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Insect toxin in spores and protein crystals of Bacillus thuringiensis
TIBS-
108 from two distant discrete segments of the complementary D N A [26] (Fig. 4). The diameter of branched supercoils at physiological ionic strength is 13 nm, the same order of magnitude as the size of the RNA polymerase molecule. The enzyme could 'short circuit' under conditions of appreciable superhelical stress and transcribe normally on relaxed DNA. The crossover position would depend on the presence of a favourable receptor sequence. Such a concept is attractive when compared to that of post transcriptional processing, as the number of bases in the intervening unused region of DNA is considerable [26]. Fortunately, most of the predictions can be experimentally verified and the 'active site' concept can be extended in experiments with eukaryote DNA so that a detailed model of the human chromosome may be obtained in the forseeable future.
References I Cook. P.R. and Brazell, I.A. {1975)J. Cell ScL 19, 261-279 2 Colman. A. and Cook. P. R. [ 1977) Eur. J. Biochem. 76. 63 78 3 Cook, P. R. and BrazelL I., in press 4 Benyajati, C. and Worccl. A. (1976) Cell 9, 393 397 5 Ide. T., Nakane. M., Anzai, J. and Andon, T. (1975) Nature (London) 258. 445 447 6 Worcel, A. and Burgi, E. (1972) J. Mol. Biol. 71, 127 147
7 Wang, J.C. (1971) J. MoL Biol. 55, 523-533 8 Champoux, J.J. and Dulbecco, R. (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 143 146 9 Gcllert, M., Mizucchi. K., O'Dea, M.H. and Nash, H.A. (1976) Proc. Natl. Aead. Sci. U.S.A. 73, 3872 3876 10 Finch, J. and Klug, A. (1976) Proe. NatL Acad. Sci. U.S.A. 73. 1897 1901 I I Nature News and l'iews, (1977) 266, 120-12I 12 Beard. P.. Morrow, J.F. and Berg, P.J. (1973) J. Virol. 12. 1303 1313 13 Jacob. R.L., Lebowitz, J. and Kliendschmidt, A. K. (1974) J. l'irol. 13, I 176- I 186 14 Monjardino, J. and James, A.W. (1975) Nature London) 255. 249 252 15 Brock, C., Bickle. T.A. and Yuan, R. (1975) J. MoL Biol. 96. 693 702 16 Campbell,A. M. and Eason, R. (1975) FEBS Lett. 55. 212 215 17 Campbell. A. M. (19761Biodlem. J. 155, 101 105 18 Campbell, A. M. (1976) Biochem. J. 159,615 620 19 Vollenweider, H.J.. Koller. T.. Parello. J. and Sogo. J. M. [1976) Proc. Natl. Aca~L Sci. U.S.A. 73,4125 4129 20 Van Bruggen, E. F.. Personalcommunication 21 Waring. M.J. (1970) J. Mol. Biol. 54. 247 279 22 Saucier, J. and Wang, J.C. (1972) Nature New Biol. 239. 167-170 23 Wang, J.C., Berkley, M.D. and Bourgeois. S. (1974] Nature (London) 251. 247 249 24 Lin. S, and Riggs. A.D. (1975] Cell4, 107 III 25 Flanders, M. and Swan. D.. "At the drop of a hat' E.M.I. Records, Hayes. Middlesex. U.K. 26 Williamson, R. (1977) Nature News and l'iews 270, 295 297 27 Campbell, A. M. and Cotter. R. I. ( 1977] Nuch, ic Acids Research 4. 3877 3887
M a y 1978
With B. thuringiensis the insecticidal activity was first associated with a remarkable inclusion that is formed during sporulation (Fig. 1) and that has been shown to be a true crystal. Although there is no reason to believe that the toxin in the inclusion is not essential to the effectiveness of B. thuringiensis, several other activities have been identified that could contribute to the toxicity and possible pathogenicity of B. thur#lgiensis. The extent to which death results from toxicity or pathogenicity may vary with different insect species and environmental factors. Other activity factors that may be involved include a phospholipase, protease inhibitors of the immune system [I] and a toxin located in the spore [2]. This last activity is closely similar to the toxin in the crystal. Those strains that are used commercially do not produce the fl-exotoxin, an ATP analogue of widespread biological activity that is excreted during the vegetative growth of relevant strains. Strains that lack exotoxin are without known harmful effect to mammals. Interesting recent reports have been made of biological activity of crystals, or material derived therefrom, in mammalian, systems. Whole crystals have been found to enhance the immune response of rats and guinea pigs [3], and a fraction derived from crystals has been shown to have both anti-tumour and insecticidal activities. The nature of these effects on mammals awaits elucidation.
