MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 92(1) Jan/Feb 1997
PAGES: 107-108 DOI: Technical notes
Bacillus sphaericus Entomocidal Potential Determined by Polymerase Chain Reaction

Koko Otsuki, Thania V Guaycurús*, Ana Carolina P Vicente +

Departamento de Genética,
*Departamento de Bacteriologia, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil

RESEARCH NOTE

The mosquitocidal activity of Bacillus sphaericus toxic strains is related to the capacity of some strains to produce a binary toxin composed of 51 and 42 kDa peptides which in combination form a crystal (P Baumann et al. 1987 J Bacteriol 169: 4061-4067). Some toxic strains also produce the 100 kDa toxin encoded by the mtx gene. This gene was first cloned from the weakly entomocidal strain SSII-1 (T Thanabalu et al. 1991 J Bacteriol 173: 2776-2785). This toxin shows regional homology to ADP-ribosyltransferase-type toxins and seems to be membrane located (T Thanabalu et al. 1993 J Bacteriol 175: 2314-2320). The well known, highly toxic strains harbour the 42, 51 and 100 kDa genes.

The detection of binary toxin is often done by phase contrast microscopy through the presence of crystalline inclusions in a culture grown between 24 to 48 hr. The presence of 100 kDa toxin can be determined by the cytotoxic effect (Thanabalu et al. 1993 loc. cit.) or by dot blot hybridization (L Liu et al. 1993 Appl Environ Microbiol 59: 3470-3473). In this paper, we present polymerase chain reaction (PCR) analysis to determine in a rapid and accurate way the presence of the genes related to the B. sphaericus entomocidal potential.

B. sphaericus 2362, 1593, 2297 and SSII-1 were from Culture Collection of Genus Bacillus-CCGB (TV Guaycurús & L Rabinovitch 1992 Catalog of Culture Collection Genus Bacillus, p. 89-92, Instituto Oswaldo Cruz). B. sphaericus isolated from different Brazilian soils were identified by morphological characteristics, i.e., rod-shaped cells with round, terminal spores.

The primers used in the PCR were derived from the sequence of the 51 and 42 kDa peptide genes (L Baumann et al. 1988 J Bacteriol 170: 2045-2050) and100 kDa toxin mtx gene (Thanabalu et al. 1991 loc. cit.). For the binary toxin genes we designed two sets of primers used for the nested PCR reaction. Sequences and positions are listed in Table. A loopful of cells from a B. sphaericus colony on an overnight nutrient yeast salt mineral (NYSM) agar plate (AA Yousten & EW Davidson 1982 App Env Microbiol44: 1449-1455) was transferred to a tube with 500 ml destilled water. It was boiled for 10 min, frozen and thawed twice. To a total volume of 50 ml containing 1.25 U of Taq DNA polymerase and 3mM MgCl2, was added 5ml of DNA suspension and 20 pmol of each primer. The mixtures were subjected to 30 temperature cycles (94oC 30 sec, 55oC 30 sec and 72oC 1 min) on a programmable heating block. After the first PCR with the BSN1/BSN2 and BSN3/BSN4 pairs of primers was acomplished, 1 ml from these reactions was used as template for the nested PCR stage under the same first round conditions.

Knownhighly toxic B. sphaericus strains 2362, 2297, 1593 and the low toxic SSII-1 strain were used for the PCR conditions. Using both sets of primers of binary toxin genes we got the specific size band, as expected, based on the sequence gene data. Primers BS1/BS2 gave a 523 bp fragment and BSN1/BSN2 a 1053 bp fragment, both corresponding to 51.4 kDa toxin gene. Primers BS3/BS4 and BSN3/BSN4 amplified respectively a 478 and 720 bp fragments both from 41.9 kDa toxin gene. The set of primers 100.1/100.2 are homologus to mtx gene with a 700 bp PCR product (Fig.). The PCR was positive for all pairs of primers using DNA from 2362, 2297 and 1593. In the SSII-1 strain only 100 kDa toxin gene was detected. Dot blots were carried out confirming the PCR results (data not shown). For improving the sensitivity and specificity of the binary gene detection, the nested PCR was applied. In some cases there were either no detectable amplified products or no specific products, after the first round. However, after the second round using inner primers, a clear DNA band with the expected size was then amplified. The nested PCR makes also the verification of amplified products by hybridization with inner probes or by restriction enzyme analysis unnecessary.

In some preliminary experiments we checked out this procedure with 12 newly isolated strains of B. sphaericus from Brazilian soil. All samples produced the specific PCR products to the three target genes. The PCR analysis was shown to be a reliable method for a prescreen of B. sphaericus isolates which should then be tested in a subsequent biological assay.

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