Risks for public health related to the presence of Bacillus cereus and other Bacillus spp. including Bacillus thuringiensis in foodstuffs

A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments. Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified. The results showed that cry1I-type genes appeared in 95 of 115 B. thuringiensis isolates and 7 of 13 standard strains. A novel cry1I-type gene was found in one standard strain and six isolates. The novel cry1I gene was cloned from B. thuringiensis isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in Escherichia coli BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella). However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays. Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B. thuringiensis δ-endotoxin nomenclature committee.

Identification of cry1I-Type Genes from Bacillus thuringiensis Strains and Characterization of a Novel cry1I-Type Gene

Characterization of cry1, cry2, and cry9 genes in Bacillus thuringiensis isolates from China.

Aim: To evaluate the genetic and molecular diversity and insecticidal activity of Bacillus thuringiensis isolates from all the natural regions of Colombia.



Methods and Results:  A total of 445 isolates from a collection of B. thuringiensis were characterized. The parasporal crystal morphology that was most abundant was bipyramidal (60%). Almost 10% of the isolates were toxic to Spodoptera frugiperda and 5·6% against Culex quinquefasciatus larvae. cry gene content determined by PCR indicated that 10·6% of the isolates contained cry1 genes and 1·1% contained cry2, cry4 or cry11 genes. Protein content of the parasporal crystal was determined by SDS–PAGE; 25 and 18 different protein profiles were found in isolates active against S. frugiperda and C. quinquefasciatus, respectively.



Conclusions: Bacillus thuringiensis presents great genetic and molecular diversity even in isolates from the same soil sample. Moreover, the diversity and activity of the isolates might have a relationship with the geographical origin of the samples.



Significance and Impact of the Study:  The results obtained here indicate that some of the B. thuringiensis isolates characterized in this study are potential control agents that could be used in programmes against mosquitoes and S. frugiperda.

Diversity of Colombian strains of Bacillus thuringiensis with insecticidal activity against dipteran and lepidopteran insects.

Characterization of Lepidoptera-active cry and vip genes in Iranian Bacillus thuringiensis strain collection

An extended PCR method was established to rapidly identify and classify Bacillus thuringiensis strains containing cry (crystal protein) genes toxic to lepidopteran, coleopteran, and dipteran pests (Ben-Dov et al., Appl. Environ. Microbiol. 63:4883–4890, 1997). To optimize identification of all reported cry genes, this methodology needs a complete PCR set of primers. In the study reported here, a set of universal (Un9) and specific primers for multiplex rapid screening for all four known genes from the cry9 group was designed. PCR analyses were performed for cry9 genes on 16 standard strains and 215 field isolates of B. thuringiensis. Among the standard strains, only B. thuringiensis subsp. aizawai HD-133, which harbors cry1 and cry2 genes, was positive with Un9 but negative to all four specific primers for cry9 genes. DNA of 22 field-collected isolates was also found to be positive with Un9. These isolates were classified into three cry9 profiles using specific primers; all of them harbor cry1 and cry2. This newly designed set of primers complements the existing PCR methodology for most currently known cry genes.

https://www.bgu.ac.il/life/Faculty/Zaritsky/pdf/AEM,'99PCR.pdf

Victor Glupov

Papers

Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains.