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Contribution of the individual components of the δ-endotoxin crystal to the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis
ELSEVIER
FEMS Microbiology
Letters 131 (1995) 249-254
Contribution of the individual components of the Sendotoxin crystal to the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis Neil Crickmore
a,b,Eileen J. Bone a, Juliet A. Williams
a, David J. Ellar a,*
a Department of Biochemistry, Universiry of Cambridge, Cambridge CB2 lQW, UK b School of Biological Sciences, Universiry of Sussex, Falmer, Brighton BNI 9QG, UK Received 9 June 1995; revised 6 July 1995; accepted
11 July 1995
Abstract The Sendotoxin crystal of the mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis contains four major 6-endotoxins. Expression systems were devised to synthesize each of the four toxins at concentrations at which they formed inclusion bodies in an acrystalliferous mutant of Bacillus thuringiensis. The relative activities of these inclusions were then determined against Aedes aegypti larvae. Bioassays of mixtures of the individual toxins revealed a number of synergistic interactions which explained in part why the native crystal is considerably more toxic than any of the individual toxins. Keywords: Synergism;
Bacillus thuringiensis israelensis; GEndotoxin;
1. Introduction The bacterium Bacillus thuringiensis subsp. israelensis is highly toxic to dipteran larvae, and is widely used to control pest species such as mosquitoes and blackflies. During sporulation, this bacterium synthesises a cytoplasmic, parasporal inclusion body (or crystal) whose main constituents are the insecticidal proteins (Sendotoxins) CryIVA, CryIVB, CryIVD, and CytA with molecular masses of 130 kDa, 135 kDa, 65 kDa and 27 kDa, respectively. The genes encoding these toxins are located on a single high molecular mass plasmid and have been cloned individually and sequenced [l-4]. When
* Corresponding author. Tel.: +44 (1223) 333 651; Fax: +44 (1223) 333 345; E-mail: [email protected]. 037%1097/95/$09.50 0 1995 Federation SSDI 0378-1097(95)00264-2
of European
Microbiological
Mosquito
the genes were expressed in Escherichia coli, B. subtilis, or B. thuringiensis, the resulting toxins were all mosquitocidal, with CytA having additional haemolytic and cytolytic activities in vitro. Reports differ, however, concerning the relative contribution of each toxin to the overall toxicity of B. thuringiensis subsp. israelensis. Notwithstanding the large differences in reported activities for any given toxin, it is clear that no single toxin is as active as the native crystal. This has led a number of workers [S-8] to investigate the possibility of synergism between two or more toxins. Here we have compared the toxicities of all four Gendotoxins, synthesised and bioassayed under identical conditions, determined their relative activities and have found new synergistic interactions between them. Societies. All rights reserved
N. Crickmore
250
et ul. / FEMS Microbiology
2. Materials and methods
Letters 131 (1995) 249-254
purified by discontinuous tion .
sucrose density centrifuga-
2.1. Bacterial strains and plasmids 2.3. Mosquito bioassay The B. thuringiensis strain IPS78/11 is an acrystalliferous mutant of B. thuringiensis subsp. israeZensis; the E. coli strain TGl was used for all routine procedures. Plasmids used in this study are shown in Fig. 1. The fragments shown were subcloned on a number of shuttle vectors containing pUC 12, 18 or 19 and all, or part of, pC194. The use of these plasmids in E. coli/B. thuringiensis shuttle vectors has been described before [9]. 2.2. Transformation
of B. thuringiensis
Aedes aegypti larvae were hatched from eggs supplied by ICI Agrochemicals. A filter paper containing the eggs was added to 6 1 deionised water (pre-warmed to 30°C) along with 1 g of crushed rat maintenance diet pellets (supplied by Special Diet Services). The eggs were incubated at 3O”C, with constant lighting, for 8 h. The filter paper was then removed and the hatched larvae were maintained under the same conditions for a further 64 h. For the bioassay, five larvae (in 0.5ml) were transferred to a 1.5-ml Eppendorf tube to which 100 ~1 of suspended toxin was then added. The tubes were capped, a single hole pierced in the lids and maintained at 3O”C, with constant lighting, for 24 h, after which mortality was determined against a control background. Thirty to 60 larvae were used for each concentration, and a range of 4-8 concentra-
and purifica-
tion of inclusions B. thuringiensis was transformed by electroporation. Transformants were selected and maintained on LB plates, and grown to sporulation in CCY medium [7] containing 5-10 pug ml-’ chloramphenicol. Inclusions produced by the sporulating cultures were
H
AC
P
AC N POkDa
T
CiylVD
cam27 cam2027
RV
CvtA
_
Rl
lmlusion 0Fonaam
--)llc+ w-
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w+
pSVlVD
+
cam130
camp271 30
T
e-
camtaq
cam201 30
I3
------c7Qc
7 T
+ B
cam135 C
+
Fig. 1. Structure of the Gendotoxin expression vectors. The top line represents the region of the B. thuringiensis subsp. israelensis 72-MDa plasmid that encodes the cytA, cr/ND. and 20-kDa genes. The subsequent lines represent the fragments of B. fhuringiensis subsp. israelensis DNA that are present on each expression vector. Structural genes are shown as pointed boxes, promoter regions by triangles. Whether or not a transformant of IPS78/11 carrying each of these plasmids synthesises an inclusion body during sporulation is indicated by a plus or minus in the right hand column. For clarity not all of the following restriction sites are shown: H3, HindUI; AC, AccI; N, NlaIV; P2, PuuII; RV, EcoRV; T, TaqI; B, BarnHI; Rl, EcoRI; C, Clal.
N. Crickmore et al. /FEMS Microbiology
tions was used for each toxin or toxin combination tested. The data were subjected to probit analysis.
Letters 131 (1995) 249-254
pletely upstream of the TuqI site that was used to subclone the cytA gene into camtaq. A possible explanation for the lack of expression of cyti in B. thuringiensis, therefore, was the absence of an intact promoter. The plasmid cam27 was therefore constructed in which the region upstream of cyti in camtaq was extended to include the putative promoter. When this construct was introduced into IPS78/11, the result was similar: expression was poor and no obvious inclusions were observed. McLean and Whiteley [lo] demonstrated that a 0.8-kb region located approximately 4 kb 5’ of cyyt was required for cytA expression in E. cofi. This region was later shown to encode a 20-kDa polypeptide Ill]. This 5’ region was apparently not required for the expression of cytA in B. subtilis since it was not present on camtaq, although it remained a possibility that it was required in B. thuringiensis. The plasmid cam2027 was constructed containing cyrA and the 20-kDa gene, and introduced into IPS78/11.
3. Results 3.1. Expression of cytA Ward et al. [9] found that expression of cyt4 in a Bacillus subtilis strain transformed with the toxinencoding plasmid camtaq resulted in the formation of small irregular inclusions. When, however, we introduced camtaq into an acrystalliferous strain of Bacillus thuringiensis subsp. isruelensis (IPS78/ 111, expression was poor and no inclusion bodies were observed. Ward and Ellar [l] mapped the transcription initiation sites of cytA in B. subtilis and B. thuringiensis and found differences between the two strains. In particular the putative promoter in B. thuringiensis was predicted to lie partially or com-
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Log Concentration Fig. 2. Probit analysis on the toxicity of the individual B. thuringiensis subsp. israelensis toxins. The graph represents the regression lines of probit mortality of Aedes aegypti larvae against the log,, concentration of toxin. Percentage mortality levels are indicated on the right hand ordinate.
N. Crickmore
252
er al. / FEMS Microbiology
Transformants containing this plasmid produced large lemon-shaped inclusions (data not shown) which, when purified on discontinuous sucrose density gradients, consisted predominantly of CytA. Finally the plasmid cam20taq was constructed in which the 20kDa gene was added to camtaq. This, too, directed the synthesis of CytA inclusions when introduced into IPS78/11. The structures of all of these constructs are shown in Fig. 1. 3.2. Expression
of CryIVA
Ward and Ellar [2] found that expression of their cloned cryNA gene was poor in both E. coli and B. subtilis, unless the promoter-containing region of cyti was added immediately upstream of the cryIVA gene. Yoshisue et al. [12] claimed that the 20-kDa protein was required for expression of cryZVA, as well as cyti, in E. coli. In B. thuringiensis we observed that the cloned gene alone (cam130) did not direct the synthesis of inclusion bodies in IPS78/11, whereas camp27130 did (Fig. 1). The addition of the 20-kDa gene to cam130 (cam20130, Fig. 1) was not sufficient to induce inclusion formation. Ward and Ellar reported that the addition of an inverted repeat structure downstream of crylVA further increased the level of expression of this gene in B. subtilis. We could find no significant difference in expression in the presence or absence of this inverted repeat in B. thuringiensis (data not shown). 3.3. Expression
of cryIVD and cryIVB
In contrast to cytA and cryIVA, crylVB required no obvious additional factors in order to form inclusions in IPS78/11. The construction of the cryZVBcontaining plasmid cam135 has been described before [13], and this sub-clone does not contain the inverted repeat normally found downstream of cryIVB. The cryIVD gene was subcloned on an EcoRV-NlaIV fragment (Fig. 1) that does not contain any of the neighbouring cytA or 20-kDa genes, although it does contain the p19 ORF described by Dervyn et al. .[14]. 3.4. Mosquito-laroicidal Inclusion individually
bioassays
bodies containing the single toxins were tested against 3-day old Aedes aegypti
Letters 131 (1995) 249-254
Table 1 Toxicity of the B. fhuringiensis Aedes aegvpti
subsp. israelensis
toxins against Expected h
Toxin(s)
LCW d
CryIVA CryIVB CryIVD CytA Bti ’ CryIVA + CytAd CryIVB + CytA’ CryIVD + CytAd CryIVB + CryIVD ’ CryIVA + CryIVB + CrylVD ’
1125 467 224 1209 10 75 62 118 173 125
CryIVA + CryIVB + CytA’
77
(66-89)
788
CryIVA + CryIVB + CryIVD + CytA’
85
(75-97)
477
(955-1349) (392-554) t 170-277) (1110-1324) (X-12) (56-93) (48-75) (97-139) (107-242) (110-144)
1163 677 372 300 401
’ LC,, values are expressed in ng ml-‘, 95% confidence limits are shown in parentheses. ’ Expected LC,,, values of the toxin combinations are based on the toxicities of the individual toxins and were calculated using the formulae derived by Tabashnik [20]. ’ B. thuringiensis subsp. israelensis native toxin. ’ Toxins were combined in equal amounts (w/w).
