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1 Department of Life Sciences, Ben-Gurion University of the Negev, PO Box 653, Be'er-Sheva 84105, Israel
2 Cardiff School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3US, UK
3 Department of Chemical Engineering and Biotechnology, College of Judea and Samaria, Ariel 44837, Israel
Correspondence
Arieh Zaritsky
ariehz{at}bgu.ac.il
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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-trefoil carbohydrate-binding motif, as found in several ricin-like toxins. The gene was PCR-amplified from pBtoxis and cloned in several vectors, allowing high-level expression in Escherichia coli. Cyt1Ca was purified by nickel-nitrilotriacetic acid affinity chromatography, characterized, and its biological activity was determined. Toxicity against larvae of Aedes aegypti of Cyt1Ca in recombinant E. coli cells was compared with that of Cyt1Aa and Cyt2Ba, and the ability of these proteins to enhance the activity of Cry4Aa was assessed. Although Cyt2Ba appeared able to interact with Cry4Aa, no activity for Cyt1Ca was observed, even when produced in truncated form. Furthermore, in contrast to Cyt1Aa, Cyt1Ca did not lyse sheep erythrocytes, and it was not bactericidal to the host cell.
These authors contributed equally to this work. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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-endotoxins (Schnepf et al., 1998
), which are toxic against larvae of diverse groups of insects. These proteins belong to two unrelated families: receptor-specific Cry toxins, active against insects, and Cyt toxins that lyse a broad range of cells, including bacteria, by binding to phospholipids. The ICPs of B. thuringiensis subsp. israelensis are specific against larvae of mosquitoes and black flies (Goldberg & Margalit, 1977), and are composed of four major polypeptides, Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa, encoded by respective genes located on the 128 kb plasmid pBtoxis (Berry et al., 2002). Cyt1Aa is the most prominent and least specific toxin, showing haemolytic and cytotoxic activities in vitro (Thomas & Ellar, 1983; Hofte & Whiteley, 1989). Its mosquito larvicidal activity is low, but it acts synergistically to potentiate the activity of Cry4Aa, Cry4Ba or Cry11Aa (Wu et al., 1994; Crickmore et al., 1995). These Cyt/Cry synergies are significantly greater than any synergistic interactions between the Cry toxins themselves (Crickmore et al., 1995; Khasdan et al., 2001). The role of Cyt1Aa in retarding resistance to the Cry proteins is crucial (Wirth et al., 1997, 2005). In addition, recombinant Escherichia coli (Douek et al., 1992) loses its colony-forming ability upon expressing Cyt1Aa, a lethal action that is circumvented by the accessory protein P20 (Manasherob et al., 2001).
Seven cytolytic toxins (Cyt1Aa, -1Ab, -1Ba, -2Aa, -2Ba, -2Bb and -2Bc) have been characterized in mosquitocidal subspecies of B. thuringiensis (Waalwijk et al., 1985; Thiery et al., 1997; Delécluse et al., 2000; Koni & Ellar, 1993; Guerchicoff et al., 1997; Cheong & Gill, 1997; Juárez-Pérez et al., 2002), the most studied of which is Cyt1Aa (Margalith & Ben-Dov, 2000), but the crystal structure of Cyt2Aa from B. thuringiensis subsp. kyushuensis is the only one elucidated to date (Li et al., 1996). Since Cyt1Aa is 38 % identical to Cyt2Aa (with many of the amino acid differences being conservative in nature), it is believed that the former is likely to adopt similar 3D folding (Li et al., 1996; Gazit et al., 1997).
The sequence of pBtoxis (Berry et al., 2002) identifies a previously unknown gene encoding a putative protein of 
60 kDa (pBt054) with an N-terminal half that is 72 % homologous to Cyt1Aa, hence it was named Cyt1Ca. The C-terminal 280 aa of Cyt1Ca are 50 % homologous to the -trefoil modules found in various natural toxins that contain ricin-B-like domains, such as Clostridium botulinum neurotoxin, Pieris brassicae pierisin-b and the mosquitocidal toxin protein Mtx1 from Bacillus sphaericus (Berry et al., 2002). Cyt1Ca is about twice the size of the other Cyt proteins (26–28 kDa), and may represent a novel two-domain fusion toxin.
