J. Cell Sci. 84, 221-236 (1986)
Printed in Great Britain © The Company of Biologists Limited 1986
221
STRUCTURALLY RELATED BACILLUS
THURINGIENSIS (5-ENDOTOXINS DISPLAY MAJOR
DIFFERENCES IN INSECTICIDAL ACTIVITY IN VIVO
AND IN VITRO
BARBARA H. KNOWLES, PHILIPPA H. FRANCIS
AND DAVID J. ELLAR
Department of Biochemistry, University of Cambridge, Tennis Court Road,
Cambridge CB2 1QW, UK
SUMMARY
Many strains within the 22 serotypes of Bacillus thuringiensis produce crystal <5-endotoxins with
slight differences in their insecticidal toxicity spectrum in vivo. Since the basis of this specificity
is unknown, we chose to compare the activity of (5-endotoxins from three strains: B. thuringiensis
var. kurstaki HD-1, var. aizawai HD-249 and var. thuringiensis HD-3S0, both in vivo and on
insect cell lines in vitro. Immunoblotting with antisera to activated var. kurstaki PI lepidopteran
toxin revealed antigenic cross-reaction with the 130xl0 3 M r toxin of var. aizawai, and with
polypeptides of 130 and 138 (XlO3)A/r from var. thuringiensis. In addition, crystals from var.
kurstaki and var. aizawai contained an antigenically related 63xlO 3 M r protein that did not
cross-react with antisera to the 130X103Afr component.
Bioassays on Pieris brassicae larvae (Lepidoptera) and Aedes aegypti larvae (Diptera) indicated
that the 130xl0 3 Af r protein of var. kurstaki, and the 138 plus 130(Xl(P)Mr components of var.
thuringiensis killed only P. brassicae, while the \30x\(rMr protein of var. aizawai and the
63xlO 3 M r proteins of var. aizawai and var. kurstaki were toxic to both P. brassicae and
A. aegypti.
Activation of the 130 and 138(XlO3)Afr proteins of the three varieties of B. thuringiensis with
insect gut proteases yielded active products of 50— 60 (X 103)Afr. Assay of these products on a range
of lepidopteran and dipteran cell lines revealed very different toxicity spectra: var. kurstaki killed
only one lepidopteran line, var. thuringiensis killed two lepidopteran lines, while var. aizawai was
cytolytic to all of the lepidopteran and most of the dipteran cell lines tested, reflecting its broader
spectrum in vivo.
Thus we have shown that antigenic cross-reaction of B. thuringiensis <5-endotoxins does not
necessarily imply a similar toxicity spectrum in vivo or in vitro.
INTRODUCTION
The intracytoplasmic protein crystal synthesized during sporulation of the grampositive bacterium Bacillus thuringiensis is the major cause of toxicity of strains of
this organism for insects. On the basis of their flagellar H-antigens, the 33 varieties
of B. thuringiensis have been divided into 22 serotypes (de Barjac et al. 1985). For
many of these varieties there are differences in the type of insect that is killed by the
<5-endotoxin crystals. Many of the serotypes produce 6-endotoxins pathogenic to
caterpillars (Lepidoptera); others kill mosquito and blackfly larvae (Diptera). One
Key words: Bacillus thuringiensis, biological insecticide, insect cell lines, toxin specificity,
6-endotoxin.
222
B. H. Knowles, P. H. Francis and D. J. Ellar
recent isolate, B. thuringiensis var. tenebrionis kills beetles (Coleoptera; Krieg et al.
1983), while others have not yet been shown to be toxic to any insect tested.
The crystal 6-endotoxin is a protoxin, activated after larval ingestion by the high
pH and proteases of the larval gut (Lecadet & Martouret, 1967). Once activated, the
toxin attacks the cells of the midgut epithelium, which rapidly swell and lyse
(Ebersold et al. 1978; de Barjac, 1978; Endo & Nishiitsusuji-Uwo, 1980; Percy &
Fast, 1983). Similar morphological changes are observed in larval cell lines in vitro
(Murphy et al. 1976; Nishiitsusuji-Uwo et al. 1979; Thomas & Ellar, 1983a).
