced Biocontrol System Co., Ltd., Shindang-Dong 1000, Daegu 704-701, South Korea Received 26 April 2004 Available online 1 June 2004
Abstract We have identified and cloned a novel toxin gene (tccC1/xptB1) from Xenorhabdus nematophilus strain isolated from Koreaspecific entomophagous nematode Steinernema glaseri MK. The DNA sequence of cloned toxin gene (3048 bp) has an open reading frame encoding 1016 amino acids with a predicted molecular mass of 111058 Da. The toxin sequence shares 50–96% identical amino acid residues with the previously reported tccC1 cloned from X. nematophilus (AJ308438), Photorhabdus luminescens W14 (AF346499) P. luminescens TTO1 (BX571873), and Yersinia pestis CO92 (NC_003143). The toxin gene was successfully expressed in Escherichia coli, and the recombinant toxin protein caused a rapid cessation in mortality of Galleria mellonella larvae (80% death of larvae within 2 days). Conclusively, the heterologous expression of the novel gene tccC1 cloned into E. coli plasmid vector produced recombinant toxin with high insecticidal activity. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Toxin gene (tccC1/XptB1); Xenorhabdus nematophilus; Heterologous expression; High insecticidal activity
Recent strategies for the control of insect pests in agriculture have relied on the development of transgenic crops expressing insecticidal protein toxins. Currently, the most successful protein toxin applied to develop transgenic corps is based on the bacterium Bacillus thuringiensis [1,2] that produces insecticidal crystalline toxins during sporulation [3]. The wide use of B. thuringiensis on a large scale and the development and use of transgenic plants expressing these toxin genes [4,5] may enhance the development of resistant insect populations. New protein toxins are therefore required to provide a greater diversity of genes for use in pest control [6–8]. Entomophagous nematodes have long been used as biological control agents, and the insecticidal bacteria that they carry have also been studied in detail [9]. However, relatively we have had little idea of the active ingredients that these bacteria such as Xenorhabdus ne* Corresponding author. Fax: +82-2-926-6102. E-mail address: [email protected] (J. Lee).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.04.203
matophilus and Photorhabdus luminescens use to attack insects and of the novel genes that encode them. Xenorhabdus and Photorhabdus are two genera of bacteria that symbiotically associate with specific nematodes. The nematode–bacterium pair is capable of invading and killing the larval stage of numerous insects [10]. Both Xenorhabdus and Photorhabdus spp. are motile gram-negative bacteria belonging to the family Enterobacteriaceae [11,12]. Ensign et al. [13] have identified protein toxins from the bacterium P. luminescens, but when the toxin genes from P. luminescens were cloned and expressed in Escherichia coli they did not display insecticidal activity [14]. Since the direct use of X. nematophilus and P. luminescens as a biopesticide is severely limited due to their incapability in surviving in water or soil for long, it is important that the insecticidal toxicity of these bacteria can be reproduced in other bacteria, microorganisms, or plants for their exploitation. To enable this and to study the toxins in detail, these toxin proteins should be able to be expressed in
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heterologous host and their insecticidal activity be maintained. Recently, Morgan et al. [15] discovered a toxin gene from X. nematophilus and isolated an E. coli clone expressing a toxin protein that showed a high insecticidal activity against larvae of Pieris brassicae. In the search for new toxin genes, we have identified and cloned a novel toxin gene (tccC1/XptB1) from X. nematophilus strain isolated from Korean insecticidal nematode Steinernema glaseri MK. The toxin gene was successfully expressed in E. coli, and the recombinant toxin protein caused a rapid cessation in mortality of Galleria mellonella larvae. We describe here the identification of thediscovered strain of X. nematophilus, the sequence of the toxin gene cloned into the E. coli expression vector, and the insecticidal activity of the toxin gene product in E. coli.
