Recombinant Enterobacter amnigenus Highly Toxic to Anopheles dirus Mosquito Larvae

The mosquito-larvicidal binary toxin of Bacillus sphaericus 2297 was expressed in Enterobacter amnigenus, a Gram-negative bacterium isolated from Anopheles dirus larvae gut. The toxin was placed under the regulation of various promoters in order to improve the expression level of the toxin. Amongst the recombinants obtained, E. amnigenus harboring pBS373, a plasmid which contains the toxin genes under the control of the native B. sphaericus promoter, expressed a significant amount of protein, comparable to that found in B. sphaericus 2297. In addition, this recombinant provided approximately twenty times higher toxicity against second-instar Anopheles dirus larvae when compared to B. sphaericus 2297. The procedure of obtaining this environmentally isolated bacterium from larvae gut and introducing the system for mosquito-larvicidal toxin synthesis is noteworthy. The promising result presented here provides a substantial degree of confidence for further field studies.     

Co-expression of Bacillus thuringiensis Cry4Ba and Cyt2Aa2 in Escherichia coli revealed high synergism against Aedes aegypti and Culex quinquefasciatus larvae

Cry4Ba is a delta-endotoxin produced by Bacillus thuringiensis subsp. israelensis and Cyt2Aa2 is a cytolytic delta-endotoxin produced by B. thuringiensis subsp. darmstadiensis. Cry4Ba produced in Escherichia coli was toxic to Aedes aegypti larvae (LC(50)=140 ng ml(-1)) but virtually inactive to Culex quinquefasciatus larvae. Cyt2Aa2 expressed in E. coli exhibited moderate activity against A. aegypti and C. quinquefasciatus larvae with LC(50) values of 350 and 250 ng ml(-1), respectively. Co-expression of both toxins in E. coli dramatically increased toxicity to both A. aegypti andC. quinquefasciatus larvae (LC(50)=7 and 20 ng ml(-1), respectively). This is the first report to demonstrate that Cry4Ba and Cyt2Aa2 have high synergistic activity against C. quinquefasciatus larvae.     

Biosynthesis of 130-kilodalton mosquito larvicide in the cyanobacterium Agmenellum quadruplicatum PR-6.

The 130-kilodalton mosquito larvicidal gene, cloned from Bacillus thuringiensis var. israelensis, was introduced into the cyanobacterium Agmenellum quadruplicatum PR-6 by plasmid transformation. Transformed cells synthesized 130-kilodalton delta-endotoxin protein and showed mosquito larvicidal activity. Results demonstrate a potential use of a cyanobacterium for biological control of mosquitoes.     

Specific mutations within the α4–α5 loop of the bacillus thuringiensis Cry4B toxin reveal a crucial role for Asn-166 and Tyr-170

The widely accepted model for toxicity mechanisms of the Bacillus thuringiensis Cry δ-endotoxins suggests that helices α4 and α5 form a helix-loop-helix hairpin structure to initiate membrane insertion and pore formation. In this report, alanine substitutions of two polar amino acids (Asn-166 and Tyr-170) and one charged residue (Glu-171) within the α4–α5 loop of the 130-kDa Cry4B mosquito-larvicidal protein were initially made via polymerase chain reaction-based directed mutagenesis. As with the wild-type toxin, all of the mutant proteins were highly expressed in Escherichia coli as inclusion bodies upon isopropyl-β-d-thiogalactopyranoside induction. When E. coli cells expressing each mutant toxin were assayed against Aedes aegypti mosquito larvae, the activity was almost completely abolished for N166A and Y170A mutations, whereas E171A showed only a small reduction in toxicity. Further analysis of these two critical residues by induction of specific mutations revealed that polarity at position 166 and highly conserved aromaticity at position 170 within the α4–α5 loop play a crucial role in the larvicidal activity of the Cry4B toxin.     

Cloning and Characterization of a Cytolytic and Mosquito Larvicidal δ-Endotoxin from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">darmstadiensis</Emphasis>

The cytolytic δ-endotoxin gene from Bacillus thuringiensis subsp. darmstadiensis was amplified from genomic DNA by PCR by using primers designed from the sequence of cyt2Aa1 gene of B. thuringiensis subsp. kyushuensis. DNA sequence analysis revealed an open reading frame translating to a 259-amino acid sequence. The cloned gene was designated cyt2Aa2. This gene was highly expressed in Escherichia coli as inclusion bodies that could be solubilized in 50 mM Na₂CO₃, pH 10.5. Activation of the solubilized protoxin by proteinase K (1% wt/wt, proteinase K/protoxin) yielded the active fragment of about 23 kDa. Cyt2Aa2 showed high hemolytic activity against sheep erythrocytes (hemolytic end- point 0.25 μg/ml) and was toxic to Culex quinquefasciatus and Aedes aegypti larvae (LC₅₀ 0.5–1.0 μg/ml). Received: 27 March 2002 / Accepted: 30 April 2002     

Production of a biologically active growth hormone from giant catfish (Pangasianodon gigas) in Escherichia coli

Giant catfish growth hormone (gcGH) cDNA was cloned and expressed in E. coli. The expected 20.5 kDa protein corresponded to the mature gcGH and was efficiently expressed. This protein was produced as inclusion bodies and comprised about 20% of total cellular proteins. The recombinant hormone promoted growth when injected intramuscularly or intraperitoneally into goldfish (Carassius auratus) at 0.1 or 1 microg soluble gcGH per g fish body wt per week. In addition, the recombinant gcGH inclusions had growth-promoting activity similar to that of the soluble form when the fish was received either by intraperitoneal injection or by oral administration.     

A Tudor staphylococcal nuclease from Penaeus monodon: cDNA cloning and its involvement in RNA interference

RNA interference (RNAi) plays an important role in an antiviral defense in shrimp. RNAi technology has been extensively used for inhibition of viral replication and studying gene function. However, the mechanism of shrimp RNAi pathway is still poorly understood. In this study, we identified and characterized an additional protein in the RNAi pathway, Tudor staphylococcal nuclease from Penaeus monodon (PmTSN). The full-length cDNA of PmTSN is 2897 bp, with an open reading frame encoding a putative protein of 889 amino acids. Phylogenetic analysis and domain structure comparison revealed that PmTSN is more closely related to vertebrate TSN by sharing the amino acid sequence identity of 57% with TSN of zebrafish. This represents a new type of TSN proteins by exhibiting the four tandem repeat of staphylococcal nuclease-like domain (SN), followed by a Tudor and a partially truncated C-terminal SN domain. Knockdown of PmTSN by dsRNA targeting SN3 domain resulted in the impairment of dsRNA targeting PmRab7 gene to silence PmRab7 expression. In addition, the efficiency of dsRNA targeting YHV-protease gene inhibiting yellow head virus replication was decreased in the PmTSN-knockdown shrimps. Our results imply that PmTSN is involved in dsRNA-mediated gene silencing in shrimp and thus we identified the additional protein involved in shrimp RNAi pathway.