Biosynthesis of the crystal
Insect toxin in spores and protein crystals of
Bacillus thuringiensis Hugh J. Somerville Bacillus thuringiensis produces an actit'e toxin specific to lepidopterous lart,ae. The activi O" is associated with a protein crystal p r o d t w e d during sporulation a n d a related actit'ity is f o u n d #1 the outer layers o f .wores. Although many attempts have been made to use micro-organisms in the control of insect and plant pests, relatively few have been successful. Perhaps the most notable successes have been achieved with two species of Bacillus: Bacillus popilliae which has been used in infection and control of Japanese beetle in North America, and H.J.S. is at Shell Research Limited, Shell Bioseienees Laboratory. Sittmgbourne Research Centre, Sittingbourne. Kent, U.K.
Bacillus thuringiensis which has been commercially successful as an insecticide active against the larvae of many lepidopterous pests. B.popilliae is difficult to grow in vitro; consequently studies on the factors involved in its activity have been hindered and it is not known whether there is any common factor linking the activities of these two bacteria. B. thuringiensis is used in the field with formulation and application methods similar to those for chemical insecticides.
The crystal is formed during stages II and III of sporulation and the time of appearance of crystal antigens coincides with the appearance of the crystal as seen in the electron microscope. Apart from crystal formation, the morphological changes associated with crystal formation follow a similar pattern to those in other spore formers (Fig. 2). In one study [4] convincing evidence has been obtained for an association between the developing crystal and the exosporium membrane, although a separate study using different procedures failed to confirm this observation [5]. No soluble crystal antigens can be detected
Fig. I. Negatively stained crystal of Bacillus thur-
ingiensis. Magnification × 80,000.
109
TIBS - May 1978
[
l-I
I c EQ
-I ©11
,/
Fig. 2. Morphogenetic changes durhlg.~porulution aJul co.stal formation in Bacillus thuringiensis. E. exo.~7~orhon; C. crystal.
during sporulation and it appears that the exosporium may serve as a template for assembly, and possibly for synthesis, of the subunits. The crystal does not seem to grow in three dimensions from a central nucleus; in thin sections the edges of developing crystals in the sporangium are rectilinear whereas edges along the interface with the exosporium are often irregular. This suggests that as synthesis progresses the finished surfaces are pushed out into the sporangium. It is not clear what limits crystal growth. One possible explanation is that growth of the crystal may be stopped, or there may be some reversal of assembly mechanism, when the growing crystal extends across the internal diameter of the cell.
Biochemistry of the crystal The crystal is insoluble at physiological pH and will dissolve only in reagents such as 0.1 M NaOH or 8 M urea-mercaptoethanol. There is some confusion in the literature as to the precise number of polypeptide components in the crystal. If confirmed, a recent report that crystal preparations contain substantial amounts of carbohydrate [6] might lead to an explanation of the interactions between the dissolved peptides that aggregate to form complexes of high molecular weight. Just as reports of the number of crystal components vary, so do those on the molecular weight of the toxin itself. Reports of toxic material of low molecular weight remain to be substantiated and the best evidence points to a single polypeptide of molecular weight about 60,000 [7]. Such a component was isolated in low yield by ion-exchange chromatography of dissolved crystals. Recently, material indistinguishable from this toxin has been isolated in high yield from the products of tryptic digestion of dissolved crystals at pH 10, conditions similar to those in the insect gut [8]. It would appear therefore that the toxin is a polypeptide, possibly a glycoprotein, of substantial molecular weight.
