Structure and function of the <i>Bacillus thuringiensis</i> protein crystal

Nickerson, Kenneth W.

doi:10.1002/bit.260220704

Cited by 55 publications

(38 citation statements)

References 100 publications

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“…Hofte and Whitely 131 have suggested that the C-terminal region is con-served because it is essential for crystal formation. It has been established that disulfide linkages in the C-terminal region are responsible for stablilizing the protein crystal [21]. The domain structure which gives rise to the sequential proteolysis may be a consequence of the structural constraints imposed by the requirement for the formation of a regular crystal lattice involving interchain disulfide bridges.…”

Section: Discussionmentioning

confidence: 99%

Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis

Choma

Surewicz

Carey³

et al. 1990

European Journal of Biochemistry

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Trypsin is shown to generate an insecticidal toxin from the 130-kDa protoxin of Bacillus thuringiensis subsp. kurstaki HD-73 by an unusual proteolytic process. Seven specific cleavages are shown to occur in an ordered sequence starting at the C-terminus of the protoxin and proceeding toward the N-terminal region. At each step, C-terminal fragments of approximately 10 kDa are produced and rapidly proteolyzed to small peptides. The sequential proteolysis ends with a 67-kDa toxin which is resistant to further proteolysis. However, the toxin could be specifically split into two fragments by proteinases as it unfolded under denaturing conditions. Papain cleaved the toxin at glycine 327 to give a 34.5-kDa N-terminal fragment and a 32.3-kDa C-terminal fragment. Similar fragments could be generated by elastase and trypsin. The N-terminal fragment corresponds to the conserved N-terminal domain predicted from the gene-deduced sequence analysis of toxins from various subspecies of B. thuringiensis, and the C-terminal fragment is the predicted hypervariable sequence domain. A double-peaked transition was observed for the toxin by differential scanning calorimetry, consistent with two or more independent folding domains. It is concluded that the N-and C-terminal regions of the protoxin are two multidomain regions which give unique structural and biological properties to the molecule.Bacillus thuringiensis is an insect pathogen with an unusual but highly specific mode of action. During the sporulation cycle it lays down a parasporal protein crystal which is rendered toxic on ingestion by susceptible insect larvae. The major component of crystals toxic to lepidoptera is a protein (protoxin) with a molecular mass of approximately 130 kDa [l -31. Treatment with thiol reagents at basic pH solubilizes the protoxin by cleaving the disulfide bonds which stabilize the crystal. Incubation of the solubilized protoxin with proteolytic enzymes or insect gut juice produces a 58 -70-kDa proteinaseresistant toxin derived from the N-terminal portion of the molecule [4,5]. The toxin then binds to receptors in the midgut epithelium, causing cell lysis and eventual larval death [6 -81. The details of the lytic mechanism are not yet established but it appears that the toxin generates small pores or localized perturbations in the plasma membrane, causing disruption of homeostatic ion regulation [9].Large proteins are generally organized into distinct structural units referred to as domains or subdomains, but the criteria used for this classification is somewhat subjective. It is clear that the protoxin is divided into at least two major domains: the carboxyl half of the molecule which is readily attacked by proteinases, and the toxin derived from the Nterminal half which is proteinase-resistant. The toxins from

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Section: Discussionmentioning

confidence: 99%

Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis

Choma

Surewicz

Carey³

et al. 1990

European Journal of Biochemistry

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“…2). The greater stability of intrachain disulfide bonds had been predicted on thermodynamic grounds (24), and these expectations were confirmed by the lower levels (1 MM) of dithioerythritol necessary for reduction of the 52-kDa dimer. Confirmation of the stability of the 26-kDa protein intrachain disulfide bond conmes from its formation by disulfidesulfhydryl interchange and its appearance as a Hg(II) crosslinked product (Fig.…”

Section: Methodsmentioning

confidence: 61%

“…israelensis, even though the 2.1% halfcystine composition reported for native B. thuringiensis subsp. israelensis crystals (33) is greater than that (1.1 to 1.9%) for crystals from all other B. thuringiensis varieties (24). In B. thuringiensis subsp.…”

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confidence: 75%

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Structural disulfide bonds in the Bacillus thuringiensis subsp. israelensis protein crystal

Couche¹,

Pfannenstiel²,

Nickerson³

1987

J Bacteriol

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“…Inclusion bodies such as those formed from defective or overexpressed foreign proteins (13,27,33) or the toxic protein crystals of B. thuringiensis (11,22) and enterotoxin inclusion bodies of C. perfringens (20) …”

Section: Discussionmentioning

confidence: 99%

Protein inclusions produced by the entomopathogenic bacterium Xenorhabdus nematophilus subsp. nematophilus

Couche¹,

Gregson²

1987

J Bacteriol

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The entomopathogenic bacterium Xenorhabdus nematophilus subsp. nematophilus produces two types of intracellular inclusion bodies during in vitro culture. Large cigar-shaped inclusions (designated type 1) and smaller ovoid inclusions (designated type 2) were purified from cell lysates, using differential centrifugation in discontinuous glycerol gradients and isopycnic density gradient centrifugation in sodium diatrizoate. The inclusions, composed almost exclusively of protein, are readily soluble at high and low pH values and in the presence of cation chelators such as EDTA, anionic detergents (sodium dodecyl sulfate), or protein denaturants (urea, NaBr). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified inclusions revealed a single 26-kilodalton protein (IP-1) in type 1 inclusions and a 22-kilodalton protein (IP-2) in type 2 inclusions. Analysis of these proteins by isoelectric focusing in the presence of 8 M urea showed that IP-1 is acidic and IP-2 is neutral. Furthermore, each protein occurred in multiple forms differing slightly in isoelectric point. Other variations in peptides released by trypsin digestion, immunological properties, and amino acid composition revealed significant structural differences between IP-1 and IP-2. Kinetic studies using light microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting procedures showed that inclusion protein synthesis occurs only during the second half of exponential culture growth. Synthesis of inclusion proteins and their aggregation to form inclusions occurred concurrently. Possible functions for these abundant proteins are discussed.Xenorhabdus species are entomopathogenic bacteria symbiotically associated with insect parasitic nematodes of the families Heterorhabditidae and Steinernematidae (1,3,30). The bacterial symbionts are asporogenous gram-negative rods, which are carried monoxenically in the intestine of nonfeeding infective-stage nematodes. On penetration of an insect host, the bacteria are released into the hemocoel. Colonization by the bacteria kills the host and establishes a suitable environment for reproduction of the nematodes by providing nutrients and inhibiting the growth of other microorganisms (1,24).When cultured in vitro, Xenorhabdus bacteria occur in two forms designated primary and secondary, which can be distinguished according to colony morphology and pigmentation on various bacteriological media, or on the basis of antibiotic production (1, 2). Both forms are pathogenic to insects, but only the primary form is isolated from infective nematodes (1). Xenorhabdus spp. will grow in a wide variety of artificial media which are rendered suitable for nematode reproduction, thus providing the basis for economical mass production of nematodes. Our nematophilus, in contrast to X. luminescens in which both primary and secondary forms produced inclusions (10). Intracellular inclusion bodies have also been described in several ultrastructural studies of Xenorhabdus spp. (4,7,15,18).In this repor...

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Structure and function of the Bacillus thuringiensis protein crystal

Cited by 55 publications

References 100 publications

Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis

Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis

Structural disulfide bonds in the Bacillus thuringiensis subsp. israelensis protein crystal

Protein inclusions produced by the entomopathogenic bacterium Xenorhabdus nematophilus subsp. nematophilus

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