Materials and methods Strain isolation and identification. Killed larva of G. mellonella by the Korea-specific insecticidal nematode, S. glaseri MK, was sterilized and washed 3–4 times with 95% ethanol and sterile distilled water, respectively. After dissecting the sterilized dead larva, the hemolymph was spread out on NBT-agar (nutrient agar 20 g/L, bromothymol blue 25 mg/L, and triphenyltetrazolium 40 mg/L) plate and incubated for 3– 5 days at 28 °C. From the formed colonies, a pure and single colony was finally isolated through several stages of culturing in BUGM media (Biolog Universal Growth agar 57 g/L, maltose 2.5 g/L). Stock cultures were subcultured several times to increase their levels of metabolic activity. Also, a plastic disposable loop was used to collect colonies carefully so that there would be minimum carryover of nutrients from the agar when the growth was suspended in 0.85% saline. The turbidimeter was blanked with a tube of uninoculated saline. The suspension was then adjusted to fall within the low- and high-limit GN MicroPlate (Biolog, Hayward, CA) turbidity standards supplied by the manufacturer. The inoculum for Biolog system (Biolog, Hayward, CA) which was always used within 10 min of preparation was poured into a disposable plastic reservoir just prior to use. MicroPlates were inoculated with an eight-channel multipipette, with 150 ll of the inoculum being dispensed per well; plates were then generally incubated at 28 °C for 24 h. The metabolic profile of the bacterial strain was then compared automatically, by using the MicroLog software, with the MicroLog GN database (release 3.01A), and similarity index was calculated for strain identification. PCR conditions and PCR based cloning of toxin gene (tccC1). Polymerase chain reaction (PCR) was carried out in a total volume of 100 ll with the following reagents: 20 mM Tris–HCl, pH 8.3, 50 mM KCl, 2 mM MgSO4 , 10 mM (NH4 )2 SO4 , 0.1% Triton X-100, 0.25 mM dNTPs, 50 pmol of each primer, and Taq DNA polymerase (Takara, Japan). PCR cycling was as follows: 30 cycles of 94 or 95 °C (30 s or 1 min), 52 or 55 °C (30 s or 1 min), and 72 °C (1 min), where applicable. In order to amplify the core region and four fragments of genomic DNA encoding tccC1, we designed the following primer: (1) for core region amplification, 50 -GATTACAAAACCGTGCGT-30 (sense oligonucleotide), 50 -GGGGTTATTCCTGCACAT-30 (antisense oligonucleotide); (2) for amplification of fragment 1 (674 bp), 50 -TGCATATG A-AGAATTTCGTTC-30 (sense oligonucleotide), 50 -ACAGAAGTG AAGCTTTCCGGC-30 (antisense oligonucleotide); (3) for amplification of fragment 2 (1062 bp), 50 -GCCGGAAAGCTTCACTTCTGT-30 (sense oligonucleotide), 50 -TGATCATTGCTTGAT-ATCCG-30 (antisense oligonucleotide); (4) for amplification of fragment 3 (875 bp), 50 -C
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GGATATCAGCAATGATCA-30 (sense oligonucleotide), 50 -GCTGA CATTCCCGGGCCT-GA-30 (antisense oligonucleotide); and (5) for amplification of fragment 4 (495 bp), 50 -CTCAGG-CCCGGGAATGT CAGC-30 (sense oligonucleotide), 50 -TCATCGATTTATGCTTCGG AT-30 (antisense oligonucleotide) (the underlined sequences denote the restriction sites). The fragment was gel purified, digested with corresponding restriction enzymes, and ligated first into pGEM-T Easy vector system1 (Promega, Mannheim, Germany). After transformation into E. coli DH5a, each fragment clone was sequenced by Macrogen (Seoul, Korea) using appropriate primers. Each pGEM-T Easy vector containing fragment insert was cut with the corresponding restriction enzyme, and the fragments 1 and 2 were together ligated into a pGEM-T Easy vector again. Similarly, the fragments 3 and 4 were together ligated into a pGEM-T Easy vector, too. After properly digested, the two halffragments 1–2 and 3–4 were ligated into pGEM-T Easy vector, resulting in the construction of whole gene clone of tccC1. The resulting pGEM-T Easy vector-tccC1 was cut with NdeI and ClaI, and the 3056 bp insert was cloned into pET3a, and the resulting clone was named pET3a-TOX. Vectors and bacterial strains used for cloning and expression. Restriction digestion of PCR products and vectors as well as ligation of DNA was performed according to standard protocols [16]. PCR products were first cloned into pGEM-T Easy vector system1 (Promega), and pET3a (Novagen, USA) was used as the expression vector. For cloning and for propagation of plasmids, E. coli DH5a was applied. For expression of the cloned tccC1, E. coli Rosetta(DE3) (F . R ompT hsdSB (r B mB ) gal dcm (DE3) pRARE (Cm )) (Novagen) was used, which supplies tRNAs for the rare codons AUA, AGG, AGA, CUA, CCC, and GGA on a compatible chloramphenicol-resistant plasmid pRARE. LB-agar plates [16] were supplemented with ampicillin (100 mg/L) and chloramphenicol (100 mg/L), where applicable. Heterologous expression of pET3a-TOX. Escherichia coli Rosetta(DE3) cells harboring pET3a-TOX were grown at 37 °C in jar fermenter (Kobiotech, Incheon, Korea) (containing 3 L of LB medium with ampicillin (100 mg/L) and chloramphenicol (100 mg/L), pH 6.75), to an OD600 of 0.5. Then isopropyl-b-D -thiogalactopyranoside (IPTG) was added to a concentration of 1 mM and the cells were cultivated further for 14 h. Bacteria were harvested by centrifugation (6000 rpm, 10 min), washed with 10 ml of 1% NaCl, and resuspended in 5 ml distilled water. After cell disruption by using Branson Sonifier (Branson Ultrasonics, Danbury, CT), the cell-free supernatant and insoluble protein aggregates were separated at 13,000 rpm for 10 min, and the cell-free extracts were subjected to reducing SDS–PAGE analysis. Estimation of insecticidal activity of recombinant toxin. Cell-free extracts prepared from the induced cultures of E. coli Rosetta(DE3)/ pT7-TOX were 5-times concentrated using Microcon (MWCO 10 kDa, Amicon, MA, USA). Using microsyringe, 3 ll of the concentrated extracts was injected into each of 10 larvae of G. mellonella. Negative control (cell-free extracts from E. coli Rosetta(DE3) cells transformed with pET3a without insert) was prepared and injected to larvae using the same procedure. The larvae used for the toxicity test had been incubated for 25 days at 25 °C with the solid feed consisting of rice and wheat powder (500 g of each), 200-ml mixture of glycerol and honey, and dried yeast powder (3 g). The larvae injected with the recombinant toxin were incubated further for 5 days at 25 °C. The larva viability was monitored everyday for 5 days.
Results and discussion Identification of insecticidal X. nematophilus strain A symbiotic bacterium was isolated from the hemolymph of killed larva of G. mellonella by the Koreaspecific insecticidal nematode, S. glaseri MK. For the
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identification of the symbiotic bacterium, the Biolog system (Biolog, Hayward, CA) was used, which reportedly performs best with oxidase-positive fermenters and biochemically active nonfermenters [17]. The Biolog system is based on tests for the oxidation of 95 substrates in a 96-well microtiter plate (MicroPlate). Each well contains a redox dye, tetrazolium violet, that permits colorimetric determination of the increased respiration that occurs when cells are oxidizing a carbon source. The inoculum of the symbiotic bacterium (in 0.85% saline) was dispensed per well of the MicroPlate where 95 different carbon sources (Table 1) were already coated on each well surface. After 4- or 24-h incubation, the metabolic profile of the symbiotic bacteria was then compared automatically, by using the MicroLog software, with the MicroLog GN database (release 3.01A) comprising 569 taxa. Since the similarity index of the genus/species was less than 0.750 as a result of similarity and distance calculation at 4 h, the MicroPlate was read
again after 24-h incubation. With metabolic profile obtained at 24 h (shown in Table 1), the similarity index of 0.728 was calculated by metabolic fingerprinting, resulting in a computer report that identified the symbiotic bacterium as X. nematophilus. Cloning and sequencing of insecticidal gene tccC1 from X. nematophilus strain The toxin complex gene of X. nematophilus consists of tcdA1, tcaC1, tccC1, and tcdA2 genes with an additional chitinase gene (tccB1), and among the three Tc elements, TccC is presumably the active delivered toxin while the others encode a delivery system [18]. According to phylogeny of the predicted TccC-like proteins, TccC1 (or XptB1: another homologous name) consists of three domains, i.e., core, extended core, and extension regions, and the ‘core’ represents the most highly conserved domain (>75%). Through database search in GenBank
Table 1 Metabolic responses to 95 different carbon sources dispensed per well of the MicroPlate for the identification of the symbiotic bacterium, using the Biolog system (Biolog, Hayward, CA) C-source
Response
C-source
Response
C-source
XR-MK
