Enterotoxigenicity and cytotoxicity of Bacillus thuringiensis strains and development of a process for Cry1Ac production.
Bacillus thuringiensis is indistinguishable from Bacillus cereus except for the production of insecticidal crystal proteins (ICPs). B. thuringiensis strains may show enterotoxin profiles and toxin levels similar to those of B. cereus strains isolated from food-poisoning cases. It is important for the food industry and farmers to consider that with the application of B. thuringiensis strains to crops, their spores may be introduced into the human food chain. In this study, 59 B. thuringiensis strains were assayed for their hemolysin BL (HBL) using a BCET-RPLA kit and their cytotoxicity to Chinese hamster ovary (CHO) cells. The enterotoxin titer was as high as that of B. cereus diarrheal-type strain ATCC 49064. In an attempt to obtain a food safety strain for bioinsecticide use, in this study, a 3.5-kb cry1Ac DNA fragment was amplified with PCR from the total DNA of B. thuringiensis subsp. kurstaki CCRC 11502 and cloned into the Bacillus expression vector pHY300PLK. The alpha-amylase promoter, amyE, was then introduced into the promoter region and, afterward, the recombinant plasmid pHYe1Ac35 was introduced into a non-enterotoxigenic and non-cytotoxic B. thuringiensis subsp. kurstaki Tt14 strain. The transformant, without any detectable enterotoxigenicity or cytotoxicity, produced Cry1Ac toxin properly, and its insecticidal activity against Trichoplusia ni larvae was found to be satisfactory.
Yang
Chi-Yea
CY
Department of Food Science, National Chung-Hsing University, Taichung, Taiwan.
Pang
Jen-Chieh
JC
Kao
Suey-Sheng
SS
Tsen
Hau-Yang
HY
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001427:Bacterial Toxins; D016466:CHO Cells / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D006224:Cricetinae; D004731:Endotoxins / Q000096:biosynthesis* / Q000235:genetics; D004768:Enterotoxins / Q000096:biosynthesis; D015870:Gene Expression; D005822:Genetic Vectors; D006460:Hemolysin Proteins; D007306:Insecticides; D010572:Pest Control, Biological; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins; D000516:alpha-Amylases / Q000235:genetics
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D004768:Enterotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D000516:alpha-Amylases