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Genetics and Molecular Biology

Print version ISSN 1415-4757On-line version ISSN 1678-4685

Genet. Mol. Biol. vol.25 no.2 São Paulo  2002 

Identification of new isolates of Bacillus thuringiensis using rep-PCR products and d-endotoxin electron microscopy


A.S.G. Lima1, A.M.Guidelli2, I.L. Abreu2 and M.V.F. Lemos2
1Departamento de Genética, USP, Campus de Ribeirão Preto.
2Departamento de Biologia Aplicada à Agropecuária, UNESP, Jaboticabal, SP, Brazil.
Send correspondence to Manoel Victor Franco Lemos. Departamento de Biologia Aplicada à Agropecuária, UNESP, Campus de Jaboticabal, 14884-900 Jaboticabal, SP, Brazil. E-mail: mvictor@




PCR has been used to analyze the distribution of REP (Repetitive Extragenic Palindromic) and ERIC (Enterobacterial Repetitive Intergenic Consensus) sequences (rep-PCR) found within the genome of the bacterium Bacillus thuringiensis, with the purpose to analyze the genetic similarities among 56 subspecies samples and 95 field isolates. The PCR products were analyzed by EB-AGE (ethidium bromide-agarose electrophoresis) and then submitted to banding comparisons, based on the Phyllip software algorithm. When the banding similarities were considered for comparison purposes among all the strains, the phylogenic tree patterns varied according to the rep-PCR primers considered, but, from a broader point of view, the ERIC sequences produced better results, which, together with electron microscopy analysis of the released parasporal bodies and colony morphology characteristics, allowed to detect two possible new subspecies of B. thuringiensis.

Key words: REP, ERIC, crystal protein, parasporal bodies.

Received: April 19, 2002; accepted: May 27, 2002.




Many organisms of the Lepidoptera, Diptera, Coleoptera and Nematoda orders still cause frequent and serious problems to the production of a variety of important crops. Chemical control based on organochlorated compounds, among others, has shown little efficiency due to several factors: the rising of resistant or tolerant organisms; environmental contamination problems, and international restriction of use (Van Rie et al. 1990a,b; Ferré et al. 1991). Due to insect resistance, environmental pollution, and other events, it became a general issue to look for alternative ways of control, such as the use of bioinsecticides, which act based on specific control mechanisms of the major crop pests, without causing biological impact problems similar to those generated by the use of chemical insecticides.

Bacillus thuringiensis is seen as a biological control bacterium that presents several advantages over the use of chemical control agents, since the parasporal bodies released by such bacteria during their growth are highly specific for some of the major agronomical targets (entomopathogenic action) and with no effect on other non-target insects, plants and domestic animals. These entomopathogenic proteins named d-endotoxins, are produced as protoxins that can be solubilized and activated by the action of alkaline proteases along the sporulation events (Ferré et al. 1991). The activated toxin molecules are found linked to specific sites located on the apical microvilosities of susceptible larvae intestinal cells (Van Rie et al. 1960a,b). After the toxin induction, its molecules are inserted within the cell’s plasmatic membrane and by doing so they punch holes in the cell surface, changing the osmotic equilibrium and determining the cell’s death (Van Rie et al., 1989).

It is important to look for new B. thuringiensis strains, and great efforts are presently being undertaken by many different research centers. One of the most recent advances in this area is the use of PCR for such purposes (Bravo et al. 1992). This technique has been used for strain identification and for target prevision, without the need to carry out all the tedious and time-consuming bioassays which are, thus allowing faster detection of new Cry protein coding genes (Carozzi et al. 1991). The use of repetitive DNA sequences such as REP and ERIC, also referred to as rep-PCR, for bacterial classification is becoming frequent, and has allowed comparisons of possible genetic similarities among different bacterial genomes (Versalovic et al. 1991; Louws et al. 1994; Selenska-Pobell et al. 1995).

