Although insects lack the basic entities of the vertebrate immune system, such as lymphocytes and immunoglobulins, they have developed alternative defence mechanisms against infections. Different types of peptide factors, exhibiting bactericidal activity, have been detected in some insect species. These humoral factors are induced upon infection. The present report describes the discovery of the apidaecins, isolated from lymph fluid of the honeybee (Apis mellifera). The apidaecins represent a new family of inducible peptide antibiotics with the following basic structure: GNNRP(V/I)YIPQPRPPHPR(L/I). These heat‐stable, non‐helical peptides are active against a wide range of plant‐associated bacteria and some human pathogens, through a bacteriostatic rather than a lytic process. Chemically synthesized apidaecins display the same bactericidal activity as their natural counterparts. While only active antibacterial peptides are detectable in adult honeybee lymph, bee larvae contain considerable amounts of inactive precursor molecules.

No abstract

A plasmid-encoded crystal protein gene (bt2) has been cloned from Bacillus thuringiensis berliner 1715. In Escherichia coli, it directs the synthesis of the 130-kDa protein (Bt2) which is toxic to larvae of Pieris brassicae and Manduca sexta. Comparison of the deduced amino acid sequence of this Bt2 protein with the B. thuringiensis kurstaki HD1 Dipel, B. thuringiensis kurstaki HD73 and B. thuringiensis sotto crystal protein sequences suggests that homologous recombination between the different genes has occurred during evolution.Treatment of the Bt2 protein with trypsin or chymotrypsin yields a 60-kDa protease-resistant and fully toxic polypeptide. The minimal portion of the Bt2 protein required for toxicity has been determined by analysing the polypeptides produced by deletion derivatives of the bt2 gene. It coincides with the 60-kDa protease-resistant Bt2 fragment and it starts between amino acids 29 and 35 at the N-terminus and terminates between positions 599 and 607 at the C-terminus.

The complete nucleotide sequence of a cloned gene encoding a 130-kDa crystal protein of Bacillus tlzuringiensis (B.t.) subspecies israelensis has been determined. The recombinant protein (Bt8) was purified and shown to be a mosquito-specific toxin with a LCso value of 43 ng/ml to third-instar larvae of Aedes aegypti. Bt8 is processed by proteases or midgut extracts of mosquito larvae into toxic fragments of 68 -78 kDa. Deletion mapping indicated that the active fragment of Bt8 is localized in the N-terminal half of the protoxin molecule. The deduced amino acid sequence of Bt8 has been compared with that of Bt2, a Lepidoptera-specific toxin, previously cloned from Bacillus thuringiensis berliner. Highly homologous amino acid stretches are present in the C-terminal half of the proteins. The N-terminal parts show much less sequence homology but they display a strikingly similar distribution of hydrophilic and hydrophobic amino acids. In addition, Bt8 and Bt2 show a significant immunological crossreaction. The data indicate that although these B.t. delta endotoxins exhibit a different insect-host specificity, they are structurally related and might use a similar mechanism to interact with insect cell membranes. B. thuringiensis subspecies israelensis produces a crystal that is highly toxic to the larvae of mosquitoes and blackflies [3, 51. The solubilized crystal proteins of B.t. isruelensis also exhibit hemolytic activity and non-specific cytotoxicity towards insect and mammalian cells in vitro [6]. The B.1. israelensis crystal is composed of at least three major polypeptides of 28 kDa, 65 kDa and 130 kDa [7], which differ in their antigenic structure [8].It is still unclear which crystal protein is responsible for the strong mosquitocidal activity. through cloning and expression of their genes. The availability of purified recombinant protein also allows one to investigate in detail the structural and functional characteristics of these insect-specific toxins.In this paper we present the complete nucleotide sequence of a gene (bt8), encoding a 130-kDa mosquitocidal protein of B.t. israelensis. The recombinant protein (Bt8) was purified from the E. coli clone. Its properties were compared to those of Bt2, a previously described [I 91 Lepidoptera-specific B.t. toxin. MATERIALS AND METHODS Strains and plasmidsB. thuringiensis subspecies israelensis strain 442-72 was obtained from the Bacillus Genetic Stock Center (Columbia, Ohio). E. coli JM107 carrying plasmid pMU388 has been generated previously [18]. E. coli K514 (pGI502), containing a B.t. herliner toxin gene and expressing a 130-kDa protein (Bt2), has been described [19]. Crystal purificationCrystals of B.t. israelensis strain 442-72 and B.t. berliner 171 5 were purified from sporulating cultures as described by Mahillon and Delcour [20]. Purification of' cloned B.t. toxinThe cell pellet from 1 1 saturated overnight culture of E. coli JM107 (pMU388) was suspended in 100 ml50 mM Tris/ HC1, pH 7.9/50 mM EDTA/15% (massivol.) sucrose and frozen at -20°C. After thaw...

We have investigated the protein composition and the insecticidal spectrum of crystals of 29 Bacillus thuringiensis strains active against lepidopteran larvae. All crystals contained proteins of 130 to 140 kilodaltons (kDa) which could be grouped into three types by the molecular weight of the protoxin and the trypsin-activated core fragment. Proteins of the three types showed a characteristic insecticidal spectrum when tested against five lepidopteran species. Type A crystal proteins were protoxins of 130 or 133 kDa, which were processed into 60-kDa toxins by trypsin. Several genes encoding crystal proteins of this type have been cloned and sequenced earlier. They are highly conserved in the N-terminal half of the toxic fragment and were previously classified in three subtypes (the 4.5-, 5.3-, and 6.6-kilobase subtypes) based on the restriction map of their genes. The present study shows that different proteins of these three subtypes were equally toxic against Manduca sexta and Pieris brassicae and had no detectable activity against Spodoptera littoralis. However, the 4.5-, 5.3-, and 6.6-kilobase subtypes differed in their toxicity against Heliothis virescens and Mamestra brassicae. Type B crystal proteins consisted of 140-kDa protoxins with a 55-kDa tryptic core fragment. These were only active against one of the five insect species tested (P. brassicae). The protoxin and the trypsin-activated toxin of type C were 135-and 63-kDa proteins, respectively. Proteins of this type were associated with high toxicity against S. littoralis and M. brassicae. A panel of 35 monoclonal antibodies was used to compare the structural characteristics of crystal proteins of the three different types and subtypes. Each type of protein could be associated with a typical epitope structure, indicating an unambiguous correlation between antigenic structure and insect specificity.