Parasporal Proteins Research Articles

Toxicity of the crystalline polypeptides of Bacillus thuringiensis subsp. israelensis in Japanese quail

The solubilized parasporal crystalline proteins (SPCP) of Bacillus thuringiensis subsp. israelensis when administered to Japanese quail by intraabdominal (IA) injection were toxic with an LD 50 at 24 hr of 22.8 mg/kg. No deaths were noted by intranasal, subcutaneous, and intravenous administration. SPCP were highly hemolytic against quail red blood cells but this SPCP-induced hemolysis was inhibited by preincubation of SPCP with quail serum. Electrocardiographic analyses of IA-injected quail demonstrated cardiac dysfunction consistent with bradycardia. Hypothermia was also positively correlated with heart rate reduction. SPCP IA injection reduced serum lipid levels and alkaline phosphatase, and increased serum glucose, creatine phosphokinase, and lactate dehydrogenase. Induction of a septic shock-like condition may be the toxic mode of action of IA-injected SPCP in Japanese quail.

Occurrence of Non-Insecticidal Bacillus thuringiensis Flagellar Serotype 14 in the Soil of Japan

Summary Three spore-forming isolates obtained from a soil sample of Hokkaido Island, Japan, were referable to Bacillus thuringiensis flagellar (H) serotype 14 (subsp. israelensis). Parasporal inclusions of these isolates were morphologically very similar to those produced by the type strain of B. thuringiensis H serotype 14. The type strain of H serotype 14 was highly virulent to aedine and culicine mosquito larvae, while the three isolates were avirulent to mosquito larvae. Immunologically, there was no relationship between parasporal inclusion proteins of the type strain and the three avirulent isolates. A great difference was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of parasporal inclusion proteins. Parasporal inclusions of the type strain contained proteins with molecular masses of 130 kD, 70 kD, 38 kD, and 24 kD, while those of the avirulent isolates contained proteins of 47 kD, 36 kD, 30 kD, and 24 kD and a few minor proteins. The results suggested that the avirulent property of the H serotype 14 isolates was attributable to the lack of 130-kD protoxin in their parasporal inclusions.

Isolation of a relatively nontoxic 65-kilodalton protein inclusion from the parasporal body of Bacillus thuringiensis subsp. israelensis.

Ultrastructural studies of the mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis revealed that the parasporal body contained three major inclusion types, designated types 1, 2, and 3, which could be differentiated on the basis of electron opacity and size and, to some extent, shape. The type-2 inclusion, which was of moderate electron density and often appeared as a bar-shaped polyhedral body, was isolated on NaBr gradients from purified parasporal bodies and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transmission electron microscopy, and bioassays against neonate larvae of Aedes aegypti. Purified inclusions averaged 150 to 200 nm by 500 to 700 nm in transverse sections and consisted almost exclusively of a 65-kilodalton (kDa) protein contaminated with minor quantities of 38- and 28-kDa proteins. Lethal concentration values at the 50% level for preparations of the purified parasporal body and the type-2 inclusion were, respectively, 0.66 and 43 ng/ml, indicating that the 65-kDa protein is only slightly toxic to mosquitoes in comparison to the intact parasporal body. Analysis of the type-2 polyhedral inclusion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and bioassays during different stages of purification demonstrated a positive correlation between the toxicity of the preparation and the degree of contamination with the 28-kDa protein. These results indicate that the 65-kDa protein is not the primary larvicidal toxin, although it may act in conjunction with other parasporal body proteins to produce the high mosquitocidal toxicity characteristic of this bacterium.

Two different parasporal inclusions are produced by Bacillus thuringiensis subsp. finitimus.

Bacillus thuringiensis subsp. finitimus produced at least two parasporal inclusions. One inclusion was formed within the exosporium and remained with the spore after mother cell lysis. A second inclusion formed somewhat later exterior to the exosporium. Each inclusion contained a major polypeptide of about 135,000 daltons with unique antigenic determinants. This subspecies contained only two plasmids, of 98 and 77 megadaltons (MDa). Strains cured of these plasmids produced only the free inclusion. Since the plasmid-cured strains did not contain DNA sequences homologous to plasmid DNA, the gene for the free-inclusion protein must be encoded in the chromosome. In contrast, the enclosed parasporal inclusion was produced only when the plasmid of 98 MDa was present. In addition, transfer of the 98-MDa plasmid to Bacillus cereus resulted in transcipients that produced small inclusions enclosed within the exosporium, and the protein extracted from these inclusions reacted with antibody specific for enclosed inclusion protein of B. thuringiensis subsp. finitimus. Genes in both the chromosome and a plasmid function in the synthesis of distinct parasporal proteins in this subspecies.

An improved enzyme-linked immunoassay for the detection and quantification of the entomocidal parasporal crystal proteins of Bacillus thuringiensis subsp. kurstaki and israelensis.

An improved and simplified enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of parasporal crystalline toxins from Bacillus thuringiensis subsp. kurstaki. The improved procedure involved pretreatment of the polystyrene cuvettes with glutaraldehyde before antibody coating. A direct comparison of treated and untreated cuvettes is provided. ELISAs were then used for the analysis of the entomocidal crystalline proteins in commercial and experimental formulations of B. thuringiensis subspp. kurstaki and israelensis.