Sporulation and crystal formation The close temporal relationship between crystal formation and sporulation suggests that the two may be interdependent and considerable evidence has accumulated to indicate a relationship between polypeptide components in the outer layers of the spore and those in the crystal [9,10]. Crystal-negative, spore-forming mutants (cr) are readily isolated following exposure to mutagens. Spores of cr strains lack toxicity but are otherwise indistinguishable from wild type spores. Asporogenic, crystal-forming mutations (sp) are relatively rare [4] and are blocked at stage III of sporulation (Fig. 2). These observations suggest that sporulation must be initiated for crystal formation to occur and that the latter is incidental to sporulation. As indicated above, spores of the wild type contain a toxin, some at least of whose activity is located in two membrane fractions [2], identified as exosporium and spore coat, that can be isolated from spores without loss of refractility. These fractions contain about 5°~ of the dry weight as cysteine. Antigens, specific to one of these fractions from the cr, non-toxic strain, appear at the same time as the synthesis of crystal antigens in the wild type. Crystal antigens have been separately shown to appear coincidentally with the toxin [11]. Although non-toxic, the membrane fractions from a cr strain could not be distinguished by density or amino acid analysis from the corresponding fractions of the wild type. However, they are immunologically distinct in that the ability to crossreact with crystal solutions in diffusion against anti-crystal serum is lost with the cr mutation. The molecular size and nature of the toxin in these fractions has not been established. The relationship between exosporum and spore coat, and the crystal has been demonstrated using both the ferritinlabelling and peroxidase-anti-peroxidaselabelling techniques for locating crystal
antigens in thin sections of spores [12] (Fig. 3). Unfortunately, no system has been developed for genetic analysis in B . t h u r h l g i e n s i s and consequently it is unknown whether there is one or more cr determinants or whether the genes coding for the cr character are located on the chromosome. The large size of the crystal relative to that of the parent micro-organisms (crystals are bipyrimidal, about 1 l~m long and weigh about one third of the spore by dry weight) has prompted interest in the mechanism of synthesis of the polypeptide components. Although crystal formation and sporulation are sensitive to low concentrations of actinomycin and rifampin, some results suggest that crystal formation may involve stable messenger RNA (mRNA). Studies [13] on the incorporation of [~aC]valine in high concentration in the presence of rifampin have suggested that a stable mRNA fraction is formed with a half-life of 10 min. Cell-free synthesis of polypeptides related to those in the crystal has been reported using both normal mRNA [14] and mRNA selected for stability [13]. However, identification of the products as crystal material cannot be regarded as established as the antisera used were prepared against extracts of crystal suspensions containing spores and it is known that this procedure results in extraction of some spore components. Crystal antigens appear over a similar time-scale to that of other sporulation associated characteristics such as dipicolinic acid and development of resistance. Although the involvement of stable mRNA in synthesis of other spore proteins has been proposed, there are conflicting reports and more detailed evidence is required to confirm the involvement of stable mRNA in crystal synthesis.
Fig. 3. Thin section through partially germinated spore of Bacillus thuringiensis showing spore coat reaction with ferritin-labelled antibodies to dissolved crystals. (Courtesy J. Short and P. Walker [12].) Magnification x 100.000.
T I B S - M a y 1978
110
Mode of action of crystal Relatively little is known about the mode of action of the endotoxin at the molecular level. It is clear that crystals (and fractions from spores) dissolve in the alkaline gut o f susceptible insects. Depending on the dose and the species of insect, the resulting effect can vary from temporary cessation of feeding to complete paralysis. Intoxication occurs within minutes o f ingestion. A number o f workers have reported effects on the midgut, and microscopic examination reveals that the epithelium is disrupted and may eventually break down completely. Unfortunately, microscopic examinations generally have been made with insects after prolonged exposure to the toxins. It remains to be demonstrated whether deterioration of the mid-gut is a primary effect; it is perhaps unlikely in view of the similar toxicity of dissolved crystal material by injection and ingestion. Both spores and crystals can contribute to the entomocidal effect of preparations x~f B. thuringiensis and spores may be of importance in the field [15]. Although it is speculative to point to similarities in the nature and mode of action of cholera toxin, it might be profitable to investigate the effects of the spore and crystal endotoxins on insects with the known modes of action of mammalian toxins in mind, particularly in view of the relatively fragmentary knowledge o f insect physiology. Perhaps the outstanding question of crystal formation is the physiological reason for this remarkable phenomena. Most, if not all, cultures of B. thuringiensis have been isolated from insect cadavers; this might indicate an ecological niche as an insect pathogen. However, a perceptive eye i~ needed to distinguish B. thuringiensis from B. cereus and a more detailed survey coupled with survival studies could indicate whether B. thuringiensis is a normal member of the soil microflora. It has been postulated that crystal formation results from oversynthesis of some normal and essential spore component [9]. Certainly there is considerable evidence to indicate that polypeptides in the crystal are related to a substantial fraction of the protein in the spore coat and exosporium, although the toxin may not form a major part of this common material. Circumstantial support for this hypothesis is provided by the formation of crystalline inclusions in a RNA polymerase mutant of B. subtilis [16], and by the occurrence of crystalline inclusions in other sporulating bacteria [171. However, the loss o f toxicity in the cr strains suggests that an alternative, or additional, explanation is necessary.