Since it became important to overcome the growing need to avoid resistance by the insect pests, it was the objective of the present work to analyze 56 subspecies samples and 95 field isolates of B. thuringiensis using rep-PCR to compare and identify probable new genes coding for Cry toxins.



Bacterial strains

The 56 B. thuringiensis subspecies used in this work were obtained from the BACILLUS STOCK CENTER of the Ohio State University, Columbus, USA. The 95 field isolates were obtained from various locations throughout the Brazilian territory (States of Alagoas, Amazônia, Bahia, Ceará, Goiás, Minas Gerais, Mato Grosso, Mato Grosso do Sul, Pará, Pernambuco, Piauí, Paraná, Rio Grande do Norte, Roraima, Rio Grande do Sul, Sergipe, São Paulo). All bacterial samples are being kept at the Laboratory of Bacterial Genetics of the Department of Applied Biology of UNESP, Campus of Jaboticabal, Brazil.

Bacterial growth, genomic DNA extraction and PCR conditions

Both the subspecies and the field isolates were grown on Petri dishes containing Difco Nutrient Agar, at 30 °C during 12-18 h, before harvesting for experimental purposes. DNA was extracted using Bio-Rad Instagene Matrix (Bio-Rad), following the procedures recommended by the manufacturer.

The amplification reactions were conducted as follows: 0.8 mL Taq-polymerase (4U), 0.8 mL MgCl2 (1 mM), 0.5 mL of dNTPs (200 mM) (Amersham Pharmacia Biotech), 2.0 mL 10X buffer (KCl 500 mM, MgCl2 15 mM, and Tris-HCl 100 mM, pH 9.0), 0.8 mL gelatin (0.001%), 3.0 mL REP primers (30 hg) or 2.5 mL ERIC primers (25 hg) (GIBCO/BRL Custom Primers), 6.0 mL genomic DNA (20 hg), and Milli Q purified water to complete 20 mL of reaction mixture.

The primer sequences (DE-Bruijn, 1992) were the following:

• REP1 R-I (5’ - I I I I C g I C g I C A T C I g g C - 3’)

• REP2 I (5’ - I C g I C T T A T C I g g C C T A C - 3’)

• ERIC1 R (5’ - A T g T A A g C T C C T g g g g A T T C A C - 3’)

• ERIC2 (5’ - A A g T A A g T g A C T g g g g T g A g C g - 3’)

The amplifications were carried out in a MJ Res model PTC 100 thermocycler, using the following program for REP primers: 1 min at 94 °C, 1 min at 45 °C, and 2 min at 72 °C, 41 cycles with a final temperature of 20 °C. For the ERIC primers, the program was: 1 min at 94 °C, 1 min at 50 °C, and 2 min at 72 °C, 41 cycles with a final temperature of 20 °C.

Analysis of PCR products

2 mL of loading dye were added to the amplified samples, and 11 mL of each sample were submitted to EB-AGE (1.5% gel) (Sambrook et al. 1989). Electrophoresis was performed in HORIZON 58 and 11-14 chambers (GIBCO/BRL), using TEB 1X as running buffer (Tris-HCl 890 mM, EDTA, 25 mM, and H3BO3 890 mM, pH 8.2), at 72 V during 2-3 h. The amplification products were analyzed under UV transillumination, and filed using a Bio-Rad GEL-DOC 2000 photodocumentor device.

Phylogenetic analysis

The amplification products were transformed into binary bidimentional matrixes and submitted to the Phylip software algorithm (Phylogeny Inference Package, release 3.6a2.1;

Identification of parasporal bodies in the field isolates

After a long incubation period on Nutrient Agar plates, all the 95 field isolates were examined under optical microscopy (1,000X), for parasporal body morphology analysis (Smirnoff, 1962). The parasporal bodies produced by field isolates PI2 and SP6 were purified, lyophilized, observed under electron microscopy (JEOL Scan Electron Microscope of the UNESP/Jaboticabal Electron Microscopy laboratory) and photographed using ILFORD FP4-125 black-and-white film, processed according to the manufacturer’s instructions. Afterwards, the parasporal bodies were grouped according to their morphology.