In this study, cyt1Ca and the previously identified cyt2Ba were cloned and their products characterized. Their toxicity against larvae of Aedes aegypti and their ability to enhance the activity of Cry4Aa were compared with those of Cyt1Aa.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), pGEM-T (Promega), pUHE-24S and its derivatives cloned with cyt1Ca (complete and truncated) and cyt2Ba (Table 1), pRM4-C containing cyt1Aa (Manasherob et al., 2001), and pHE4-A containing cry4Aa (Ben-Dov et al., 1995). The vector pGEM-T was introduced into strain BL-21, while the others were hosted in strain XL-Blue MRF' (Stratagene).
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) under the original cyt1Aa promoter. The cloned derivatives of the vector were hosted for expression in the acrystalliferous plasmid-less derivative strain IPS78/11, kindly obtained from D. Ellar (Cambridge, UK). The toxin-coding pBtoxis was isolated as described previously (Ben-Dov et al., 1996) from B. thuringiensis subsp. israelensis strain 4Q2-72 (kindly supplied by D. R. Zeigler of the Bacillus Genetic Stock Center, Columbus, OH, USA), and was used as a template for PCR amplification of cyt1Aa, cyt2Ba and cyt1Ca.
PCR.
The primers used to amplify the three cyt genes, the cyt1Aa promoter and p20 from pBtoxis are depicted in Table 1
. Taq DNA polymerase (New England BioLabs) was employed for cloning into E. coli, and Vent DNA polymerase (New England BioLabs) for cloning into B. thuringiensis subsp. israelensis, both in a DNA thermal cycler (T-gradient; Biometra) for 30 cycles at the following conditions: 1 min at 94 °C, 50 s at 55 °C and 1–2 min at 72 °C.
Plasmid construction.
The blunt-end PCR products (Table 1) were purified from agarose gel by a GFX purification kit (Amersham), digested by the appropriate restriction enzymes (Table 1), and further purified from gels with the same GFX kit. The amplicons for cyt2Ba and a different version of cyt1Ca were double-ligated into NcoI/XbaI-digested pUHE-24S or NcoI/BglII-digested pQE-60 for cloning into E. coli. For expression in B. thuringiensis subsp. israelensis, we used either triple ligation of the amplicons for the cyt1Aa promoter, with cyt2Ba or cyt1Ca, into SphI/XbaI-digested pHT315, or double ligation of the amplicon of cyt1Aa with its own promoter. When p20 was added, the constructs were subsequently ligated into the XbaI–SacI sites of cyt-containing pHT315. Verification of all cloned genes was performed by sequencing.
DNA sequences.
Sequencing was performed by ABI PRISM dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase FS and the ABI model 373A DNA sequencer system (Perkin–Elmer).
Transformation.
The ligated DNA (0.5 µg) was mixed in a 0.2 cm cuvette with a suspension of E. coli XL-1 Blue MRF', and introduced into the bacteria by electroporation (using a Bio-Rad mini apparatus set) at 2.5 kV and 186 
. Cloned derivatives of shuttle vector pHT-315 were further electroporated into the acrystalliferous strain IPS78/11 of B. thuringiensis subsp. israelensis. Screening for transformants was performed on Luria–Bertani (LB) plates, with either 100 µg ampicillin ml–1 at 37 °C (for E. coli) or 20 µg erythromycin ml–1 at 30 °C (for B. thuringiensis subsp. israelensis).
Gene expression.
Cultures of E. coli were grown at 37 °C in LB medium supplemented with 100 µg ampicillin and 10 µg tetracycline ml–1, and induced by IPTG (0.5 mM) at OD600 0.2–0.3 (2x108 cells ml–1). Cultures of B. thuringiensis subsp. israelensis were grown overnight in 5 ml LB medium, transferred to 500 ml CCY sporulation medium (Stewart et al., 1981), with 20 µg erythromycin ml–1 at 30 °C for 4 days (Nisnevitch et al., 2006).
E. coli cells and B. thuringiensis subsp. israelensis spores and crystals were harvested by centrifugation at various times after induction or sporulation, respectively, resuspended in distilled water, and boiled for 10 min in sample treatment buffer (62.5 mM Tris/HCl, pH 6.8, 2 % SDS, 10 % glycerol, v/v, 0.01 % bromophenol blue and 0.1 M DTT) with protease inhibitor PMSF (Sigma P7626; 5 mM). Samples were analysed by SDS-PAGE (Laemmli, 1970). The gels were stained with 0.1 % Coomassie Blue R-250. Protein concentrations were determined with BSA standards (Bradford, 1976).
Western blot analysis.
