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.

Isolation and characterization of Synechococcus PCC7942 promoters: tRNApro gene functions as a promoter.

Promoter-active fragments of Synechococcus PCC7942 were isolated by transcriptional gene fusion to the promoterless beta-glucuronidase (GUS) gene of E. coli, which was used as a reporter gene. Several of the isolated promoter-active fragments expressed GUS activity in Synechococcus comparable to that of the lambdaPR promoter. Only 10% of the isolated promoter-active fragments also functioned in E. coli. The transcription initiation sites of the two promoter-active fragments, D13 and E3, were identified. The major transcription initiation sites of D13 and E3 in Synechococcus were located within the nucleotides TTTG and TTG respectively, which were identical to those corresponding to E. coli. The inferred -10 and -35 regions of D13 were TAAACT and TTGTAG respectively, which conformed to the E. coli sigma70 promoter. Immediately upstream of the E3 transcription initiation sites was the tRNApro (GGG) gene, which contained two regions exhibiting strong homology to the major promoter elements in eukaryotic tRNA genes, but did not contain the E. coli promoter element. Thus, the tRNApro gene can act as a promoter.

Efficient expression of mosquito-larvicidal proteins in a gram-negative bacterium capable of recolonization in the guts of Anopheles dirus larva.

The gram-negative bacterium, An11/2 G1, isolated from the guts of Anopheles dirus mosquito larvae, was identified as Enterobacter amnigenus. The E. amnigenus was able to recolonize in the gut of An. dirus larva but not in those of Aedes aegypti and Culex quinquefasciatus larvae. It was able to float in water for a longer period than Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus. These are desirable characteristics for a delivery vehicle of mosquito-larvicidal toxins for the control of mosquito larvae, and E. amnigenus was therefore used as a host to express the cryIVB gene of B. thuringiensis subsp. israelensis and the binary toxin genes of B. sphaericus. The recombinant E. amnigenus produced a high level of CryIVB protein, which was toxic to larvae of Ae. aegypti and An. dirus. Another E. amnigenus producing the 51-kDa protein of B. sphaericus was toxic to larvae of An. dirus and Cx. quinquefasciatus. The recombinant plasmids were stable in E. amnigenus without the presence of selective pressure for at least 23 generations. The recombinant E. amnigenus should represent a desirable biological agent for controlling mosquito larvae.

Common features of Bacillus thuringiensis toxins specific for Diptera and Lepidoptera.

The complete nucleotide sequence of a cloned gene encoding a 130-kDa crystal protein of Bacillus thuringiensis (B.t.) subspecies israelensis has been determined. The recombinant protein (Bt8) was purified and shown to be a mosquito-specific toxin with a LC50 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 cross-reaction. 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.

Cloning and expression of 130-kd mosquito-larvicidal delta-endotoxin gene of Bacillus thuringiensis var. Israelensis in Escherichia coli.

Five recombinant E. coli clones exhibiting toxicity to Aedes aegypti larvae were obtained from a library of 800 clones containing XbaI DNA fragments of 110 kb plasmid from B. thuringiensis var. israelensis. All the five clones (pMU 14/258/303/388/679) had the same 3.8-kb insert and encoded a major protein of 130 kDa which was highly toxic to A. aegypti larvae. Three clones (pMU 258/303/388) transcribed the 130 kD a gene in the same direction as that of lac Z promoter of pUC12 vector whereas the transcription of the other two (pMU 14/679) was in the opposite direction. A 1.9-kb fragment of the 3.8 kb insert coded for a protein of 65 kDa. Partial DNA sequence of the 3.8 kb insert, corresponding to the 5'-terminal of the 130 kDa gene, revealed a continuous reading frame, a Shine-Dalgarno sequence and a tentative 5'-regulatory region. These results demonstrated that the 3.8 kb insert is a minimal DNA fragment containing a regulatory region plus the coding sequence of the 130 kDa protein that is highly toxic to mosquito larvae.