Susceptible cells become rapidly leaky to small ions, dyes and internal markers (Fast
&Donaghue, 1971; Ebersold et al. 1978, 1980; Gupta et al. 1985).
The biochemical basis of the specificity of these toxins is one of the many
unanswered questions in the study of these commercially important organisms.
Among the possible explanations for this specificity are: the ability of larval gut
proteases to solubilize and activate the protoxin, the presence or accessibility of
specific toxin receptors in the insect gut, the composition of the insect diet (Luthy
et al. 1985) or the structure of the <5-endotoxin itself. It has been shown that in the
case of B. thuringiensis var. colmeri, the source of gut proteases plays a critical role
in determining the pattern of proteolytic processing of the protoxin and hence the
insect specificity of the 6-endotoxin (Haider et al. 1986). The (5-endotoxin of
B. thuringiensis var. israelensis has certain ubiquitous phospholipids as its receptors
(Thomas & Ellar, 19836) but even when activated it is not toxic to lepidopteran
larvae, whose gut cells apparently contain the receptors in a form or location
inaccessible to the toxin. Crystal (5-endotoxins from different serotypes may vary in
their polypeptide composition (Tyrell et al. 1981; Ellar et al. 1985). At least three
antigenically distinct toxic proteins have been described: B. thuringiensis var.
kurstaki HD-1 contains a 130xl0 3 M r lepidopteran toxin (PI) and a 63xlO 3 M r
protein (P2) toxic to both Lepidoptera and mosquitoes (Yamamoto & McLaughlin,
1981) and B. thuringiensis var. israelensis contains a 27xlO 3 M r mosquito toxin
(Ward etal. 1984).
Analysis of the genes of a number of 130x 103Mr lepidopteran toxins has revealed
extensive protein sequence homology with the PI toxin of HD-1 (Schnepf et al.
1985; Shibano et al. 1985; Adang et al. 1985). Despite this homology, bioassays
show that these proteins differ substantially in specificity and potency. These data
suggest that minor modifications in the sequence of the toxins may yield major
differences in their ability to recognize and bind to insect-specific receptors. To
investigate this possibility we have compared the specificity and potency of three
antigenically cross-reacting <5-endotoxins. The results demonstrate that when these
similar toxins are activated under identical conditions they do indeed show very
different toxicity spectra.
MATERIALS AND METHODS
Sources and growth of microorganisms
Bacillus thuringiensis var. kurstaki HD-1 was obtained from Dr H. D. Burges (Glasshouse
Crops Research Institute, England); B. thuringiensis var. aizawai HD-249 and B. thuringiensis
B. thuringiensis 8-endotoxin specificity
223
var. thuringiensis HD-350 were from Dr H. T. Dulmage (USDA, Brownsville, Texas). Conditions for growth and sporulation were as described by Stewart et al. (1981) for B. megaterium
KM. The crystals were separated from spores and vegetative cell debris by ultracentrifugation on
discontinuous sucrose gradients as described by Thomas & Ellar (1983a) forB. thuringiensis var.
kurstaki.
Protein estimation
Protein concentration was measured by the method of Lowry et al. (1951) using bovine serum
albumin (Sigma) as a standard.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE was carried out by the method of Laemmli & Favre (1973) as modified by Thomas
& Ellar (1983a).
Preparation of antisera
Solubilized PI (5-endotoxin from B. thuringiensis var. kurstaki, processed by endogenous
proteases to 54xlO 3 M r (Knowles et al. 1984) was mixed with Freund's complete adjuvant and
administered by subcutaneous injection into New Zealand White rabbits. In subsequent injections
at 10-day intervals Freund's incomplete adjuvant was used. Blood was obtained from the ear vein
and serum collected after clotting. Antiserum towards var. kurstaki P2 protein, purified by
preparative PAGE, was kindly donated by Mr F. Drobniewski (University of Cambridge).