Proteins were electro-transferred from the gel onto nitrocellulose membranes, and exposed to specific antiserum directed against Cyt1Aa (kindly provided by Sarjeet Gill, University of California, Riverside, CA, USA). Protein A–alkaline phosphatase conjugate was the primary antibody detector. Visualization of the antigen was achieved using Sigma Fast 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium tablets (Sigma), the chromogenic substrate for alkaline phosphatase.
Purification of His6-tagged Cyt1Ca.
Purification was performed by nickel-nitrilotriacetic acid (Ni-NTA) column affinity chromatography from the lysate of transgenic E. coli cells expressing cyt1Ca–His. Cells harbouring pQE-cyt1CaHis were harvested after 4 h induction, washed twice, incubated with lysozyme [10 mg (g wet weight of cells)–1] for 30 min, and broken up by sonication in 50 mM Tris/HCl (pH 8.0). The mixture was centrifuged, and the supernatant loaded into an Ni-NTA column. Loosely bound proteins were washed from the resin in the above Tris/HCl buffer containing 20 mM imidazole, while the recombinant His6-tagged Cyt1Ca was eluted by buffer containing 400 mM imidazole, according to the standard procedures of the manufacturer (Qiagen).
Proteolysis of Cyt1Ca–His.
Purified Cyt1Ca–His was solubilized at about 1 mg ml–1 in 50 mM Na2CO3, pH 10.5, at 37 °C for 1 h. Lower pH values were ineffective for solubilization. The solubilized protein was treated with 10 % (w/w) trypsin, chymotrypsin, thermolysin or proteinase K at 37 °C for 1 h.
Haemolytic assay.
PBS-suspended sheep erythrocytes were incubated overnight at 37 °C with activated Cyt1Ca (100 µg ml–1), and OD570 of the supernatant was recorded. Incubation with double-distilled water, PBS or Cyt1Aa was used as a control for haemolytic activity.
Mosquito larvicidal activity.
Twenty third- or fourth-instar Ae. aegypti larvae, in duplicate, were incubated at 28 °C in 100 ml sterile tap water, with appropriate dilutions of E. coli expressing cyt1Ca, cyt2Ba and cyt1Aa. Larval mortality was scored after 24 h. Synergistic interactions between Cyt1Aa, Cyt1Ca, Cyt2Ba and Cry4Aa were tested by feeding with bacterial mixtures in a 1 : 1 ratio by cell number.
Molecular mass determination.
The molecular mass of purified His6-tagged Cyt1Ca, dissolved in a mixture of propanol, double-distilled water and formic acid (2 : 3 : 1, by vol.), was determined using a Reflex IV MALDI-TOF mass spectrometer (Bruker), with an 
-cyano-4-hydroxycinnamic acid (CHCA) matrix.
Bacterial viability.
Viability was determined by colony-forming ability on LB plates (with 100 µg ampicillin and 10 µg tetracycline ml–1) following appropriate dilutions. The number of colonies was counted after 24 h incubation at 37 °C. Each viability value was calculated from the mean of duplicate values for three different dilutions.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Khasdan et al., 2001), and delays or prevents the selection for resistance of target insects (Wirth et al., 1997, 2005). Discovering a potentially cytolytic protein from the Cyt family with a binding domain may indicate that it is targeted to the cell via a receptor, as for Cry family toxins. The questions thus arise whether Cyt1Ca is toxic at all; if so, what is its mode of action, and could it be exploited to enhance the biological control of mosquitoes?
To address these issues, cyt1Ca was PCR-amplified and cloned into E. coli in three vectors, pGEM-T Easy, pUHE-24S and pQE-60, to produce clones designated pGMCB-1C, pUH-cyt1Ca and pQE-cyt1CaHis, respectively. The recombinant E. coli strains were grown in LB medium and induced with 0.5 mM IPTG for 4 h, and the protein content analysed by SDS-PAGE (Fig. 1A). An additional polypeptide, not observed in un-induced cultures, was detected only in cells transformed with the latter two plasmids (Fig. 1A, lanes 2 and 4), but its electrophoretic mobility corresponded to a molecular mass significantly lower (50 kDa) than the expected 60 kDa. Western blot analysis demonstrated that Cyt1Ca did not cross-react with polyclonal anti-Cyt1Aa antibodies (data not shown), despite the 49 % identity between Cyt1Aa and the N-terminal domain of Cyt1Ca.
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) was also used. All three genes (cyt1Aa, cyt2Ba and cyt1Ca) were thus overexpressed in the same E. coli strain (XL-Blue MRF'), from the same vector (pUHE-24S), under the control of the same promoter (T7 early), and in the same growth conditions. Each of these was mixed in a 1 : 1 ratio by cell number with clone pHE4-A (expressing cry4Aa), and possible synergy in toxicity against Ae. aegypti larvae was compared (Fig. 2). No toxicity was displayed by any of these three cyt-expressing strains alone at the maximum cell concentration used, 3x107 cells ml–1 (data not shown). Cry4Aa-producing cells caused 90 % larval mortality at 1x107 cells ml–1; this was enhanced to 6.3x105 cells ml–1 in combination with an equal concentration of Cyt1Aa-producing cells, and to a lesser extent (5x106 cells ml–1) in combination with Cyt2Ba-producing cells (Fig. 2). Cyt1Ca-producing cells, however, were unable to supplement the activity of Cry4Aa, even at a ratio of Cyt1Ca : Cry4Aa as high as 4 : 1 (Itsko et al., 2005).
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; Khasdan et al., 2001). Moreover, higher synergy values (between 16.6 and 70.5) were obtained when resistant strains of Culex quinquefasciatus were tested, thus demonstrating the significance of Cyt1Aa in suppressing resistance (Wirth et al., 1997). Cyt2Ba has been demonstrated to synergize with B. sphaericus (Wirth et al., 2001), but synergy with Cry4Aa has never been demonstrated. The difficulty in expressing cyt1Aa in the heterologous host E. coli leads to very low levels of production. This makes it hard to compare the synergism of the individual Cyt proteins accurately in these experiments. However, cyt1Aa-expressing cells are able to produce significant enhancement of Cry4Aa activity, despite the fact that protein induction can hardly be detected by immunoblotting (Manasherob et al., 2001). In contrast, Cyt2Ba could be visualized by SDS-PAGE (data not shown), yet produced less enhancement of Cry4Aa activity, and Cyt1Ca, also clearly induced in the cultures tested (Fig. 1A, lane 2), produced no effect.
The level of enhancement of Cry4Aa activity by each of the Cyt proteins reflects their relative abundance in B. thuringiensis subsp. israelensis. Cyt1Aa composes up to 50 % of the crystal, Cyt2Ba is present in very low quantities, and Cyt1Ca is undetectable, although transcript from cyt1Ca has been detected (Stein et al., 2006). Perhaps the high-level expression of the most synergistic toxin was selected in this B. thuringiensis strain.
In the case of Cyt1Aa, effects on the growth and viability of the heterologous host E. coli have been observed within minutes of the induction of protein expression (Manasherob et al., 2001). Production of Cyt1Ca showed no such effects (data not shown), indicating that this protein is not toxic either to mosquito larvae or to E. coli cells, although Cyt1Ca mutants have been produced that do display effects on the host bacterium, but not on mosquitoes (Itsko et al., 2005).
Cloning of three cyt genes for expression in B. thuringiensis subsp. israelensis
To rule out the possibility that Cyt1Ca is not properly folded in E. coli, and hence loses its presumed activity, the three cyt genes, cyt1Aa, cyt2Ba and cyt1Ca, were cloned under the strong cyt1Aa promoter in the expression vector pHT315 into acrystalliferous B. thuringiensis subsp. israelensis. While cyt1Aa and cyt2Ba displayed substantial expression, cyt1Ca did not produce Cyt1Ca at all (Fig. 3). Moreover, the accessory protein P20, known to raise the levels of Cyt1Aa (Wu & Federici, 1993) and of Cyt2Ba (Nisnevitch et al., 2006) in acrystalliferous strains of B. thuringiensis subsp. israelensis, did not assist in cyt1Ca expression (Fig. 3). Similarly, another gene of B. thuringiensis subsp. israelensis, p19, presumed to encode an accessory protein, has been shown to be expressed in E. coli (Manasherob et al., 2001) but not in B. thuringiensis (unpublished data). Transcripts of cyt1Ca and cyt2Ba have recently been detected in strain 4Q5 (Stein et al., 2006), without their products; this observation is explained by instability of the transcript or of the resultant protein, or failure in translation. Here, replacing the original respective promoters with that of cyt1Aa resulted in high production of Cyt2Ba but none of Cyt1Ca. Consistently, haemolytic activity was detected in the former clone (Nisnevitch et al., 2006) but not in the latter (data not shown).
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, lane 4); thus, the low concentration of soluble Cyt1Ca may have been a reason for the lack of lethal activity. To test this possibility, the cells were heat-treated (41 °C for 2 h) before induction with IPTG to raise the quantity of chaperones (Baneyx, 1999). Higher levels of soluble Cyt1Ca were thus obtained (Fig. 1B, compare lanes 8 and 7). The amount was even greater when cells were transferred to 28 °C after induction (Fig. 1B, compare lane 9 with 8 and 7). Nevertheless, no change in either toxicity to the host bacterium or larvae, or in the ability to enhance the activity of Cry4Aa, was observed in bioassays repeating those described in Fig. 2 (data not shown).
For further characterization of Cyt1Ca, cyt1Ca was cloned into pQE-60 to encode a fusion protein with six His residues at the C terminus, and the chimera was purified on a Ni-NTA column. The SDS-PAGE mobility of the purified polypeptide was indeed significantly lower (50 kDa) than the 60 kDa predicted from the gene sequence (Fig. 4, inset). The sequence of the cyt1Ca gene in this clone was verified to rule out mutation during PCR amplification, and was identical to the published sequence (Berry et al., 2002). Sequencing the N-terminal amino acids (MAQSEF) confirmed that the protein was Cyt1Ca, and analysis by MALDI-TOF MS yielded a size of 61.259 kDa (Fig. 4), almost exactly that (61.398 kDa) of the chimeric Cyt1Ca–His6. Thus, the lack of toxicity was not a result of Cyt1Ca degradation while expressed in E. coli. The faster migration of Cyt1Ca in SDS-PAGE thus seems to derive from some unusual intrinsic structure.
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), but no significant effect was detected. Proteolytic activation of Cyt1Ca with proteinase K, chymotrypsin and trypsin yielded only a marginal effect (Fig. 5), confirming that Cyt1Ca is neither bacteriolytic (when expressed in E. coli) nor cytolytic (when added to larvae and erythrocytes). The low haemolytic activity observed may have resulted from the high pH (10.5) used for solubilization, but the Cyt proteins did not dissolve at the pH range (7–9) optimal for the proteases, a pH range at which Cyt proteins precipitate (Du et al., 1999).
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-trefoil module in its C-terminal region. This ricin B-like domain may interfere with the insertion and organization of the Cyt-like part of Cyt1Ca into the membrane of target cells. Cyt1Aa loses its lethal activity when fused at its C terminus to green fluorescent protein (GFP) (Manasherob et al., 2003), indicating that the organization of the C terminus of Cyt1Aa may be crucial for its activity. In attempts to obtain a toxic variant of Cyt1Ca, the 3' end of cyt1Ca was genetically truncated at positions 722 and 744 bp (to generate proteins truncated at E228 and F237, respectively) (Table 1, Fig. 6A), to mimic Cyt1Aa and Cyt2Aa proteinase K activation sites (Al-Yahyaee & Ellar, 1995; Du et al., 1999). A new set of bioassays was performed with the truncated clones using fourth-instar mosquito larvae (Fig. 6B), but no significant differences in mortality (Itsko et al., 2005) or synergism with cry4Aa-expressing cells were observed with the truncated Cyt1Ca.
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-trefoil domain, or that Cyt1Ca has acquired such a domain, after the evolution of Cyt toxins. In the case of cyt1Aa, a convergent coding sequence (pBt020) exists on pBtoxis (accession no. AL731825) and is separated from cyt1Aa by only 2 nt, a situation indicating genetic rearrangement in this region that could have deleted an ancestral C-terminal domain. However, this feature of a close, convergent coding sequence does not appear to occur with other cyt sequences. The facts that Cyt1Aa is adversely affected by C-terminal fusions (Manasherob et al., 2003), and that Cyt1Ca appears inactive, indicate that the ancestor is rather of the single Cyt domain type. In summary, a novel protein, Cyt1Ca from B. thuringiensis subsp. israelensis, has been characterized. This protein was composed of two domains, Cyt- and lectin-like, suggesting a receptor-binding ability not recognized in any previously known Cyt protein. However, the protein and its genetically engineered truncated version were neither toxic nor able to enhance the toxicity of Cry4Aa, in contrast to the other Cyt proteins, Cyt2Ba and Cyt1Aa.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 9 March 2006;
revised 15 May 2006;
accepted 22 May 2006.
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