References I Edlund, T., Siden, 1. and Boman, H.G. (1976) Infect. lmmun. 14, 934-941 2 Scherrer, P.S. and Somerville, H.J. (1977) Eur. J. Biochem. 72, 479490 3 Prasad, S.S.S.V. and Shethna, V.I. (1975) Biochem. Biophys. Res. Commun. 62, 517 4 Somerville,H.J. (1971) Eur. J. Biochem. 18, 226237 5 Bechtel,D. B. and Bulla, L. A. (1976)J. BacterioL 127, 1472-1481 6 Bulla, UA., Kramer, K.J. and Davidson, L.I. (1977) J. Bacteriol. 130, 375-383 7 Herbert, B.N., Gould. H.J. and Chain, E.B. (1971) Eur. J. Biochem. 24, 366-375 8 Lilley,M. and Somerville,H.J. (1975) Proc. Soc. Gen. Microbiol. 3, 10 9 Delafield,F. P,, Somerville,H.J, and Rittenberg, S.C. (1968)J. BacterioL 96, 713-720
10 Lecadet, M. M., Chevrier, G. and Dedonder, R. (1972) Eur. J. Biochem. 25, 348-349 I 1 Norris, J.R. (1969) Spores IV (Campbell, L.L., ed.) Amer. Soc. Microbiol.,45~18 12 Short, J., Walker, P., Thomson, R.O. and Somerville, H.J. (1974) J. Gen. Microbiol. 84, 261-276 13 Glatron, M. F. P. and Rapoport, G. (1976) Biochimie 58, 119-129 14 Herbert, B. N. and Gould, H. J. (1973) Eur. J. Biochem. 37, 441448 15 Burges, H.D., Thomson, E.M. and katchford, R.A. (1976) J~ lnvertebr. Pat/sol.27, 87-94 16 Santo, L.Y. and Doi, R.H. (1973)J. Bacteriol. 116, 479~,82. 17 Duncan, C.L., King, G.J. and Frieben, W.R. (1973) J. BaeterioL 114, 845-859
I II
Protein-bound oligosaccharides of cell membranes Johan Jhrnefelt, Jukka Finne, Tom Krusius and Heikki Rauvala Membrane glycoproteins contain several different types o f carbohydrate side chain. Fractionation reveals f o u r major classes, each having characteristic structural features. The relative abundance o f the various carbohydrate structures is different in different tissues. This supports the v&u, that glycoproteins, through their carbohydrate portions, express some o f the cellomembrane specificity.
The outer surface of cells is covered with various carbohydrates, which are covalently linked either to a lipid or to a protein. Such glycolipids and glycoproteins are integral parts of cell membranes in most organisms and are believed to take part in interactions between cells or between cells and external molecules [1]. So-called receptor functions have often been attributed to carbohydrates. Evidence that the receptors of several biologically active substances, such as thyrotropin and related glycoprotein hormones [21, serotonin [3], interferon [4], cholera toxin [3] and transferrin [5], are glyeolipids or glycoproteins has been forthcoming. The structural multiplicity and the small amounts of each specific carbohydratecontaining molecular species occurring in cell membranes has made isolation and 'analysis of these molecules very difficult. Glycolipids have been easier to characterize because the lipid carrier has little influence in the fractionation methods conventionally used, whereas membrane glycoproteins have been isolated only in a few cases. For soluble glycoproteins, occurring J.J., J.F.. T.K. and H.R. are at the Department of Medical Chenlistry. Universityof Helsinki, Siltavuorenpenger tO A, SF-OOI70Helsioki 17. Finland.
in blood serum or in various secretions, more complete structural information is available, the obvious reason being simpler isolation procedures and consequently the ease of obtaining pure samples in large quantities. Even when the isolation of a membrane glycoprotein has been successful, the analysis of its carbohydrate parts is a difficult task, since the molecule may contain several different carbohydrate side chains, and the glycoprotein is mostly available in minute amounts. The aspects to be described in this article are primarily the nature of the oligosaccharide side chains of membrane glycoproteins. In order to work out analytical and separation methods for the various oligosaccharides, representative samples in sufficient quantity are required. These are obviously not obtainable through the preliminary isolation of glycoproteins. The amounts available would be too small, and each glycoprotein would have only a selection o f carbohydrate side chains. Obtaining all the membrane proteinbound carbohydrate o f a given tissue, free o f other components, would be a distinct advantage from the viewpoint of the carbohydrate chemistry.
