Somsak Pantuwatana

The comparative persistence of toxicity of Bacillus sphaericus strain 1593 and Bacillus thuringiensis serotype H-14 against mosquito larvae in different kinds of environments☆

Abstract Bacillus sphaericus strain 1593 and B. thuringiensis serotype H-14 were evaluated for persistence of toxicity against two species of mosquito larvae, Culex quinquefasciatus and Aedes aegypti , in a selected simulating plot in Bangkok. Both strains of bacteria demonstrated larvicidal activity towards both species of mosquito larvae. In tap water, the toxicity of B. sphaericus strain 1593 was found to be greater towards C. quinquefasciatus larvae than A. aegypti larvae, whereas the toxicity of B. thuringiensis serotype H-14 was found to be greater towards A. aegypti larvae than C. quinquefasciatus larvae. The persistence of toxicity of these two bacteria was found to be different. The lethal concentration of B. thuriengiensis H-14 against A. aegypti decreased from LC 90 to below LC 50 in about 15 weeks when tested in tap water. The decrease was faster in polluted water. The toxicity of B. sphaericus 1593 towards C. quinquefasciatus larvae persisted for at least 9 months in tap water and 6 months in polluted water. The multiplication of bacteria was indicated only in populations of B. sphaericus 1593 tested with C. quinquefasciatus larvae.

Multiple chitinase enzymes from a single gene of Bacillus licheniformis TP-1

Three chitinase activity bands in a culture supernatant of Bacillus licheniformis TP-1 were detected by non-denaturing PAGE. They were designated chitinase bands 1, 2, and 3 in order from the gel origin. Analysis by immunodiffusion and immunoelectrophoresis using polyclonal antibody raised against chitinase band 3 indicated that these chitinases were antigenically similar. B. licheniformis TP-1 and Escherichia coli harboring the cloned chitinase gene (pCHIL3) from strain TP-1 expressed 3 chitinase bands by non-denaturing PAGE and SDS-PAGE which gave estimated molecular masses of 68, 62, and 50 kDa (named Chi68, Chi62, and Chi50, respectively). All three chitinases had the same N-terminal amino acid sequences, strongly suggesting that Chi62 and Chi50 were derived from Chi68 by a processing step(s) at the C-terminus. The deduced C-terminal amino acid sequence of Chi68 showed homology to amino acid sequences of known chitin and cellulose binding domains. Chi62 and Chi50 lacked this domain (judging from their deduced amino acid sequences and calculated molecular masses) and they were unable to bind chitin, suggesting that they were generated from Chi68 by cleavage of the chitin binding domain at the C-terminus. Comparison of chitinase activities indicated that this binding domain was important for complete hydrolytic activity towards colloidal chitin.

Toxicity of Bacillus thuringiensis toward Aedes aegypti larvae.

Abstract Among six strains of Bacillus thuringiensis and five other species of Bacillus, only two strains of B. thuringiensis, strains HD-1 and BA-068, were toxic to Aedes aegypti larvae within 24 hr. The LC50s were 5.6 × 104 and 2.4 × 105 spores/ml for strains HD-1 and BA-068, respectively. The toxic factor(s) was heat sensitive and γ ray resistant and preliminary evidences indicated that it was associated with the crystalline body of B. thuringiensis.

Comparison of development of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus in mosquito larvae

Immunofluorescent staining was used with thin sections of paraffin-embedded specimens to detect the development of Bacillus thuringiensis var. israelensis and Bacillus sphaericus in the gut of mosquito larvae. The third- and fourth-instar larvae of Aedes aegypti, Anopheles maculatus, and Culex quinquefasciatus were fed either vegetative cells or spores of the bacteria. Spore germination, multiplication, and sporulation were studied in the larvae of each species. The spores of B. thuringiensis var. israelensis and B. sphaericus strain 2297 could germinate and cells could sporulate in the larval body. The vegetative cells of B. sphaericus strain 810428 were also able to produce spores in the mosquito larval gut, but the germination of spores could not be detected in the larvae. Multiplication of all bacterial species was observed after the larvae died. Growth of the bacteria in distilled water containing crude extracts of larvae made from each species was compared with that in synthetic medium (nutrient broth). They could produce spores and toxins in all the media used and the toxins had larvicidal activity against the target mosquitos Ae. aegypti, An. maculatus, and C. quinquefasciatus.