REP primers

Figure 1A shows the amplified material from the field isolates, when REP primers were used. Based on an overall view, most of the isolates seemed to show a common amplification pattern, with only two samples not sharing this homology. These samples correspond to the field isolates PI2 and SP6, which exhibited different amplification patterns. With regard to the B. thuringiensis subspecies, it was observed that most of the amplification products of the fifty-six samples used in this work had the same patterns as those of the field isolates. For a group of fourteen subspecies strains, however, it was not possible to detect any amplification product (data not shown).



Figure 1B shows a comparative analysis based on PCR-amplified fragment bands observed on the electropherograms of the 56 subspecies, compared to the 95 field isolates; the amplification products were obtained using primers REP1-R and REP2. It can be seen that two clearly separated groups of clustered subspecies and field isolate samples were formed; only a very small amount of cross-linking was observed (comparisons made using Phylip software).



ERIC primers

Figure 2A shows that, when ERIC primers were used, the banding patterns of most of the field isolates were not as similar as when this comparison was made with the amplification products obtained with REP primers, but the differences between the PI2 and SP6 field isolates and the fifty-six B. thuringiensis subspecies were kept.



Figure 2B shows a comparative analysis based on PCR-amplified fragment bands observed on the electropherograms of the 56 subspecies, compared to the 95 field isolates; the amplification products were obtained using ERIC1-R and ERIC2 primers. It also can be seen that two clearly separated groups of clustered subspecies and field isolates samples were formed, only a greater amount of cross-linking was found (comparisons made using Phylip software).



Using these two kinds of conserved bacterial sequences for phylogenetic comparisons according to Versalovic et al. (1994), Louws et al. (1994) and Selenska-Pobell et al. (1995), this work attempted to find out if some information regarding the type of d-endotoxin produced by the isolated field samples could be pinpointed using the PCR-amplified material, after the reaction with these types of repetitive sequences. Indeed, a certain degree of cross-linking between the subspecies and the isolated field samples was detected, whether REP or ERIC primers were used, and the field isolates PI2 and SP6 were part of the cross-linked samples, but with different banding patterns. It must be said, however, that from a phylogenetic point of view, the ERIC primers produced a phylogram (Figure 2B) with better spreading, generating a tree that shows more strain differences. This result might be due to the fact that, the ERIC primers being longer, their chances to anneal to their target sequences are greater. Since neither the REP nor the ERIC repetitive sequences have any relation to the types of d-endotoxin produced by these bacterial strains, we decided to evaluate their types of parasporal bodies, analyzing their shapes and comparing them with the described types.

Colony and parasporal body morphology

The colony and parasporal body morphology analysis revealed that the field isolates were made of general smooth colonies with an irregular shape, except for isolate PI2 that produced dark yellowish colonies.

When crystal morphology was considered and compared between the subspecies strains and the field isolates, almost all of them were found to produce similar bipiramidal crystals, as did all the subspecies in which the CryI proteins are expressed, such as kurstaki-HD73, aizawai and tolworthi; the exceptions were isolates PI2 and SP6. For these two field isolates (PI2 and SP6), the crystal shapes were determined by scanning electron microscopy, after special crystal purification by an ultracentrifugation procedure (Fast, 1972), and can be seen in Figures 3 and 4. In isolate SP6, the common bipiramidal shape was detected, but in PI2 the crystals were amorphous. As far as shape is concerned, the bipiramidal crystals are related to lepidopteran targets, but without a pathogenesis level description. In field isolate PI2, the size of the parasporal bodies (0.4 to 0.5 mm) was considered quite small.




Based on the above mentioned differences among the bacterial samples analyzed in this study, we propose that field isolates PI2 and SP6 might correspond to new B. thuringiensis strains. Further studies of their d-endotoxin specificity and mode of action need to be carried out in the future.



The authors would like to thank Dr. Eliana G.M. Lemos for her assistance with critical manuscript reading, and CAPES which supported ASGL with a scholarship.



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