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Detection of the mosquitocidal toxin genes encoding Cry11 proteins from Bacillus thuringiensis using a novel PCR-RFLP method

D. H. Sauka^*, R. H. Monella, G. B. Benintende

Área Bioinsumos Microbianos, Instituto de Microbiología y Zoología Agrícola (IMYZA), Instituto Nacional de Tecnología Agropecuaria (INTA). De los Reseros y Las Cabañas s/nro. (1712) Castelar, Buenos Aires, Argentina
* Correspondence: E-mail: dsauka@cnia.inta.gov.ar

ABSTRACT

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for detection of cry11 genes from Bacillus thuringiensis was established. Based on the analysis of conserved regions of the cry11 genes, 2 oligonucleotide primers were designed to amplify a 1459-bp fragment of the cry11Aa gene, and a 1471-bp of the cry11Ba and cry11Bb genes. The amplification products were digested with restriction endonuclease HinfI. Exotic B. thuringiensis strains and native isolates collected from soils, leaves and stored product dust of Argentina were analyzed to study the distribution of cry11 genes. The PCR-RFLP patterns revealed the detection of cry11 genes in 3 of 64 exotic strains and in 10 of 107 native B. thuringiensis isolates tested. Just the cry11Aa gene subclass was detected among these bacteria. Since the methodology was also developed to detect cry11Ba and cry11Bb genes, an experimental future confirmation will be required. Based on the results obtained, the PCR-RFLP method presented may be a valuable tool for specific detection of the mosquitocidal toxin genes encoding Cry11 proteins from B. thuringiensis.

Key words: Bacillus thuringiensis; Cry11; PCR-RFLP

RESUMEN

Detección de genes que codifican proteínas mosquitocidas Cry11 de Bacillus thuringiensis mediante un método de PCR-RFLP novedoso. En el presente estudio se estableció una estrategia basada en la amplificación génica (PCR) y el posterior análisis de restricción (RFLP) para detectar todos los genes cry11 de Bacillus thuringiensis informados hasta ahora. De acuerdo con el análisis de las regiones conservadas en los genes cry11, se diseñaron dos cebadores para amplificar un fragmento de 1459 pb de los genes cry11Aa y un fragmento de 1471 pb de los genes cry11Ba y cry11Bb. Los productos de la amplificación fueron digeridos con la enzima de restricción HinfI. Se analizaron cepas exóticas de B. thuringiensis y aislamientos nativos de Argentina obtenidos a partir de muestras de suelos, hojas y polvillo de silos, para estudiar la distribución de los genes cry11. Los patrones de PCR-RFLP revelaron la presencia de genes cry11 en 3 de las 64 cepas exóticas y en 10 de los 107 aislamientos nativos de B. thuringiensis ensayados. Sólo se detectó la subclase cry11Aa entre estas bacterias. Ya que esta metodología fue también desarrollada para detectar genes cry11Ba y cry11Bb, se requeriría una futura confirmación experimental. Los resultados obtenidos nos permiten inferir que el método de PCR-RFLP constituiría una herramienta valiosa para la detección específica de genes que codifican proteínas mosquitocidas Cry11 de B. thuringiensis.