larvae. Fig. 2 shows the result of probit analysis on the four toxins; LC,,, values are shown in Table 1. Under the conditions used, CryIVD is the most toxic with an LC,, value of 224 ng ml-‘, compared with CryIVB (470 ng ml-‘), CryIVA (1120 ng ml-’ ) and CytA (1210 ng mll’). The slope of the CytA regression line is considerably steeper than those of the other toxins, such that at higher toxin concentrations CytA becomes the most effective. None of the four individual toxins is as active as the native B. thuringiensis subsp. israelensis crystal (Table 1). This suggests that certain properties of the native crystal make it more active and/or that the individual components are acting synergistically with each other. In order to test the latter possibility, a series of toxin combinations was tested (Table 1). Although CytA was the least toxic, whenever it was combined with any of the other toxins the resulting activity was greater than that expected from the individual activities. Individually CytA and CryIVA had similar LC, values of approximately 1.2 pg ml-‘, yet when they were mixed the combination had an activity of 7.5 ng ml I, which represents a
N. Crickmore et al. / FEMS Microbiology Letters 131 (1995) 249-254
15-fold increase over the expected value. Similar effects were seen when CryIVB/CytA, CryIVD/ CytA and CryIVB/CryIVD combinations were tested, whilst combinations of CryIVA, CryIVB and CytA resulted in activities some lo-15fold greater than that expected. A number of other combinations of all four toxins with ratios more closely related to the native crystal were also tested and each showed a synergistic effect, although none of the mixtures tested was significantly more active than those described above (data not shown).
4. Discussion There have been many reports concerning the toxicities of the individual components of the B. thuringiensis subsp. israelensis crystal which present widely differing views. In the case of CytA, LC,, values against Aedes aegypti larvae range from 115 ng ml-’ [6] to > 25 mg ml-’ [15]. Much of the reason for this variation may be caused by the form in which the toxin is presented to the mosquito. Where the toxin has been separated biochemically from the B. thuringiensis subsp. israelensis crystal (often contaminated with other toxins), it is usually fed to the larvae (natural filter feeders) in a soluble or re-precipitated form. Where cloned genes have been used, inclusion bodies are usually produced in either E. coli or Bacillus but are of variable size, contain varying numbers of spores (in the case of Bacillus), and have been shown to have other straindependent differences such as solubility 171. Spores have previously been shown to synergise the activity of Gendotoxins against certain other insect species [16]. Despite being purified by density centrifugation, the toxin preparations used here contained low levels of contaminating spores. However, this low level, and the speed at which the toxic effect was seen (trends were often observed within an hour), suggests that spore germination with resulting septicaemia was not contributing significantly to our results. Another major cause of variation is in the bioassay itself, the LC,, value obtained being dependent on a number of factors including larval age, larval diet and the particular batch of mosquitoes used. Natural variation in various insect populations has
2.53
recently been shown to complicate the attainment of reliable bioassay data [17]. For the above reasons the comparison of results from different workers has proved difficult, and only when assays of individual toxins and toxin mixtures are performed under the same conditions can a true comparison be made. By performing such an experiment we have found that CryIVD is the most toxic of the four when using concentrations that result in the death of around 50% of the larvae, while at higher concentrations CytA becomes the most toxic. It is clear from Fig. 2 that the CytA dose-response curve is significantly steeper than those of the other toxins, which may indicate different mechanisms of pore formation between Cyt and Cry toxins. This may also be reflected in the general in vitro cytolytic activity associated with the Cyt toxins. None of the four toxins proved to be as active against Aedes aegypti larvae as the native B. thuringiensis subsp. israelensis crystal, and there are several possible explanations for this. There might be additional factors associated with the native crystal which are important for toxicity. For example, it has yet to be established whether the gene product of c@VC [18] is present in the inclusion and contributes to the overall toxicity. Alternatively, the nature of the native crystal might mean that it is ingested or solubilised more efficiently than those from the recombinant strains. The presentation of all four toxins in a single crystal might be more efficient than a mixture of four inclusions or, finally, the toxins might be acting synergistically with each other. There have been several reports claiming synergism between various B. thuringiensis subsp. israelensis toxins [5-81, in particular between CryIVD and CytA and between CryIVA and CryIVB. We have found that any combination of the four toxins appeared to have a synergistic effect, the effect being greatest when CytA was one of the components. This may be related to the possible differences in the mechanism of action of CytA and the Cry toxins. The interactions involved in the toxic mechanism are clearly complex. A strain in which the cytA gene was genetically inactivated was found to be as active as the native strain [19], implying that CytA was not essential for mosquitocidal activity. But as the mutant strain was not more toxic than the native strain (as might have been expected on the basis of the
254
N. Crickmore et al. /FEMS
Microbiology Letters I31 (1995) 249-254
individual toxicities) it is clear that CytA is playing an important role in the toxicity of B. fhurirzgiensis subsp. isruelensis.This was confirmed by the activity of the combinations tested in this paper.
Acknowledgements We would like to thank Mr. Trevor Sawyer and other members of the biochemistry department for their input to this project.
References ill Ward, ES. and Ellar, D.J. (1986) Bacillus thuringiensis VW. israelensis Gendotoxin: nucleotide sequence and characterisation of the transcripts in Eacilhts thuringiensis and Escherichia co/i. J. Mol. Biol. 191, l-11. El Ward, ES. and Ellar, D.J. (1988) Cloning and expression of two homologous genes of Bacillus thuringiensis subsp. israelensis which encode 130-kilodalton mosquitocidal proteins. J. Bacterial. 170, 727-735. W., Hofte, H., Seurinck, J., Angsuthana[31 Chungjatupomchai, sombat, C. and Vaeck, M. (1988) Common features of Bacillus thuringiensis toxins specific for Diptera and Lepidoptera. Eur. J. B&hem. 173, 9-16. C. and Gilbert M.P. (1988) 141 Donovan, W.P., Dankocsik, Molecular characterisation of a gene encoding a 72-kilodalton mosquito-toxic crystal protein from Bacillus thuringiensis subsp. isruelensis J. Bacterial. 170, 4732-4738. 151 Wu, D. and Chang, F.N. (198.5) Synergism in mosquitocidal activity of 26 and 65 kDa proteins from Bacillus thuringiensis subsp. israelensis crystal. FEBS I&t. 190, 232-236. (61 Chilcott, C.N. and Ellar, D.J. (1988) Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro. J. Gen. Microbial. 134, 2551-2558. C., Crickmore, N. and Ellar, D.J. (1992) [71 Angsuthanasombat, Comparison of Bacillus thuringiensis subsp. israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo. FEMS Microbial. Lett. 94, 63-68. [8] Chang, C., Yong-Man, Y., Shu-Mei, D., Law, S.K. and Gill, S.G. (1993) High-level c+VD and cyti gene expression in Bacillus thuringierwis does not require the 20-kilodalton protein, and the coexpressed gene products are synergistic in their toxicity to mosquitoes. Appl. Environ. Microbial. 59, 815-821. [9] Ward, E.S., Ridley, A.R., Ellar, D.J. and Todd, J.A. (1986) BacilIus thuringiensis var. israelensis Gendotoxin: cloning
and expression of the toxin in sporogenic and asporogenic strains of Bacillus subtilis. J. Mol. Biol. 191, 13-22. [lo] McLean, K.M. and Whiteley H.R. (1987) Expression in Escherichia cob of a cloned crystal protein gene of Bacillus thurbtgiensis subsp. israeiensis. J. Bacterial. 169, 10171023. 1111 Adams, L.F., Visick, J.E. and Whiteley, H.R. (19891 A 20kilodalton protein is required for efficient production of the Bacillus thuringiensis subsp. israelensis 27-kilodalton crystal protein in Escherichia cob. J. Bacterial. 171, 521530. [12] Yoshisue, H., Yoshida, K.-l., Sen, K., Sakai, H. and Komano, T. (1992) Effects of Bacillus thuringiensis var. israeLewis 20-kDa protein on production of the Bti 130~kDa crystal protein in Escherichia cob. Biosci. Biotechnol. Biochem. 56, 1429-1433. C., Crickmore, N. and Ellar, D.J. (1991) 1131 Angsuthanasombat, Cytotoxicity of a cloned Bacillus thuringiensis subsp. israelensis CryIVB toxin to an Aedes aegypti cell line. FEMS Microbial. Lett. 83, 273-276. [141 Dervyn, E., Poncet, S., Klier, A. and Rapoport, G. (1995) Transcriptional regulation of the cryNI) gene operon from Bacillus thuringiensis subsp. israelensis. J. Bacterial. 177, 2283-2291. 1151 Hurley, J.M., Bulla, L.A. and Andrews, R.E. (19871 Purification of the mosquitocidal and cytolytic proteins of Bacillus thuringiensis subsp. israelensis. Appl. Environ. Microbial. 53, 1316-1321. [161 Miyasono, M., Inagaki, S., Yamamoto, M., Ohba, K., Ishiguro, T., Takeda, R. and Hayashi, Y. (1994) Enhancement of S-endotoxin activity by toxin-free spore of Bacillus thuringiensis against the Diamondback Moth, Plutella xylostella. J. Invert. Pathol. 63, 111-112. 1171 Robertson, J.L., Preisler, H.K., Ng, S.S., Hickle, L.A. and Gelemter, W.D. (1995) Natural variation: a complicating factor in bioassays with chemical and microbial pesticides. J. Econ. Entomol. 88, l-10. ml Thome, L., Garduno, F., Thompson, T., Decker, D., Zounes, M., Wild, M., Walfield, A.M. and Pollock, T.J. (1986) Structural similarity between the Lepidoptera-and dipteraspecific insecticidal endotoxin genes of Bacillus thuringiensis subsp. ‘kurstaki’ and ‘israelensis’. J. Bacterial. 166, 801-811. 1191 Delecluse, A., Charles, J.-F., Klier, A. and Rapoport, G. (1991) Deletion by in vivo recombination shows that the 28kilodalton cytolytic polypeptide from Bacillus thuringiensis is not essential for mosquitocidal activity. J. Bacterial. 173, 3374-3381. 1201 Tabashnik, B.E. (1992) Evaluation of synergism among Bacillus fhuringiensis toxins. Appl. Environ. Microbial. 58, 3343-3346.