Immunoblotting
Crystal proteins separated by SDS-PAGE were transferred electrophoretically to nitrocellulose
filters (Schleicher & Schiill) by the method of Towbin et al. (1979) using a BioRad 'Trans-Blot'
apparatus. Immunoblotting was carried out as described by Hawkes et al. (1982) and detection
of bound peroxidase conjugated goat anti-rabbit immunoglobulins (Sigma) was with 4-chloro-lnaphthol and H2O2.
Solubilization and activation of crystal 6-endotoxins
Crystals were dissolved by incubation in freshly prepared 50mM-Na2CO3/HCl, pH9 - 5, and
lOmM-dithiothreitol (DTT) for 60min at 37°C (Huber et al. 1981). Insoluble material was
removed by centrifugation at 10 000 # for 5 min. The fractions obtained by this method are referred
to as soluble and insoluble crystal proteins, respectively.
The soluble fraction was incubated 10:1 (v/v) with gut extract from Pieris brassicae or Aedes
aegypti larvae at 37CC for 30 min. Preparation of P. brassicae gut extract was described by Knowles
et al. (1984). A. aegypti gut extract was made from 7-day-old larvae: 50 larval guts were excised
and disrupted by sonication in l m l Na 2 CO 3 /HCl, pH9-5. Particulate material was removed by
centrifugation at 10000gfor 5min and the supernatant filtered through a 0-45/an Milipore filter.
Bioassays
Mosquito assays were conducted by the method of Tyrell et al. (1979) using 7-day-old A. aegypti
larvae hatched from eggs kindly provided by Mr D. Funnell (Shell Research Ltd, Kent).
Bioassay of 3rd instar P. brassicae larvae (obtained from Mr B. Gardiner, Cambridge Biotech
Services) was carried out as described by Burges et al. (1975).
Insect cell lines
Choristoneura fumiferana CF1 cells (Lepidoptera, spruce budworm, trypsinized larval tissue)
obtained from Dr S. S. Sohi (Canadian Forest Pest Management Institute, Ontario) were grown
in Grace's Medium.
Spodoptera frugiperda (Lepidoptera, fall army worm, pupal ovary), Heliothis zea (Lepidoptera,
cotton bollworm, adult ovary) and Mamestra brassicae cells (Lepidoptera, cabbage moth), ob-
224
B. H. Knowles, P. H. Francis and D. J. Ellar
tained from Mrs T. Lescott (NERC Institute of Virology, Oxford), were grown in TC100
medium. Aedes albopictus (Diptera, trypsinized larval tissue) obtained from Mrs T. Lescott,
A. aegyptt Aa(s) (Diptera, trypsinized larval tissue) and Culex quinquefasciatus C2 cells (Diptera,
larval tissue), obtained from Dr D. W. Roberts (Boyce Thompson Institute, Ithaca, U.S.A.),
were grown in Mitsuhashi and Maramorosch medium. Dmsophila melanogaster cells (Diptera,
ovarian tissue), obtained from Mrs T. Lescott, were grown in Schneider's Drosophila medium.
All media contained 10% foetal calf serum (Gibco), 50//gml~' gentamicin (Nicholas
Laboratories) and 50/igmP 1 fungizone (Flow Laboratories). Cells were grown at 28°C.
I n vitro assays
Activated soluble crystal protein was made 10 % in foetal calf serum (FCS, Gibco) prior to use in
order to neutralize the effect of insect gut enzymes on cell lines. Cells, at 1-2 (X106) ml" 1 in tissue
culture medium were incubated with activated toxin at 50/igml~'. Viability was assessed by ability
to exclude Trypan Blue (Thomas & Ellar, 1983a). Controls contained the appropriate volumes of
buffer, gut extract and foetal calf serum (FCS).