108 from two distant discrete segments of the complementary D N A [26] (Fig. 4). The diameter of branched supercoils at physiological ionic strength is 13 nm, the same order of magnitude as the size of the RNA polymerase molecule. The enzyme could 'short circuit' under conditions of appreciable superhelical stress and transcribe normally on relaxed DNA. The crossover position would depend on the presence of a favourable receptor sequence. Such a concept is attractive when compared to that of post transcriptional processing, as the number of bases in the intervening unused region of DNA is considerable [26]. Fortunately, most of the predictions can be experimentally verified and the 'active site' concept can be extended in experiments with eukaryote DNA so that a detailed model of the human chromosome may be obtained in the forseeable future.
References I Cook. P.R. and Brazell, I.A. {1975)J. Cell ScL 19, 261-279 2 Colman. A. and Cook. P. R. [ 1977) Eur. J. Biochem. 76. 63 78 3 Cook, P. R. and BrazelL I., in press 4 Benyajati, C. and Worccl. A. (1976) Cell 9, 393 397 5 Ide. T., Nakane. M., Anzai, J. and Andon, T. (1975) Nature (London) 258. 445 447 6 Worcel, A. and Burgi, E. (1972) J. Mol. Biol. 71, 127 147
7 Wang, J.C. (1971) J. MoL Biol. 55, 523-533 8 Champoux, J.J. and Dulbecco, R. (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 143 146 9 Gcllert, M., Mizucchi. K., O'Dea, M.H. and Nash, H.A. (1976) Proc. Natl. Aead. Sci. U.S.A. 73, 3872 3876 10 Finch, J. and Klug, A. (1976) Proe. NatL Acad. Sci. U.S.A. 73. 1897 1901 I I Nature News and l'iews, (1977) 266, 120-12I 12 Beard. P.. Morrow, J.F. and Berg, P.J. (1973) J. Virol. 12. 1303 1313 13 Jacob. R.L., Lebowitz, J. and Kliendschmidt, A. K. (1974) J. l'irol. 13, I 176- I 186 14 Monjardino, J. and James, A.W. (1975) Nature London) 255. 249 252 15 Brock, C., Bickle. T.A. and Yuan, R. (1975) J. MoL Biol. 96. 693 702 16 Campbell,A. M. and Eason, R. (1975) FEBS Lett. 55. 212 215 17 Campbell. A. M. (19761Biodlem. J. 155, 101 105 18 Campbell, A. M. (1976) Biochem. J. 159,615 620 19 Vollenweider, H.J.. Koller. T.. Parello. J. and Sogo. J. M. [1976) Proc. Natl. Aca~L Sci. U.S.A. 73,4125 4129 20 Van Bruggen, E. F.. Personalcommunication 21 Waring. M.J. (1970) J. Mol. Biol. 54. 247 279 22 Saucier, J. and Wang, J.C. (1972) Nature New Biol. 239. 167-170 23 Wang, J.C., Berkley, M.D. and Bourgeois. S. (1974] Nature (London) 251. 247 249 24 Lin. S, and Riggs. A.D. (1975] Cell4, 107 III 25 Flanders, M. and Swan. D.. "At the drop of a hat' E.M.I. Records, Hayes. Middlesex. U.K. 26 Williamson, R. (1977) Nature News and l'iews 270, 295 297 27 Campbell, A. M. and Cotter. R. I. ( 1977] Nuch, ic Acids Research 4. 3877 3887
M a y 1978
With B. thuringiensis the insecticidal activity was first associated with a remarkable inclusion that is formed during sporulation (Fig. 1) and that has been shown to be a true crystal. Although there is no reason to believe that the toxin in the inclusion is not essential to the effectiveness of B. thuringiensis, several other activities have been identified that could contribute to the toxicity and possible pathogenicity of B. thur#lgiensis. The extent to which death results from toxicity or pathogenicity may vary with different insect species and environmental factors. Other activity factors that may be involved include a phospholipase, protease inhibitors of the immune system [I] and a toxin located in the spore [2]. This last activity is closely similar to the toxin in the crystal. Those strains that are used commercially do not produce the fl-exotoxin, an ATP analogue of widespread biological activity that is excreted during the vegetative growth of relevant strains. Strains that lack exotoxin are without known harmful effect to mammals. Interesting recent reports have been made of biological activity of crystals, or material derived therefrom, in mammalian, systems. Whole crystals have been found to enhance the immune response of rats and guinea pigs [3], and a fraction derived from crystals has been shown to have both anti-tumour and insecticidal activities. The nature of these effects on mammals awaits elucidation.
