Molecular cloning of the 130-kilodalton mosquitocidal delta-endotoxin gene of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus.

A 3.7-kilobase (kb) XbaI fragment harboring the cryIVB gene (L. Thorne, F. Garduno, T. Thompson, D. Decker, M. A. Zounes, M. Wild, A. M. Walfield, and T. J. Pollock, J. Bacteriol. 166:801-811, 1986) which encoded a 130-kilodalton (kDa) mosquitocidal toxin from a 110-kb plasmid of Bacillus thuringiensis subsp. israelensis 4Q2-72 was cloned into pUC12 and transformed into Escherichia coli. The clone with a recombinant plasmid (designated pBT8) was toxic to Aedes aegypti larvae. The fragment (3.7 kb) was ligated into pBC16 (tetracycline resistant [Tcr]) and transformed by the method of protoplast transformation into Bacillus sphaericus 1593 and 2362, which were highly toxic to Anopheles and Culex mosquito larvae but less toxic to Aedes larvae. After cell regeneration on regeneration medium, the Tcr plasmids from transformants (pBTC1) of both strains of B. sphaericus were prepared and analyzed. The 3.7-kb XbaI fragment from the B. thuringiensis subsp. israelensis plasmid was shown to be present by agarose gel electrophoresis and Southern blot hybridization. In addition, B. sphaericus transformants produced a 130-kDa mosquitocidal toxin which was detected by Western (immuno-) blot analysis with antibody prepared against B. thuringiensis subsp. israelensis 130-kDa mosquitocidal toxin. The 50% lethal concentrations of the transformants of strains 1593 and 2362 against A. aegypti larvae were 2.7 X 10(2) and 5.7 X 10(2) cells per ml, respectively. This level of toxicity was comparable to the 50% lethal concentration of B. thuringiensis subsp. israelensis but much higher than that of B. sphaericus 1593 and 2362 (4.7 X 10(4) cells per ml) against A. aegypti larvae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of pathogens attacking Pomacea canaliculata

Seven microorganisms capable of killing Pomacea canaliculata were isolated from soil samples obtained from various agricultural areas of Thailand. The identification of these microorganisms was performed using microscopic examination and biochemical tests. Five strains were identified as Pseudomonas aeruginosa and were designated P. aeruginosa 19.1, 21.2.1, B1.1, P1 and P2. The other two strains were identified as Pseudomonas fluorescens and were designated P. fluorescens 13.1 and Ct1. Pathogenicity studies of these microorganisms to P. canaliculata (Lamarck) were performed and characterized by LC₅₀ levels. The LC₅₀ levels of non-autoclave-treated and autoclave-treated cell suspensions to P. canaliculata were found to be 3.56 × 10⁴–1.35 × 10⁶ c.f.u./ml and 3.09 × 10⁴ to 1.23 × 10⁶ c.f.u./ml, respectively.     

Cloning of a chitinase gene into Bacillus thuringiensis subsp. aizawai for enhanced insecticidal activity

Chitinase from a high producing strain (TP-1) of Bacillus licheniformis was used with B. thuringiensis subsp. aizawai (B.t.a.) in a combined larvicidal assay against the pest, Spodoptera exigua. With 10 mU of this chitinase, the LD(50) of B.t.a. was reduced by 7.6, 13.8 and 15 times on days 3, 5 and 7, respectively when compared to use of B.t.a. alone. In addition, a combination of chitinase (10 mU) and B.t.a. at a sub-lethal dose retarded growth and development of S. exigua. In preparation for transformation of B.t.a., the TP-1 chitinase gene was cloned in E. coli DH5alpha and sequenced to reveal a single open reading frame of 1,815 bp. This open reading frame encoded for a protein of 604 amino acids and a characteristic signal peptide sequence of 35 amino acids. The gene was subsequently introduced into B.t.a. where it was expressed constitutively. The transformed strain showed slightly improved activity against S. exigua when compared to the non-transformed strain. This was probably due to the low chitinase activity (15 mU/ml) of the transformant, which might be improved by further gene manipulation to overexpress enzyme production.     