FEMS Microbiology
Letters 131 (1995) 249-254
Contribution of the individual components of the Sendotoxin crystal to the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis Neil Crickmore
a,b,Eileen J. Bone a, Juliet A. Williams
a, David J. Ellar a,*
a Department of Biochemistry, Universiry of Cambridge, Cambridge CB2 lQW, UK b School of Biological Sciences, Universiry of Sussex, Falmer, Brighton BNI 9QG, UK Received 9 June 1995; revised 6 July 1995; accepted
11 July 1995
Abstract The Sendotoxin crystal of the mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis contains four major 6-endotoxins. Expression systems were devised to synthesize each of the four toxins at concentrations at which they formed inclusion bodies in an acrystalliferous mutant of Bacillus thuringiensis. The relative activities of these inclusions were then determined against Aedes aegypti larvae. Bioassays of mixtures of the individual toxins revealed a number of synergistic interactions which explained in part why the native crystal is considerably more toxic than any of the individual toxins. Keywords: Synergism;
Bacillus thuringiensis israelensis; GEndotoxin;
1. Introduction The bacterium Bacillus thuringiensis subsp. israelensis is highly toxic to dipteran larvae, and is widely used to control pest species such as mosquitoes and blackflies. During sporulation, this bacterium synthesises a cytoplasmic, parasporal inclusion body (or crystal) whose main constituents are the insecticidal proteins (Sendotoxins) CryIVA, CryIVB, CryIVD, and CytA with molecular masses of 130 kDa, 135 kDa, 65 kDa and 27 kDa, respectively. The genes encoding these toxins are located on a single high molecular mass plasmid and have been cloned individually and sequenced [l-4]. When
* Corresponding author. Tel.: +44 (1223) 333 651; Fax: +44 (1223) 333 345; E-mail: [email protected]. 037%1097/95/$09.50 0 1995 Federation SSDI 0378-1097(95)00264-2
of European
Microbiological
Mosquito
the genes were expressed in Escherichia coli, B. subtilis, or B. thuringiensis, the resulting toxins were all mosquitocidal, with CytA having additional haemolytic and cytolytic activities in vitro. Reports differ, however, concerning the relative contribution of each toxin to the overall toxicity of B. thuringiensis subsp. israelensis. Notwithstanding the large differences in reported activities for any given toxin, it is clear that no single toxin is as active as the native crystal. This has led a number of workers [S-8] to investigate the possibility of synergism between two or more toxins. Here we have compared the toxicities of all four Gendotoxins, synthesised and bioassayed under identical conditions, determined their relative activities and have found new synergistic interactions between them. Societies. All rights reserved
N. Crickmore
250
et ul. / FEMS Microbiology
2. Materials and methods
Letters 131 (1995) 249-254
purified by discontinuous tion .
sucrose density centrifuga-
2.1. Bacterial strains and plasmids 2.3. Mosquito bioassay The B. thuringiensis strain IPS78/11 is an acrystalliferous mutant of B. thuringiensis subsp. israeZensis; the E. coli strain TGl was used for all routine procedures. Plasmids used in this study are shown in Fig. 1. The fragments shown were subcloned on a number of shuttle vectors containing pUC 12, 18 or 19 and all, or part of, pC194. The use of these plasmids in E. coli/B. thuringiensis shuttle vectors has been described before [9]. 2.2. Transformation
of B. thuringiensis
Aedes aegypti larvae were hatched from eggs supplied by ICI Agrochemicals. A filter paper containing the eggs was added to 6 1 deionised water (pre-warmed to 30°C) along with 1 g of crushed rat maintenance diet pellets (supplied by Special Diet Services). The eggs were incubated at 3O”C, with constant lighting, for 8 h. The filter paper was then removed and the hatched larvae were maintained under the same conditions for a further 64 h. For the bioassay, five larvae (in 0.5ml) were transferred to a 1.5-ml Eppendorf tube to which 100 ~1 of suspended toxin was then added. The tubes were capped, a single hole pierced in the lids and maintained at 3O”C, with constant lighting, for 24 h, after which mortality was determined against a control background. Thirty to 60 larvae were used for each concentration, and a range of 4-8 concentra-
and purifica-
tion of inclusions B. thuringiensis was transformed by electroporation. Transformants were selected and maintained on LB plates, and grown to sporulation in CCY medium [7] containing 5-10 pug ml-’ chloramphenicol. Inclusions produced by the sporulating cultures were
H
AC
P
AC N POkDa
T
CiylVD
cam27 cam2027
RV
CvtA
_
Rl
lmlusion 0Fonaam
--)llc+ w-
cam2Otaq
w+
pSVlVD
+
cam130
camp271 30
T
e-
camtaq
cam201 30
I3
------c7Qc
7 T
+ B
cam135 C
+
Fig. 1. Structure of the Gendotoxin expression vectors. The top line represents the region of the B. thuringiensis subsp. israelensis 72-MDa plasmid that encodes the cytA, cr/ND. and 20-kDa genes. The subsequent lines represent the fragments of B. fhuringiensis subsp. israelensis DNA that are present on each expression vector. Structural genes are shown as pointed boxes, promoter regions by triangles. Whether or not a transformant of IPS78/11 carrying each of these plasmids synthesises an inclusion body during sporulation is indicated by a plus or minus in the right hand column. For clarity not all of the following restriction sites are shown: H3, HindUI; AC, AccI; N, NlaIV; P2, PuuII; RV, EcoRV; T, TaqI; B, BarnHI; Rl, EcoRI; C, Clal.