Palabras clave: Bacillus thuringiensis; Cry11; PCR-RFLP

Bacillus thuringiensis is a Gram-positive bacterium characterized by its ability to produce proteinaceous crystalline inclusions during sporulation. These proteins (Cry proteins) along with the spores constitute the basis of the most widely applied biological pesticides used to manage insects that affect agriculture, forestry and which transmit human and animal pathogens (1).
Cry proteins have been classified into 56 groups divided into classes and subclasses according to their amino acid similarity (B. thuringiensis toxin nomenclature website at http://www.biols.susx.ac.uk/ home/Neil_Crickmore/Bt/) (2). Genes coding for the Cry proteins (cry genes), follow the protein classification. The cry11 genes encode 67-94 kDa proteins highly active against different species of mosquito larvae, which are vectors of tropical diseases such as yellow fever, malaria and dengue (3-5). Cry11Aa, Cry11Ba and Cry11Bb proteins are very active toxins found in the B. thuringiensis svar israelensis, jegathesan and medellin crystals respectively (3, 5, 6).
It was of interest to study the cry11 gene content of exotic and native B. thuringiensis with the objective of finding novel strains that could be used as tools for effective control and chemical resistant management of important dipteran species. Firstly, it is important to have a reliable method for cry11 gene detection. Some polymerase chain reaction (PCR)-based methods have been developed to detect cry11A genes (7-9, 11). However, to our knowledge, none of the works has described the distribution of different cry11 gene profiles of B. thuringiensis. This study aimed to establish a PCR-restriction fragment length polymorphism (RFLP) method for detecting cry11 genes from B. thuringiensis strains and native isolates.
Sixty four exotic B. thuringiensis strains were kindly provided by the United States Department of Agriculture - Agricultural Research Service (Peoria, IL), Institut Pasteur (France), Bacillus Genetic Stock Center (Columbus, OH) and the stock collection of Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (Irapuato, Mexico). One hundred seven native B. thuringiensis isolates collected from soils, leaves and stored product dust from different regions of Argentina were obtained from the bacterial collection of the Instituto de Microbiología y Zoología Agrícola-Instituto Nacional de Tecnología Agropecuaria (IMYZA-INTA) . Novel specific primers for the detection of cry11 genes were designed based on the analysis of conserved regions by multiple alignments of DNA sequences in the B. thuringiensis toxin nomenclature website using ClustalW (available at: http://www.ebi.ac.uk/clustalw/) and Oligoanalyzer 3.0 (available at: http://scitools.idtdna.com/scitools/Applications/OligoAnalyzer/). Primers used for amplification of a 1459-bp DNA fragment of cry11Aa, and a 1471-bp of cry11Ba and cry11Bb were as follows: 11F (forward; 5'-CCAGCATTAATAGCAGTAGCTCC-3') and 11R (reverse; 5'-TGCCRTCTGTTGCTTGATC-3'). The DNA templates for PCR were obtained as previously described (10). The reactions were performed with a final volume of 25 ml containing final concentrations of 50 mM KCl, 2.0 mM MgCl₂, 10 mM Tris-HCl (pH 8.3), 200 mM each deoxynucleoside triphosphate (dATP, dTTP, dGTP, and dCTP), 16 pmol each primer, and 2.5 U of Taq polymerase (Invitrogen). The PCR amplification consisted of DNA denaturation at 94 °C for 3 min followed by 25 cycles of amplification with a thermocycler (Eppendorf Mastercycler gradient). Each cycle consisted of a denaturation step at 94 °C for 1 min, an annealing step at 50 °C for 1 min, and a chain elongation step at 72 °C for 2 min. The final elongation step was extended for an additional 10 min. Finally, 10 ml PCR product was analyzed by 1.0% agarose gel electrophoresis. For the identification of different cry11 genes, 10 ml of positive PCR product was digested with HinfI (Promega) according to the manufacturer's instructions, analyzed by 10% polyacrylamide gel electrophoresis and stained with ethidium bromide. The expected restriction fragment sizes of the known cry11 genes were determined by in silico digestion of their available sequences in the B. thuringiensis toxin nomenclature website with the software ‘Restriction Mapper' (Table 1).

Table 1. Expected restriction fragment sizes of digested cry11 genes

The novel specific primers for cry11 genes produced amplification in 3 exotic B. thuringiensis strains (Fig. 1), and 10 native isolates. B. thuringiensis svar kurstaki HD-1 and HD-73, used as negative controls, failed to produce any amplification. The identification of cry11 genes was determined in these bacteria according to restriction analysis of PCR products. Just one subclass of cry11 genes was detected during this study. The cry11Aa gene was identified in the 3 B. thuringiensis strains (Fig. 1), and in the 10 native isolates too. The polyacrylamide gels showed 3 main bands of 760, 231 and 183/182 when the product was digested with HinfI. We did not find any strain or native isolate that harbored cry11Ba, cry11Bb or combinations between any one of this cry gene class. The cry11Aa gene content of B. thuringiensis strains is listed in Table 2. We just detected cry11 genes in the mosquitocidal B. thuringiensis strains that belong to svar israelensis. This finding is in agreement with the knowledge that Cry11Aa's are part of ovoid crystal inclusions typical of svar israelensis strains (11), and strongly suggests that their encoding genes are restricted to this B. thuringiensis svar.

Figure 1. PCR amplification with oligonucleotide primers 11F and 11R (A), and PCR-RFLP patterns of cry11Aa genes of B. thuringiensis svar israelensis strains (B). Lanes: 1, HD-567; 2, T14001; 3, IPS-82. MW, molecular weight marker with sizes indicated on the right (bp).

Table 2. cry11 gene content of exotic B. thuringiensis strains.

When we analyzed the cry11 content of native B. thuringiensis isolates according to the sample source, we found that the rate of cry11Aa positive isolates did not depend on it. These genes were found in 9 of 22 and in 1of 70 native isolates collected from soils and leaves respectively, all of them producers of ovoid crystal inclusions that resembled B. thuringiensis svar israelensis crystals (data not shown). We did not find any isolate collected from stored product dust harboring cry11 genes.
We herein presented the establishment of a novel PCR-RFLP method that could detect already existing cry11 genes. The method was initially developed using in silico design of PCR primers and predictions of restriction fragment sizes. The expected PCR product size and restriction fragment patterns of cry11Aa genes were experimentally confirmed in exotic B. thuringiensis strains and native to Argentina isolates. Since the methodology was also developed to detect cry11Ba and cry11Bb genes, an experimental future confirmation will be required. It might be suggested that the in silico analysis of the different cry11 genes should be corroborated in the lab, by amplifying and then digesting the respective amplicon from strains containing each type of gene. However, it should be understood that it is very difficult to get all the strains containing such poorly studied genes and sometimes protected by a patent. Besides increasing our general understanding of their distribution, these results suggest that this methodology is a valuable tool for detecting mosquitocidal toxin genes encoding Cry11 proteins from B. thuringiensis.

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Recibido: 3/03/09
Aceptado: 24/22/09