Biosynthesis of the crystal
Insect toxin in spores and protein crystals of
Bacillus thuringiensis Hugh J. Somerville Bacillus thuringiensis produces an actit'e toxin specific to lepidopterous lart,ae. The activi O" is associated with a protein crystal p r o d t w e d during sporulation a n d a related actit'ity is f o u n d #1 the outer layers o f .wores. Although many attempts have been made to use micro-organisms in the control of insect and plant pests, relatively few have been successful. Perhaps the most notable successes have been achieved with two species of Bacillus: Bacillus popilliae which has been used in infection and control of Japanese beetle in North America, and H.J.S. is at Shell Research Limited, Shell Bioseienees Laboratory. Sittmgbourne Research Centre, Sittingbourne. Kent, U.K.
Bacillus thuringiensis which has been commercially successful as an insecticide active against the larvae of many lepidopterous pests. B.popilliae is difficult to grow in vitro; consequently studies on the factors involved in its activity have been hindered and it is not known whether there is any common factor linking the activities of these two bacteria. B. thuringiensis is used in the field with formulation and application methods similar to those for chemical insecticides.
The crystal is formed during stages II and III of sporulation and the time of appearance of crystal antigens coincides with the appearance of the crystal as seen in the electron microscope. Apart from crystal formation, the morphological changes associated with crystal formation follow a similar pattern to those in other spore formers (Fig. 2). In one study [4] convincing evidence has been obtained for an association between the developing crystal and the exosporium membrane, although a separate study using different procedures failed to confirm this observation [5]. No soluble crystal antigens can be detected
Fig. I. Negatively stained crystal of Bacillus thur-
ingiensis. Magnification × 80,000.
109
TIBS - May 1978
[
l-I
I c EQ
-I ©11
,/
Fig. 2. Morphogenetic changes durhlg.~porulution aJul co.stal formation in Bacillus thuringiensis. E. exo.~7~orhon; C. crystal.
during sporulation and it appears that the exosporium may serve as a template for assembly, and possibly for synthesis, of the subunits. The crystal does not seem to grow in three dimensions from a central nucleus; in thin sections the edges of developing crystals in the sporangium are rectilinear whereas edges along the interface with the exosporium are often irregular. This suggests that as synthesis progresses the finished surfaces are pushed out into the sporangium. It is not clear what limits crystal growth. One possible explanation is that growth of the crystal may be stopped, or there may be some reversal of assembly mechanism, when the growing crystal extends across the internal diameter of the cell.
Biochemistry of the crystal The crystal is insoluble at physiological pH and will dissolve only in reagents such as 0.1 M NaOH or 8 M urea-mercaptoethanol. There is some confusion in the literature as to the precise number of polypeptide components in the crystal. If confirmed, a recent report that crystal preparations contain substantial amounts of carbohydrate [6] might lead to an explanation of the interactions between the dissolved peptides that aggregate to form complexes of high molecular weight. Just as reports of the number of crystal components vary, so do those on the molecular weight of the toxin itself. Reports of toxic material of low molecular weight remain to be substantiated and the best evidence points to a single polypeptide of molecular weight about 60,000 [7]. Such a component was isolated in low yield by ion-exchange chromatography of dissolved crystals. Recently, material indistinguishable from this toxin has been isolated in high yield from the products of tryptic digestion of dissolved crystals at pH 10, conditions similar to those in the insect gut [8]. It would appear therefore that the toxin is a polypeptide, possibly a glycoprotein, of substantial molecular weight.