Production of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus strain 1593 on media using a byproduct from a monosodium glutamate factory

Abstract Two newly developed media, H4 and H7, were found to be highly suitable for culturing Bacillus thuringiensis subsp. israelensis and B. sphaericus, respectively. These media contained 0.05% K2HPO4 and 4% HDL (H4 medium) or 0.05% K2HPO4 and 7% HDL (H7 medium); HDL is the by-product from a monosodium glutamate factory. Tests to compare endospore formation and toxicity values of B. thuringiensis subsp. israelensis in H4 medium and nutrient broth supplemented with salts and glucose (NBSG) medium were carried out in a 3-liter fermentor. The viable cell count and LC50 value of B. thuringiensis subsp. israelensis in H4 medium at 48 hr were 2.5 × 108 cells/ml and 10−7.2 (dilution), respectively, while those in NBSG medium were 1.6 × 108 cells/ml and 10−6.5, respectively. In the case of B. sphaericus grown in H7 medium, the number of cells and LC50 value were found to be 1.4 × 109 cells/ml and 10−7.8, respectively. B. sphaericus grown in nutrient broth supplemented with salt and yeast extract (NBSY) were found to produce 6.4 × 108 cells/ml and an LC50 value of 10−6.8. The toxicity of B. thuringiensis subsp. israelensis was tested against Aedes aegypti larvae, while that of B. sphaericus was tested against Culex quinquefasciatus. The cost of 10 liters of medium for production of B. thuringiensis subsp. israelensis and in B. sphaericus and H4 and H7 was

Inhibition of a conjugation-like gene transfer process in Bacillus thuringiensis subsp. israelensis by the anti-S-layer protein antibody

0.02 and

Role of the parasporal body in causing toxicity of Bacillus thuringiensis toward Aedes aegypti larvae.

0.03, respectively. The cost of these newly developed media was much less than that of NBSG medium (

Transfer of plasmids pBC 16 and pC 194 into Bacillus thuringiensis subsp. israelensis

7.05 per 10 liters) for cultivation of B. thuringiensis subsp. israelensis and NBSY medium (

High production of CryIVB protein does not lead to formation of crystalline inclusions in Bacillus thuringiensis subsp. israelensis

11.67 per 10 liters) for cultivation of B. sphaericus.

Molecular Cloning of S-Layer Protein Gene from Bacillus Thuringiensis

Extraction of the S-layer protein by treatment with 6 m urea revealed a high-molecularweight protein in the extracts obtained from Bacillus thuringiensis subsp. israelensis (B.t.i) strain 4Q2. This protein band was found to be absent in partially cured (4Q2-72) and completely cured (c4Q2-72) strains. The antibody toward this S-layer protein was prepared and used to locate its antigenic protein on B.t.i cells by using indirect immunofluorescence. Immunodiffusion reactions and Western blot analysis confirmed the specificity of the anti-S-layer protein antibody. It was found that the antibody against 4Q2 S-layer protein, inhibited plasmid transfer via a conjugationlike process between, B.t.i. strains 4Q2-16 and c4Q2-72. That is, the frequency of transfer of plasmid pBC16 was reduced from 9.7×10-6 in the absence of the antibody to less than 1.0×10-8 in the presence of the antibody. The antibody was also found to reduce the frequency of pBC16 plasmid transfer via a conjugation-like process between B.t.i. strains A084-16-194 and c4Q2-72 from 2.2×10-5 in the absence of the antibody to 1.2×10-6 in the presence of the antibody.