N. Crickmore et al. /FEMS Microbiology
tions was used for each toxin or toxin combination tested. The data were subjected to probit analysis.
Letters 131 (1995) 249-254
pletely upstream of the TuqI site that was used to subclone the cytA gene into camtaq. A possible explanation for the lack of expression of cyti in B. thuringiensis, therefore, was the absence of an intact promoter. The plasmid cam27 was therefore constructed in which the region upstream of cyti in camtaq was extended to include the putative promoter. When this construct was introduced into IPS78/11, the result was similar: expression was poor and no obvious inclusions were observed. McLean and Whiteley [lo] demonstrated that a 0.8-kb region located approximately 4 kb 5’ of cyyt was required for cytA expression in E. cofi. This region was later shown to encode a 20-kDa polypeptide Ill]. This 5’ region was apparently not required for the expression of cytA in B. subtilis since it was not present on camtaq, although it remained a possibility that it was required in B. thuringiensis. The plasmid cam2027 was constructed containing cyrA and the 20-kDa gene, and introduced into IPS78/11.
3. Results 3.1. Expression of cytA Ward et al. [9] found that expression of cyt4 in a Bacillus subtilis strain transformed with the toxinencoding plasmid camtaq resulted in the formation of small irregular inclusions. When, however, we introduced camtaq into an acrystalliferous strain of Bacillus thuringiensis subsp. isruelensis (IPS78/ 111, expression was poor and no inclusion bodies were observed. Ward and Ellar [l] mapped the transcription initiation sites of cytA in B. subtilis and B. thuringiensis and found differences between the two strains. In particular the putative promoter in B. thuringiensis was predicted to lie partially or com-
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Log Concentration Fig. 2. Probit analysis on the toxicity of the individual B. thuringiensis subsp. israelensis toxins. The graph represents the regression lines of probit mortality of Aedes aegypti larvae against the log,, concentration of toxin. Percentage mortality levels are indicated on the right hand ordinate.
N. Crickmore
252
er al. / FEMS Microbiology
Transformants containing this plasmid produced large lemon-shaped inclusions (data not shown) which, when purified on discontinuous sucrose density gradients, consisted predominantly of CytA. Finally the plasmid cam20taq was constructed in which the 20kDa gene was added to camtaq. This, too, directed the synthesis of CytA inclusions when introduced into IPS78/11. The structures of all of these constructs are shown in Fig. 1. 3.2. Expression
of CryIVA
Ward and Ellar [2] found that expression of their cloned cryNA gene was poor in both E. coli and B. subtilis, unless the promoter-containing region of cyti was added immediately upstream of the cryIVA gene. Yoshisue et al. [12] claimed that the 20-kDa protein was required for expression of cryZVA, as well as cyti, in E. coli. In B. thuringiensis we observed that the cloned gene alone (cam130) did not direct the synthesis of inclusion bodies in IPS78/11, whereas camp27130 did (Fig. 1). The addition of the 20-kDa gene to cam130 (cam20130, Fig. 1) was not sufficient to induce inclusion formation. Ward and Ellar reported that the addition of an inverted repeat structure downstream of crylVA further increased the level of expression of this gene in B. subtilis. We could find no significant difference in expression in the presence or absence of this inverted repeat in B. thuringiensis (data not shown). 3.3. Expression
of cryIVD and cryIVB
In contrast to cytA and cryIVA, crylVB required no obvious additional factors in order to form inclusions in IPS78/11. The construction of the cryZVBcontaining plasmid cam135 has been described before [13], and this sub-clone does not contain the inverted repeat normally found downstream of cryIVB. The cryIVD gene was subcloned on an EcoRV-NlaIV fragment (Fig. 1) that does not contain any of the neighbouring cytA or 20-kDa genes, although it does contain the p19 ORF described by Dervyn et al. .[14]. 3.4. Mosquito-laroicidal Inclusion individually
bioassays
bodies containing the single toxins were tested against 3-day old Aedes aegypti
Letters 131 (1995) 249-254
Table 1 Toxicity of the B. fhuringiensis Aedes aegvpti
subsp. israelensis
toxins against Expected h
Toxin(s)
LCW d
CryIVA CryIVB CryIVD CytA Bti ’ CryIVA + CytAd CryIVB + CytA’ CryIVD + CytAd CryIVB + CryIVD ’ CryIVA + CryIVB + CrylVD ’
1125 467 224 1209 10 75 62 118 173 125
CryIVA + CryIVB + CytA’
77
(66-89)
788
CryIVA + CryIVB + CryIVD + CytA’
85
(75-97)
477
(955-1349) (392-554) t 170-277) (1110-1324) (X-12) (56-93) (48-75) (97-139) (107-242) (110-144)
1163 677 372 300 401
’ LC,, values are expressed in ng ml-‘, 95% confidence limits are shown in parentheses. ’ Expected LC,,, values of the toxin combinations are based on the toxicities of the individual toxins and were calculated using the formulae derived by Tabashnik [20]. ’ B. thuringiensis subsp. israelensis native toxin. ’ Toxins were combined in equal amounts (w/w).