Water a-Cyclodestrin Dextrin Glycogen Tween 40 Tween 80 N-Acetyl-D -galactosamine N-Acetyl-D -glucosamine Adonitol L -Arabinose D -Arabitol Cellobiose i-Erythritol D -Fructose L -Frucose D -Galactose Gentiobiose a-D -Glucose m-Inositol a-D -Lactose Lactulose Maltose D -Mannitol D -Mannose D -Melibiose b-Methyl-D -glucoside D -Psicose D -Raffinose L -Rhamnose D -Sorbitol Sucrose D -Trehalose
) ) + w w w ) + ) w ) ) ) + ) ) ) + w ) ) + ) + ) ) ) ) ) ) ) +
Turanose Xylitol Methyl pyruvate Mono-methyl succinate Acetic acid cis-Acconitic acid Citric acid Formic acid D -Galactonic acid latone D -Galacturonic acid D -Gluconic acid D -Glucosaminic acid D -Glucuronic acid a-Hydroxybutyric acid b-Hydroxybutyric acid c-Hydroxybutyric acid p-Hydroxyphenylacetic acid Itaconic acid a-Keto butyric acid a-Keto glutaric acid a-Keto valeric acid D , L -Lactic acid Malonic acid Propionic acid Quinic acid D -Saccharic acid Sebacid acid Succinic acid Bromosuccinic acid Succinamic acid Glucuronamide Alaninamide
) ) + + w ) ) w ) ) ) ) ) ) ) + w ) ) ) ) w ) w ) ) ) w ) ) ) w
D -Alanine
acid acid Glycyl-L -aspartic acid Glycyl-L -glutamic acid L -Histidine Hydroxy L -proline L -Leucine L -Omithine L -Phenylalanine L -Proline L -Pyroglutamic acid D -Serine L -Serine L -Threonine D , L -Camitine c-Amino butyric acid Urocanic acid Inosine Uridine Thymidine Phenyl ethylamine Putrescine 2-Amino ethanol 2,3-Butanediol Glycerol D , L -Glycerol phosphate Glucose-1-phosphate Glucose-6-phosphate
) + w + w + ) + w ) ) ) ) w ) w + ) ) ) ) + + w w w ) ) w + + +
Similarity Result of identification
0.728 X. nematophilus
+, positive response; ), negative response; and W, weak response
L -Alanine L -Alanyl-glycine L -Asparagine L -Aspartic
L -Glutamic
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(NCBI), we found only a single case of submitted tccC1 nucleotide sequence (3000 bp) (Accession No. AJ308438) from X. nematophilus [15]. To ascertain the existence of toxin gene in extracted genomic DNA, PCR was carried out for amplifying the core region using appropriate primers designed on the basis of the GenBank sequence of tccC1. As a result of agarose gel electrophoresis (Fig. 1A), a single PCR product was apparently visualized. The sequencing of the PCR product showed about 50% homology in amino acid sequence compared to the sequence AJ308438 and also very high homology (92%) with the amino acid sequence of TccC1Xn that Waterfield et al. [18] recently reported (Fig. 1B). These sequence homologies indicate that the extracted genomic DNA contains toxin gene tccC1 with highly conserved core region. Assuming the high homology with AJ308438, we first carried out six independent PCRs to get the toxin gene clone (tccC1) using appropriate primers designed on the basis of 50 - and 30 -UTR sequences of tccC1 (AJ308438). Through the sequence analysis, the six PCR products
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showed more than 90% homology with AJ308438, but the base sequences were all slightly different from each other when compared, which is due probably to the problems of erroneous long PCR (3000 bp) (data not shown). Fortunately, however, we found three restriction enzyme sites (HindIII, EcoRV, and SmaI) in the regions showing 100% sequencing reproducibility among six PCR products. To minimize probable errors in such a long PCR, we considered another PCR strategy of synthesizing the four different tccC1-derived fragments (50 -NdeI-fragment(1)-HindIII-30 , 50 -HindIIIfragment(2)-EcoRV-30 , 50 -EcoRV-fragment(3)-SmaI-30 , and 50 -SmaI-fragment(4)-ClaI-30 ), all of which can be combined together to give a whole tccC1 gene using the three restriction enzyme sites above. That is, the joining of fragments was possible because two fragments to be joined had the common nucleotide sequence including a restriction enzyme site at 50 - or 30 -end of each fragment. With 3–4 PCR products for each tccC1 fragment, the sequence analysis was repeated about 7-times, and correct nucleotide sequence was determined for each frag-
Fig. 1. (A) Result of PCR amplification followed by electrophoresis (1% agarose gel) for core region DNA fragment, using genomic DNA from symbiosis bacteria X. nematophilus MK as PCR template. (Arrow indicates that predicted core region size (180 bp). Lambda HindIII size marker bands indicate 23,130, 9416, 6557, 4361, 2322, 2027, and 564 bp from top to bottom.) (B) Alignment of amino acid sequences corresponding to TccC core region X. nematophilus toxin. (“PCR product”: amplified PCR product shown in (A) “TccXn”: core region sequences reported by Waterfield et al. [18], and “AJ308438”: core region sequences of toxin gene deposited in the GenBank (Accession No. AJ308438)).