RESULTS
Crystal polypeptide composition and antigenic relationship
The polypeptide composition of crystal <5-endotoxins was assessed by SDS—
PAGE (Fig. 1). Crystals of var. kurstaki and var. aizawai showed almost identical
gel profiles, with a major polypeptide of 130xl0 3 M r and a minor component of
63x10 Mr only faintly visible by Coomassie Blue staining, but clearly visualized
by immunoblotting. var. kurstaki PI antiserum cross-reacted with var. aizawai
130X10 MT protein, and antiserum to var. kurstaki 63X 103Mr (P2) protein reacted
with var. aizawai 63xl0 3 M r protein, visualized by immunoblotting (Fig. 2). var.
thuringiensis had a major component of 138xlO3Mr, cross-reacting weakly to PI
antiserum, and a minor component of 130Xl03Mr, cross-reacting strongly; neither
component bound P2 antiserum.
The polypeptides of 100-60 (XlO3)Mr seen in the SDS-PAGE profiles are
proteolytic products of the 130X 103Mr component, all of which bind PI antiserum.
These are generated by the endogenous proteases associated with the crystals (Bulla
etal. 1977; Chestukhina et al. 1978).
Solubilization and activation of crystal proteins
In order to assay toxicity in vitro, the crystal protein protoxins must be solubilized
and activated. This is done by simulating the conditions in the insect gut, the site of
crystal activation in vivo. Incubation of crystals in SOmM-NazCC^/HCl, pH9-5,
and lOmM-dithiothreitol for 60min at 37°C resulted in the complete solubilization
of the 130xl0 3 M r proteins of var. kurstaki and var. aizawai, and the 130 and
138 (X 103)Mr components (i.e. the entire crystal) of var. thuringiensis (Fig. 3A-C,
track 3), leaving the 63xlO 3 M r protein of var. kurstaki and var. aizawai as an
insoluble pellet (Fig. 3A,B, track 1). All subsequent in vitro assays were carried out
using the soluble proteins, since the insoluble fractions cannot be assayed in vitro.
Activation of the soluble protoxins with P. brassicae or A. aegypti gut extracts
resulted in proteolysis of the highM r proteins, yielding products of 50—60 (X 103)Mr
(Fig. 3A-C, tracks 4 and 5).
B. thuringiensis 6-endotoxin specificity
1 2
3
225
4
Mr
x10" 3
-138
-130
66
"
-63
4536- —
29- —
2420- —
Fig. 1. SDS-PAGE of crystal (5-endotoxins, Coomassie Blue stained. Track 1, Mr
standards; 2, SOjUg var. kurstaki crystal; 3, 50jUg var. aizaiwai crystal; 4, 50/ig var.
thuringiensis crystal.
Bioassays
Purification of the crystal <5-endotoxins by discontinuous sucrose density gradient
ultracentrifugation yielded preparations with less than 0-2 % spore contamination for
var. kurstaki and var. thuringiensis, assessed by phase-contrast microscopy, but 10 %
contamination for var. aizawai. In assays in vitro the spores were removed from the
solubilized toxin preparation by centrifugation, but for in vivo assays of native
crystal and insoluble crystal fraction spores were present. For this reason bioassays
were conducted for only 24 or 48 h, since the reduction of feeding of P. brassicae
larvae over short exposure periods is caused only by crystals, and is not influenced by
the spores (Burges et al. 1975).
Results of bioassays are shown in Table 1: var. thuringiensis proteins of 130 plus
138 (X103) Afr and var. kurstaki 130X 103Mr protein killed only P. brassicae larvae,
whereas var. aizawai 130xl0 3 M r protein, and var. aizawai and var. kurstaki
63 X 103Mr proteins, were toxic to both P. brassicae and A. aegypti larvae.
Fig. 2. Immunoblotting crystal proteins separated by SDS-PAGE. A. Coomassie Blue stained gel. B. Immunoblot of an identical gel,
incubated with antisera to activated var. kurstaki P1 (see Materials and Methods). C. Immunoblot as above, incubated with antiserum to
var. kurstaki P2. A,B,C: tracks 1, 75 pg var. thun'ngiensis crystal; 2, 75 pg var. aizawai crystal; 3, 75 pg var. kurstaki crystal; 4, M ,
standards.