larvae. Fig. 2 shows the result of probit analysis on the four toxins; LC,,, values are shown in Table 1. Under the conditions used, CryIVD is the most toxic with an LC,, value of 224 ng ml-‘, compared with CryIVB (470 ng ml-‘), CryIVA (1120 ng ml-’ ) and CytA (1210 ng mll’). The slope of the CytA regression line is considerably steeper than those of the other toxins, such that at higher toxin concentrations CytA becomes the most effective. None of the four individual toxins is as active as the native B. thuringiensis subsp. israelensis crystal (Table 1). This suggests that certain properties of the native crystal make it more active and/or that the individual components are acting synergistically with each other. In order to test the latter possibility, a series of toxin combinations was tested (Table 1). Although CytA was the least toxic, whenever it was combined with any of the other toxins the resulting activity was greater than that expected from the individual activities. Individually CytA and CryIVA had similar LC, values of approximately 1.2 pg ml-‘, yet when they were mixed the combination had an activity of 7.5 ng ml I, which represents a
N. Crickmore et al. / FEMS Microbiology Letters 131 (1995) 249-254
15-fold increase over the expected value. Similar effects were seen when CryIVB/CytA, CryIVD/ CytA and CryIVB/CryIVD combinations were tested, whilst combinations of CryIVA, CryIVB and CytA resulted in activities some lo-15fold greater than that expected. A number of other combinations of all four toxins with ratios more closely related to the native crystal were also tested and each showed a synergistic effect, although none of the mixtures tested was significantly more active than those described above (data not shown).
4. Discussion There have been many reports concerning the toxicities of the individual components of the B. thuringiensis subsp. israelensis crystal which present widely differing views. In the case of CytA, LC,, values against Aedes aegypti larvae range from 115 ng ml-’ [6] to > 25 mg ml-’ [15]. Much of the reason for this variation may be caused by the form in which the toxin is presented to the mosquito. Where the toxin has been separated biochemically from the B. thuringiensis subsp. israelensis crystal (often contaminated with other toxins), it is usually fed to the larvae (natural filter feeders) in a soluble or re-precipitated form. Where cloned genes have been used, inclusion bodies are usually produced in either E. coli or Bacillus but are of variable size, contain varying numbers of spores (in the case of Bacillus), and have been shown to have other straindependent differences such as solubility 171. Spores have previously been shown to synergise the activity of Gendotoxins against certain other insect species [16]. Despite being purified by density centrifugation, the toxin preparations used here contained low levels of contaminating spores. However, this low level, and the speed at which the toxic effect was seen (trends were often observed within an hour), suggests that spore germination with resulting septicaemia was not contributing significantly to our results. Another major cause of variation is in the bioassay itself, the LC,, value obtained being dependent on a number of factors including larval age, larval diet and the particular batch of mosquitoes used. Natural variation in various insect populations has
2.53
recently been shown to complicate the attainment of reliable bioassay data [17]. For the above reasons the comparison of results from different workers has proved difficult, and only when assays of individual toxins and toxin mixtures are performed under the same conditions can a true comparison be made. By performing such an experiment we have found that CryIVD is the most toxic of the four when using concentrations that result in the death of around 50% of the larvae, while at higher concentrations CytA becomes the most toxic. It is clear from Fig. 2 that the CytA dose-response curve is significantly steeper than those of the other toxins, which may indicate different mechanisms of pore formation between Cyt and Cry toxins. This may also be reflected in the general in vitro cytolytic activity associated with the Cyt toxins. None of the four toxins proved to be as active against Aedes aegypti larvae as the native B. thuringiensis subsp. israelensis crystal, and there are several possible explanations for this. There might be additional factors associated with the native crystal which are important for toxicity. For example, it has yet to be established whether the gene product of c@VC [18] is present in the inclusion and contributes to the overall toxicity. Alternatively, the nature of the native crystal might mean that it is ingested or solubilised more efficiently than those from the recombinant strains. The presentation of all four toxins in a single crystal might be more efficient than a mixture of four inclusions or, finally, the toxins might be acting synergistically with each other. There have been several reports claiming synergism between various B. thuringiensis subsp. israelensis toxins [5-81, in particular between CryIVD and CytA and between CryIVA and CryIVB. We have found that any combination of the four toxins appeared to have a synergistic effect, the effect being greatest when CytA was one of the components. This may be related to the possible differences in the mechanism of action of CytA and the Cry toxins. The interactions involved in the toxic mechanism are clearly complex. A strain in which the cytA gene was genetically inactivated was found to be as active as the native strain [19], implying that CytA was not essential for mosquitocidal activity. But as the mutant strain was not more toxic than the native strain (as might have been expected on the basis of the
254
N. Crickmore et al. /FEMS
Microbiology Letters I31 (1995) 249-254
individual toxicities) it is clear that CytA is playing an important role in the toxicity of B. fhurirzgiensis subsp. isruelensis.This was confirmed by the activity of the combinations tested in this paper.
Acknowledgements We would like to thank Mr. Trevor Sawyer and other members of the biochemistry department for their input to this project.