Problematic Occurrence of Tyrosine Crystals in the Thai Soybean Paste Tao Chieo

In a Bangkok soy sauce factory that had recently converted to controlled inoculum and incubation for the koji stage of fermentation, a problem arose with unsightly white particles in the soybean paste condiment called tao chieo. This problem had not arisen during the previous history of the factory where the koji stage of the fermentation was uncontrolled. Microscopic examination of the particles showed that they were crystalline. Physical separation of the crystals followed by solvent extraction, recrystallization, chemical characterization, and spectroscopy showed that they consisted of tyrosine. Tao chieo prepared by using low- or high-protease strains of Aspergillus sp. indicated that the tyrosine crystals resulted from mold proteolysis of the soybean substrate. Addition of polyethylene glycol at the beginning of the moromi fermentation reduced the severity of crystal formation.     

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

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 LC₉₀ to below LC₅₀ 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.     

Purification and Characterization of Chitinase from Bacillus circulans No.4.1

Bacillus circulans No.4.1 produced a high level of chitinase when cells were grown in tryptic soy broth supplemented with 0.3% colloidal chitin at 35°C for 5 days. Purification was carried out by protein precipitation with 80% saturation ammonium sulfate, anion-exchange chromatography with DEAE-Sephacel, and gel filtration with Sephadex G-100, sequentially. The purified enzyme could be demonstrated as a single band on SDS-PAGE, estimated to be 45 kDa. This enzyme could hydrolyze colloidal chitin, purified chitin, glycol chitin, carboxymethyl-chitin (CM-chitin), and 4-methylumbelliferyl-β-D-N,N′-diacetylchitobioside [4-MU-(GlcNAc)₂]. The optimal conditions for this chitinase were pH 8.0 and 40°C. The isoelectric point of the chitinase was 5.1. The amino acid composition of the purified chitinase was determined. The initial 20 amino acid residues of the N-terminal were found to be alanine (A), proline (P), tryptophan (W), asparagine (N), serine (S), lysine (K), glycine (G), asparagine (N), tyrosine (Y), alanine (A), leucine (L), proline (P), tyrosine (Y), tyrosine (Y), arginine (R), glycine (G), alanine (A), tryptophan (W), alanine (A), and valine (V). Knowledge of these properties of chitinase from B. circulans No. 4.1 should be useful in the development of genetically engineered Bacillus sp. as biopesticides. Received: 19 March 1999 / Accepted: 30 April 1999     

Expression of chitinase-encoding genes in Bacillus thuringiensis and toxicity of engineered B. thuringiensis subsp. aizawai toward Lymantria dispar larvae

Chitinase genes from Aeromonas hydrophila and Bacillus circulans No.4.1 were cloned into the plasmid pHY300PLK and designated as pHYA2 and pHYB43, respectively. Both plasmids were introduced into various strains of B. thuringiensis by electroporation. Plasmid pHYB43 was generally structurally stable, but showed lower segregrational stability than pHYA2 in B. thuringiensis subsp. aizawai when grown under nonselective conditions. The production of chitinase from B. thuringiensis subsp. aizawai harboring pHYB43 or pHYA2 could be detected after native polyacrylamide gel electrophoresis by using 4-methylumbelliferyl -D-N,N- diacetylchitobioside as the substrate. Moreover, B. thuringiensis subsp. aizawai harboring pHYB43 gave 15 times higher chitinase activity than when harboring pHYA2, as determined by means of a colorimetric method using glycol chitin as the substrate. In addition, B. thuringiensis subsp. aizawai harboring pHYB43 was more toxic to gypsy moth larvae (Lymantria dispar) than parental B. thuringiensis subsp. aizawai or its clone harboring pHYA2.