228
B. H. Knowles, P. H. Francis and D. jf. Ellar
It should be noted that since A. aegypti larvae are filter feeders, and ingest
particulate material more efficiently than soluble material, LC50 values for soluble
toxin will be artificially high, and cannot be compared directly with values for
insoluble material.
In vitro assays
The results of in vitro assay of activated soluble toxins on a range of lepidopteran
and dipteran cell lines are shown in Table 2. The time taken for 50 % cell death
(assessed by Trypan Blue staining) on addition of SO^gmF 1 toxin was used for a
direct comparison of toxicity towards the different cell lines. The control cells,
incubated in an equal volume of buffer, gut enzymes and FCS without toxin, showed
less than 5 % mortality during the course of the assays. This is important in assays
in vitro, since cultured cell lines are sensitive to changes in pH and osmotic concentration, and to the presence of digestive enzymes, and are likely to be more
susceptible to toxins if stressed in this way.
var. kurstaki activated PI protein killed only C. fumiferana cells, var. thuringiensis activated soluble proteins lysed C. fumiferana and H. zea cells, while var.
aizawai activated soluble protein killed all of the lepidopteran and most of the
dipteran cell lines tested. Toxins activated with P. brassicae gut extract showed
qualitatively the same toxic effects as those activated by A. aegypti gut extract
Table 1. In vivo assays
P. brassicae*
IC50 (jigml" 1 )
A. aegypti}
LC50 (/^gml
var. kurstaki
Native crystal
Soluble fraction}
Insoluble fraction}
0-1-0-01
1-0-0-1
1-0-0-1
10-0-1-0
>1000
100-10
var. aizawai
Native crystal
Soluble fraction}
Insoluble fraction}
1-0-0-1
1-0-0-1
10-0-1-0
1-0-0-1
100-10
100-10
var. thuringiensis
Native crystal
Soluble fraction}
Insoluble fraction?
0-1-0-01
0-1-0-01
>1000
>1000
—
—
)
•Twenty 3rd-instar larvae. IC50 value represents toxin concentration giving 50% inhibition of
feeding after 24 h.
f Twenty five 7-day-old larvae. LC50 value represents toxin concentration giving 50 % mortality
after 48 h.
} Native crystal incubated for 60min at 37°C in 50 mM-Na2CO3/HCl, pH9-5, and lOmM-DTT
yielded a soluble fraction, used directly, and an insoluble fraction, resuspended in distilled water
by sonication.
§ There was no insoluble fraction in the case of var. thuringiensis.
B. thuringiensis 6-endotoxin specificity
229
Table 2. Toxicity spectrum of activated solubilized crystal proteins in vitro
Time for 50 % lysis* after addition of
1
50^gmr soluble toxin activated with
P. brassicae gut extract
Cell line
(1X106 cells mT 1 )
kurstaki
aizawai
thuringiensis
Choris toneura fumiferana
Heliothis zea
Mamestra brassicae
Spodoptera frugiperda
Culex quinquefasciatus
Aedes aegypti
A. albopictus
Anopheles stephensi
A. gambiae
Drosophila melanogaster
50min
No lysisf
No lysis
No lysis
No lysis
No lysis
No lysis
No lysis
No lysis
No lysis
60min
15 min
20 min
45 min
No lysis
23h
No lysis
160 min
30 min
20 h
90 min
260 min
No lysis
No lysis
No lysis
No lysis
No lysis
No lysis
No lysis
No lysis
•Lysis was assessed by inability to exclude Trypan Blue.
f No lysis indicates that no cytopathic effect was observed within 24 h.
(although A. aegypti gut-extract-activated toxins showed quantitatively lower toxicity), indicating that the source of gut extract is not important in determining the
insect specificity of these toxins.