References ill Ward, ES. and Ellar, D.J. (1986) Bacillus thuringiensis VW. israelensis Gendotoxin: nucleotide sequence and characterisation of the transcripts in Eacilhts thuringiensis and Escherichia co/i. J. Mol. Biol. 191, l-11. El Ward, ES. and Ellar, D.J. (1988) Cloning and expression of two homologous genes of Bacillus thuringiensis subsp. israelensis which encode 130-kilodalton mosquitocidal proteins. J. Bacterial. 170, 727-735. W., Hofte, H., Seurinck, J., Angsuthana[31 Chungjatupomchai, sombat, C. and Vaeck, M. (1988) Common features of Bacillus thuringiensis toxins specific for Diptera and Lepidoptera. Eur. J. B&hem. 173, 9-16. C. and Gilbert M.P. (1988) 141 Donovan, W.P., Dankocsik, Molecular characterisation of a gene encoding a 72-kilodalton mosquito-toxic crystal protein from Bacillus thuringiensis subsp. isruelensis J. Bacterial. 170, 4732-4738. 151 Wu, D. and Chang, F.N. (198.5) Synergism in mosquitocidal activity of 26 and 65 kDa proteins from Bacillus thuringiensis subsp. israelensis crystal. FEBS I&t. 190, 232-236. (61 Chilcott, C.N. and Ellar, D.J. (1988) Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro. J. Gen. Microbial. 134, 2551-2558. C., Crickmore, N. and Ellar, D.J. (1992) [71 Angsuthanasombat, Comparison of Bacillus thuringiensis subsp. israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo. FEMS Microbial. Lett. 94, 63-68. [8] Chang, C., Yong-Man, Y., Shu-Mei, D., Law, S.K. and Gill, S.G. (1993) High-level c+VD and cyti gene expression in Bacillus thuringierwis does not require the 20-kilodalton protein, and the coexpressed gene products are synergistic in their toxicity to mosquitoes. Appl. Environ. Microbial. 59, 815-821. [9] Ward, E.S., Ridley, A.R., Ellar, D.J. and Todd, J.A. (1986) BacilIus thuringiensis var. israelensis Gendotoxin: cloning
and expression of the toxin in sporogenic and asporogenic strains of Bacillus subtilis. J. Mol. Biol. 191, 13-22. [lo] McLean, K.M. and Whiteley H.R. (1987) Expression in Escherichia cob of a cloned crystal protein gene of Bacillus thurbtgiensis subsp. israeiensis. J. Bacterial. 169, 10171023. 1111 Adams, L.F., Visick, J.E. and Whiteley, H.R. (19891 A 20kilodalton protein is required for efficient production of the Bacillus thuringiensis subsp. israelensis 27-kilodalton crystal protein in Escherichia cob. J. Bacterial. 171, 521530. [12] Yoshisue, H., Yoshida, K.-l., Sen, K., Sakai, H. and Komano, T. (1992) Effects of Bacillus thuringiensis var. israeLewis 20-kDa protein on production of the Bti 130~kDa crystal protein in Escherichia cob. Biosci. Biotechnol. Biochem. 56, 1429-1433. C., Crickmore, N. and Ellar, D.J. (1991) 1131 Angsuthanasombat, Cytotoxicity of a cloned Bacillus thuringiensis subsp. israelensis CryIVB toxin to an Aedes aegypti cell line. FEMS Microbial. Lett. 83, 273-276. [141 Dervyn, E., Poncet, S., Klier, A. and Rapoport, G. (1995) Transcriptional regulation of the cryNI) gene operon from Bacillus thuringiensis subsp. israelensis. J. Bacterial. 177, 2283-2291. 1151 Hurley, J.M., Bulla, L.A. and Andrews, R.E. (19871 Purification of the mosquitocidal and cytolytic proteins of Bacillus thuringiensis subsp. israelensis. Appl. Environ. Microbial. 53, 1316-1321. [161 Miyasono, M., Inagaki, S., Yamamoto, M., Ohba, K., Ishiguro, T., Takeda, R. and Hayashi, Y. (1994) Enhancement of S-endotoxin activity by toxin-free spore of Bacillus thuringiensis against the Diamondback Moth, Plutella xylostella. J. Invert. Pathol. 63, 111-112. 1171 Robertson, J.L., Preisler, H.K., Ng, S.S., Hickle, L.A. and Gelemter, W.D. (1995) Natural variation: a complicating factor in bioassays with chemical and microbial pesticides. J. Econ. Entomol. 88, l-10. ml Thome, L., Garduno, F., Thompson, T., Decker, D., Zounes, M., Wild, M., Walfield, A.M. and Pollock, T.J. (1986) Structural similarity between the Lepidoptera-and dipteraspecific insecticidal endotoxin genes of Bacillus thuringiensis subsp. ‘kurstaki’ and ‘israelensis’. J. Bacterial. 166, 801-811. 1191 Delecluse, A., Charles, J.-F., Klier, A. and Rapoport, G. (1991) Deletion by in vivo recombination shows that the 28kilodalton cytolytic polypeptide from Bacillus thuringiensis is not essential for mosquitocidal activity. J. Bacterial. 173, 3374-3381. 1201 Tabashnik, B.E. (1992) Evaluation of synergism among Bacillus fhuringiensis toxins. Appl. Environ. Microbial. 58, 3343-3346.