Sporulation and crystal formation The close temporal relationship between crystal formation and sporulation suggests that the two may be interdependent and considerable evidence has accumulated to indicate a relationship between polypeptide components in the outer layers of the spore and those in the crystal [9,10]. Crystal-negative, spore-forming mutants (cr) are readily isolated following exposure to mutagens. Spores of cr strains lack toxicity but are otherwise indistinguishable from wild type spores. Asporogenic, crystal-forming mutations (sp) are relatively rare [4] and are blocked at stage III of sporulation (Fig. 2). These observations suggest that sporulation must be initiated for crystal formation to occur and that the latter is incidental to sporulation. As indicated above, spores of the wild type contain a toxin, some at least of whose activity is located in two membrane fractions [2], identified as exosporium and spore coat, that can be isolated from spores without loss of refractility. These fractions contain about 5°~ of the dry weight as cysteine. Antigens, specific to one of these fractions from the cr, non-toxic strain, appear at the same time as the synthesis of crystal antigens in the wild type. Crystal antigens have been separately shown to appear coincidentally with the toxin [11]. Although non-toxic, the membrane fractions from a cr strain could not be distinguished by density or amino acid analysis from the corresponding fractions of the wild type. However, they are immunologically distinct in that the ability to crossreact with crystal solutions in diffusion against anti-crystal serum is lost with the cr mutation. The molecular size and nature of the toxin in these fractions has not been established. The relationship between exosporum and spore coat, and the crystal has been demonstrated using both the ferritinlabelling and peroxidase-anti-peroxidaselabelling techniques for locating crystal
antigens in thin sections of spores [12] (Fig. 3). Unfortunately, no system has been developed for genetic analysis in B . t h u r h l g i e n s i s and consequently it is unknown whether there is one or more cr determinants or whether the genes coding for the cr character are located on the chromosome. The large size of the crystal relative to that of the parent micro-organisms (crystals are bipyrimidal, about 1 l~m long and weigh about one third of the spore by dry weight) has prompted interest in the mechanism of synthesis of the polypeptide components. Although crystal formation and sporulation are sensitive to low concentrations of actinomycin and rifampin, some results suggest that crystal formation may involve stable messenger RNA (mRNA). Studies [13] on the incorporation of [~aC]valine in high concentration in the presence of rifampin have suggested that a stable mRNA fraction is formed with a half-life of 10 min. Cell-free synthesis of polypeptides related to those in the crystal has been reported using both normal mRNA [14] and mRNA selected for stability [13]. However, identification of the products as crystal material cannot be regarded as established as the antisera used were prepared against extracts of crystal suspensions containing spores and it is known that this procedure results in extraction of some spore components. Crystal antigens appear over a similar time-scale to that of other sporulation associated characteristics such as dipicolinic acid and development of resistance. Although the involvement of stable mRNA in synthesis of other spore proteins has been proposed, there are conflicting reports and more detailed evidence is required to confirm the involvement of stable mRNA in crystal synthesis.
Fig. 3. Thin section through partially germinated spore of Bacillus thuringiensis showing spore coat reaction with ferritin-labelled antibodies to dissolved crystals. (Courtesy J. Short and P. Walker [12].) Magnification x 100.000.
T I B S - M a y 1978
110
Mode of action of crystal Relatively little is known about the mode of action of the endotoxin at the molecular level. It is clear that crystals (and fractions from spores) dissolve in the alkaline gut o f susceptible insects. Depending on the dose and the species of insect, the resulting effect can vary from temporary cessation of feeding to complete paralysis. Intoxication occurs within minutes o f ingestion. A number o f workers have reported effects on the midgut, and microscopic examination reveals that the epithelium is disrupted and may eventually break down completely. Unfortunately, microscopic examinations generally have been made with insects after prolonged exposure to the toxins. It remains to be demonstrated whether deterioration of the mid-gut is a primary effect; it is perhaps unlikely in view of the similar toxicity of dissolved crystal material by injection and ingestion. Both spores and crystals can contribute to the entomocidal effect of preparations x~f B. thuringiensis and spores may be of importance in the field [15]. Although it is speculative to point to similarities in the nature and mode of action of cholera toxin, it might be profitable to investigate the effects of the spore and crystal endotoxins on insects with the known modes of action of mammalian toxins in mind, particularly in view of the relatively fragmentary knowledge o f insect physiology. Perhaps the outstanding question of crystal formation is the physiological reason for this remarkable phenomena. Most, if not all, cultures of B. thuringiensis have been isolated from insect cadavers; this might indicate an ecological niche as an insect pathogen. However, a perceptive eye i~ needed to distinguish B. thuringiensis from B. cereus and a more detailed survey coupled with survival studies could indicate whether B. thuringiensis is a normal member of the soil microflora. It has been postulated that crystal formation results from oversynthesis of some normal and essential spore component [9]. Certainly there is considerable evidence to indicate that polypeptides in the crystal are related to a substantial fraction of the protein in the spore coat and exosporium, although the toxin may not form a major part of this common material. Circumstantial support for this hypothesis is provided by the formation of crystalline inclusions in a RNA polymerase mutant of B. subtilis [16], and by the occurrence of crystalline inclusions in other sporulating bacteria [171. However, the loss o f toxicity in the cr strains suggests that an alternative, or additional, explanation is necessary.