The effect of activated toxins on a range of cell lines is shown in Figs 4—5. The
toxins cause, sequentially, blebbing of membrane vesicles from cell processes,
rounding up, granulation, swelling and lysis. These effects are very similar to those
observed in midgut cells in vivo (Endo & Nishiitsusuji-Uwo, 1980; Percy & Fast,
1983).
Antibody neutralization of toxicity in vitro
Equal volumes of var. kurstaki PI antiserum and 50/igmP 1 solubilized, activated
6-endotoxins from all three crystal varieties were preincubated together for 30 min at
22°C before addition to insect cells in vitro. In all cases the antiserum completely
neutralized toxicity: this can be seen in the case of var. aizawai toxin assayed against
H. zea cells in Fig. 6.
When PI antiserum was added to C. fumiferana cells at varying time intervals after
addition of activated var. kurstaki toxin, the antiserum was unable to neutralize
toxicity 2 min after toxin application, and 1 min of exposure to toxin was sufficient
to cause 50% maximal cell damage. This is in agreement with the findings of
Murphy et al. (1976) using a similar system.
DISCUSSION
The results presented here illustrate the complexity of B. thuringiensis 6-endotoxin classification. Thus the similar polypeptide and antigenicity profiles of var.
kurstaki HD-1 and var. aizawai HD-249 crystals are not paralleled by a similar
toxicity spectrum. Previous workers have reported that different varieties of
230
B. H. Knowles, P. H. Francis and D. J. Ellar
i?-Y
o
&».
*f»i
^
mWmm
Figs 4, 5. Cytopathic effects of activated (5-endotoxins on insect cell lines in vitm. Assay
conditions are described in Materials and Methods. Solubilized crystal proteins were
activated with P. brassicae gut extract.
Fig. 4. C. fumiferana cells treated with activated var. thuringiensis soluble protein.
A. Control 120min; B, toxin 120min; S. frugiperda cells treated with activated var.
aizawai soluble protein. C. Control, 60min; D, toxin 60min. Bar, 25 fim.
B. thuringiensis 5-endotoxin specificity
211
IPisiiiit^
Fig. 5. An. gambiae cells treated with activated var. aizawai soluble protein.
A. Control, 180min; B, toxin, 180min; M. brasstcae cells treated with activated var.
aizawai soluble protein. C. Control, 60 min; D, toxin, 60 min. Bar, 25 fim.
Fig. 6. Neutralization of toxicity by antisera. Solubili2ed var. aizawai crystal protein
activated with P. brassicae gut extract was preincubated with antisera to activated var.
kurstaki PI (Materials and Methods); lml H. zea cells (10 6 /ml) were incubated for
60min with: A, 50 fig antiserum; B, 50 fig toxin; C, 50 fig antiserum preincubated with
50 fig toxin. Bar, 25 fun.
B. thuringiensis 6-endotoxin specificity
233
B. thuringiensis and different isolates of the same variety can produce <5-endotoxins
differing in their host spectrum in vivo (Heimpel & Angus, 1960; Dulmage, 1975).
However, these in vivo studies are open to criticism on various counts: for instance,
it is now known that many crystals comprise at least two distinct toxic moieties
(Yamamoto & Iizuka, 1983) with very different toxicity spectra, that may act
synergistically in some hosts, thus making it difficult to interpret toxicity data from
assays involving whole crystals. Also, the diet of the insect can alter its response to
a particular toxin, as seen in the case of Heliothis virescens, which is susceptible
to 6-endotoxins when fed soya, tobacco or artificial diets, but is not adequately
controlled when feeding on cotton (Luthy et al. 1985). This effect of the diet may be
due to tannins that inactivate var. kurstaki HD-1 6-endotoxin (Luthy et al. 1985),
or to the possibility that certain plant lectins may inhibit this toxin in vitro (Knowles
et al. 1984). Moreover, if the diet of larvae affects their gut pH and, or, protease
production this might alter their ability to activate the toxins.