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Fig. 2. Alignment of the protein sequences of the cloned toxin gene in this study and the previously reported protein sequences of X. nematophilus (AJ308438) [15] (“X. n XptB1”), P. luminescens W14 (AF346499) [18] (“P. 1 W14 TccC1”), P. luminescens TTO1 (BX571873) [19] (“P. 1 TTO1 TccC1”), and Y. pestis CO92 (NC_003143) [20] (“Y. p CO92 TccC1”).
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ment. The DNA sequence of cloned toxin gene (3048 bp) has an open reading frame encoding 1016 amino acids with a predicted molecular mass of 111,058 Da (Fig. 2). From the alignment of the protein sequences, the determined toxin sequence shares 96%, 51%, 50%, and 52% identical amino acid residues with the X. nematophilus (AJ308438) [15], P. luminescens W14 (AF346499) [18], P. luminescens TTO1 (BX571873) [19], and Yersinia pestis CO92 (NC_003143) [20], respectively (Fig. 2).
Fig. 3. Result of SDS–PAGE analysis of cell-free extracts from E. coli culture (Rosetta(DE3)/pT7-TOX) in 5-L jar fermenter, showing timecourse variations in soluble recombinant exotoxin. (M: size marker, lanes 1-8: cell-free extracts from recombinant E. coli cultures sampled at 0 h (lane 1), 2 h (lane 2), 4 h (lane 3), 6 h (lane 4), 8 h (lane 5), 10 h (lane 6), 12 h (lane 7), and 14 h (lane 8) after gene expression is induced by adding IPTG (1 mM).)
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Heterologous expression of tccC1 gene in E. coli and insecticidal activity of recombinant TccC1 The whole gene of tccC1 was cloned into expression vector, pET3a-TOX, that was used to transform the E. coli strain Rosetta(DE3) (Novagen, USA) that expresses rare tRNAs on a compatible chloramphenicolresistant plasmid pRARE (Materials and methods). Cultures of E. coli Rosetta(DE3)/pT7-TOX were induced with IPTG, the bacterial cells were harvested, and the cell-free extracts were separated by reducing SDS– PAGE. A prominent band of about 110 kDa was visible, which was not found in control cells (E. coli Rosetta(DE3)) transformed with pET3a without insert (Fig. 3). The size of the 110 kDa protein band, found by SDS–PAGE, is in accordance with the calculated size of 111,058 Da of the expressed protein. An equal volume (3 ll) of cell-free extracts prepared from the induced cultures of E. coli Rosetta(DE3)/ pT7-TOX was injected into 10 larvae of G. mellonella. Cell-free extracts from E. coli Rosetta(DE3) cells transformed with pET3a without insert were used as negative control. After the injection of recombinant toxin, the larva viability was monitored everyday for 5 days at 28 °C. Negative control never caused larva death until 5th day, whereas under the same assay conditions, 80% of the larvae that the cell extracts containing recombinant toxin TccC1 were applied to
Table 2 Test results of insecticidal activity of recombinant exotoxin applied to 10 larvae of G. mellonella Percentage mortality (%)a
Negative control (supernatant of host E. coli lysates) Supernatant of recombinant E. coli lysates containing recombinant exotoxin a
Day 1
Day 2
Day 3
Day 4
Day 5
0 60
0 80
0 80
0 80
0 80
(Number of dead larvae) (total number of larvae tested) 100.
Fig. 4. Test results of insecticidal activity of recombinant exotoxin at 5 days after applied to G. mellonella larvae. (A) Larvae injected with negative control (supernatant of host E. coli (Rosetta(DE3) lysates); (B) larvae injected with supernatant of recombinant E. coli (Rosetta(DE3)/pT7-TOX) lysates containing recombinant exotoxin).
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was killed within only 2 days (Table 2 and Fig. 4). We therefore conclude that the heterologous expression of the cloned gene tccC1 produced recombinant toxin with high insecticidal activity.
Acknowledgments This work was supported by Ministry of Commerce, Industry, and Energy in South Korea.
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