References I Edlund, T., Siden, 1. and Boman, H.G. (1976) Infect. lmmun. 14, 934-941 2 Scherrer, P.S. and Somerville, H.J. (1977) Eur. J. Biochem. 72, 479490 3 Prasad, S.S.S.V. and Shethna, V.I. (1975) Biochem. Biophys. Res. Commun. 62, 517 4 Somerville,H.J. (1971) Eur. J. Biochem. 18, 226237 5 Bechtel,D. B. and Bulla, L. A. (1976)J. BacterioL 127, 1472-1481 6 Bulla, UA., Kramer, K.J. and Davidson, L.I. (1977) J. Bacteriol. 130, 375-383 7 Herbert, B.N., Gould. H.J. and Chain, E.B. (1971) Eur. J. Biochem. 24, 366-375 8 Lilley,M. and Somerville,H.J. (1975) Proc. Soc. Gen. Microbiol. 3, 10 9 Delafield,F. P,, Somerville,H.J, and Rittenberg, S.C. (1968)J. BacterioL 96, 713-720
10 Lecadet, M. M., Chevrier, G. and Dedonder, R. (1972) Eur. J. Biochem. 25, 348-349 I 1 Norris, J.R. (1969) Spores IV (Campbell, L.L., ed.) Amer. Soc. Microbiol.,45~18 12 Short, J., Walker, P., Thomson, R.O. and Somerville, H.J. (1974) J. Gen. Microbiol. 84, 261-276 13 Glatron, M. F. P. and Rapoport, G. (1976) Biochimie 58, 119-129 14 Herbert, B. N. and Gould, H. J. (1973) Eur. J. Biochem. 37, 441448 15 Burges, H.D., Thomson, E.M. and katchford, R.A. (1976) J~ lnvertebr. Pat/sol.27, 87-94 16 Santo, L.Y. and Doi, R.H. (1973)J. Bacteriol. 116, 479~,82. 17 Duncan, C.L., King, G.J. and Frieben, W.R. (1973) J. BaeterioL 114, 845-859
I II
Protein-bound oligosaccharides of cell membranes Johan Jhrnefelt, Jukka Finne, Tom Krusius and Heikki Rauvala Membrane glycoproteins contain several different types o f carbohydrate side chain. Fractionation reveals f o u r major classes, each having characteristic structural features. The relative abundance o f the various carbohydrate structures is different in different tissues. This supports the v&u, that glycoproteins, through their carbohydrate portions, express some o f the cellomembrane specificity.
The outer surface of cells is covered with various carbohydrates, which are covalently linked either to a lipid or to a protein. Such glycolipids and glycoproteins are integral parts of cell membranes in most organisms and are believed to take part in interactions between cells or between cells and external molecules [1]. So-called receptor functions have often been attributed to carbohydrates. Evidence that the receptors of several biologically active substances, such as thyrotropin and related glycoprotein hormones [21, serotonin [3], interferon [4], cholera toxin [3] and transferrin [5], are glyeolipids or glycoproteins has been forthcoming. The structural multiplicity and the small amounts of each specific carbohydratecontaining molecular species occurring in cell membranes has made isolation and 'analysis of these molecules very difficult. Glycolipids have been easier to characterize because the lipid carrier has little influence in the fractionation methods conventionally used, whereas membrane glycoproteins have been isolated only in a few cases. For soluble glycoproteins, occurring J.J., J.F.. T.K. and H.R. are at the Department of Medical Chenlistry. Universityof Helsinki, Siltavuorenpenger tO A, SF-OOI70Helsioki 17. Finland.
in blood serum or in various secretions, more complete structural information is available, the obvious reason being simpler isolation procedures and consequently the ease of obtaining pure samples in large quantities. Even when the isolation of a membrane glycoprotein has been successful, the analysis of its carbohydrate parts is a difficult task, since the molecule may contain several different carbohydrate side chains, and the glycoprotein is mostly available in minute amounts. The aspects to be described in this article are primarily the nature of the oligosaccharide side chains of membrane glycoproteins. In order to work out analytical and separation methods for the various oligosaccharides, representative samples in sufficient quantity are required. These are obviously not obtainable through the preliminary isolation of glycoproteins. The amounts available would be too small, and each glycoprotein would have only a selection o f carbohydrate side chains. Obtaining all the membrane proteinbound carbohydrate o f a given tissue, free o f other components, would be a distinct advantage from the viewpoint of the carbohydrate chemistry.