The work described here is the first to compare the toxicity of different 6-endotoxins to a wide range of insect cell lines in vitro. This approach is of value in
studying specificity determinants since it enables the assay of different toxins to
be carried out in a controlled manner, unaffected by the variable conditions encountered in assays in vivo. This assay system is highly reproducible, and has already
provided information on the mechanism of action of B. thuringiensis var. israelensis
<5-endotoxin (Thomas & Ellar, 19836) and the basis of specificity of B. thuringiensis
var. cohneri (5-endotoxin (Haider et al. 1986).
However, in vitro assays do have limitations, principally stemming from the
unavailability of midgut cell lines. It has not so far proved possible to grow midgut
cells in continuous culture, perhaps because of the difficulties involved in providing
a culture system that simulates the asymmetry of growth conditions in vivo. For
example, steep K + and H + gradients are maintained across the midgut and there are
many other differences in the composition of gut contents and haemolymph (Wood,
1972). Thus in vitro assays are restricted to cell lines derived from tissue of various
sources such as those produced from ovarian tissue (H. zea, S. frugiperda, D.
melanogaster) or whole trypsinized larvae (C. fumiferana, A. albopictus, An.
stephensi, An. gambiae). In the latter case mixed cultures of unknown origin are
produced. However, it seems that for at least one of the toxins described here, the
soluble protein of var. aizawai, the source of insect tissue does not determine its
spectrum of activity in vitro, implying that the var. aizawai receptor is not confined
to midgut cells. However, unlike the 6-endotoxin of var. israelensis, which binds
phospholipids and thus kills all eukaryotic cells once activated (Thomas & Ellar,
19836), the activated soluble protein of var. aizawai does not lyse a range of
erythrocytes tested (data not shown), suggesting that in this case the range of activity
might be confined to insects.
B. thuringiensis var. thuringiensis HD-350 was isolated from an unusual source,
a dead grasshopper, and although we have not shown it to kill insects of this order
(unpublished observation), the possibility remains that the host range of this
organism is unique. The 138xlO3Mr protein present in the crystal does not have a
234
B. H. Knowles, P. H. Francis and D. J. Ellar
counterpart in other strains studied in this laboratory, but a recent report by Jarrett
(1985) indicates that crystals from var. aizawai isolates HD-112, HD-135, HD-137
and HD-282 contain 138 and 130(Xl0 3 )M r components with distinct toxicity
spectra encoded in different plasmids. Since many of the 6-endotoxin proteins are
encoded on plasmid-borne genes, crystal components may be conveniently studied
separately by generating mutants lacking one of the toxin-encoding plasmids as
described by Jarrett (1985). The assays described here do not distinguish between
the 130 and 138 (X 103)Mr proteins since they are equally soluble in the buffers used,
thus it remains possible that the toxicity observed may be due to only one of the two
proteins, or to synergistic effects.
The fact that many B. thuringiensis (5-endotoxin genes are borne on plasmids,
which might be transferred from one variety to another (Gonzalez et al. 1982),
implies that there is no a priori reason to suppose that toxins produced by different
isolates of the same serotype bear any structural or functional relationship to each
other. Thus, for example, a recently isolated strain of var. morrisoni, designated
PG-14, has a toxicity spectrum totally unrelated to that of the reference strain of var.
morrisoni (Padua et al. 1984) and var. darmstadtiensis isolate 73-E-10-2 has a
different host range to the reference strain (Padua et al. 1980). It has also become
clear from recent toxin sequence data that small changes in amino acid sequence can
lead to significant differences in toxicity spectra (Schnepf et al. 1985; Shibano et al.
1985; Adang et al. 1985). This underlines the importance of specifying the isolate
number in studies of different (5-endotoxins since the variety name is not sufficient
to define the toxin type.
We thank Mrs Jillian Clements and Mrs Margaret Bradley for secretarial assistance and Mrs
Audrey Symonds for her meticulous washing up. This work was supported by grants from SERC
and AFRC.
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(Received 13 January 1986 -Accepted 17 April 1986)