Letters in Applied Microbiology 1990,10, 141-143

DNA fingerprinting of the mosquito pathogen Bacihs sphaericus with M13 DNA as a probe A.N. ABADJIEVA, R.T. G R I G O R O V &AV.I. M I T E V AInstitute of Microbiology, Bulgarian Academy of Sciences, S o f a 1113, Bulgaria Received 2 November 1989 and accepted 3 November 1989

A B A D J I E V A A.N., , GRIGOROVA, R.T. & M I T E V A ,V.I. 1990. DNA fingerprinting of the mosquito pathogen Bacillus sphaericus with M13 DNA as a probe. Letters in Applied Microbiology 10, 141-143. Hypervariable nucleotide sequences were detected in Bacillus sphaericus by hybridization with radioactively labelled M 13 DNA. Different serotypes could be distinguished by their hybridization profiles. The appearance of bands common for mosquito-pathogenic strains and their absence in an apathogenic strain opens the probability that M13 could hybridize to specific alleles, related to insect toxicity.

The Bacillus sphaericus group encompasses a large number of strains, pathogenic or not for mosquitoes, and often only slightly related. As the antimosquito potency of this microorganism is far from well known and new strains are still being discovered, it is important to develop reliable methods for the classification of this group. Five approaches have been used for distinguishing between strains of B. sphaericusthe routine phenotypic tests (Baumann et al. 1984), serological classification (de Barjac et al. 1985), phage typing (Yousten et al. 1980), DNA homology (Krych et al. 1980) and the zymogram technique (Singer 1987). A new highly efficient method for individual identification called DNA fingerprinting was developed by Jeffreys et al. (1985). Later it was discovered that DNA from the bacteriophage M13 under appropriate conditions could be used as a probe for this purpose (Jincharadze et al. 1987; Vassart et al. 1987). Hypervariable nucleotide sequences have been found in many eucaryotic organisms and in some Gramnegative procaryotes (Ryskov et al. 1988). In this study DNA fingerprinting with M13 DNA was used for identification of B. sphaericus strains.

Materials and Methods The B. sphaericus strains studied are listed in Table 1. They were grown in nutrient broth (Difco) at 30°C. Total DNA was prepared according to Hintermann et al. (1981). Between 20 and 30 U of each restriction enzyme (Boehringer Gmb) were used to digest 10 pg of DNA for 18 h. After electrophoresis in 1% agarose gel (Sigma, Type II), denatured DNAs fragments were blotted to nitrocellulose filters (LKB). Single stranded DNA of M13mp8 was labelled with 32P to a specific activity of 1.0 x lo9 cpm/pg DNA using Amersham sequencing primer and kit. Hybridization was performed in 5 x SSC, 5 x Denhardt’s solution, 0.1% SDS, 5 mM EDTA at 57°C for 16-20 h. The filters were washed in 2 x SSC, 0.1% SDS at 57°C. Autoradiograms were exposed at -70°C with an intensifying screen.

Results and Discussion Three known strains and three local isolates, previously studied for toxicity and characterized by routine biochemical tests, phage typing and

A . N . Abadjieva et al.


Table 1. Bacterial strains Strain

Pathogenic activity


Phage group



5a, 5b* 5a, 5bt 5a, 5bt 5a, 5bt ND

3 3 3 3 ND

Origin ~~

WHO 2297 WHO 2362 Local 089 Local 099 Local 03 CCEB 640

+ + + + + -

Obtained from H. de Barjac, Inst. Pasteur, IP 25-1 IP 5-10 Grigorova (Bulgaria) Grigorova (Bulgaria) Grigorova (Bulgaria) Obtained from 0. Lysenco

* de Barjac et al. (1985).

t de Barjac (personal communication) ND, not determined. serologically (Grigorova et al. 1988) were investigated. The hybridization protocol of Ryskov et al. (1988) was followed but less stringent conditions had to be used for revealing of polymorphic fingerprint profiles in B. sphaericus. Figure l(a) illustrates that the chromosomal DNAs of the reference strains tested after total EcoRI digestion exhibit different hybridization patterns. It deserves noting that the bands in the genome of B. sphaericus 2297 were more intensive, possibly connected with the presence of tandem repeats. The M13 probe readily distinguished between 2291 and 2362 reported to belong to different phage groups and serotypes (de Barjac et aE. 1985). The presence of regions of homology with M13 DNA in B. sphaericus encouraged us to

examine our local isolates and to compare them with the reference strains. The H i n d l l l digests of DNAs of the pathogenic strains 089 and 099 gave very similar or identical hybridization patterns to the reference strain 2362 (Fig. lb). As the number of characteristic bands was small we tried, and were successful in obtaining a variety of DNA fragments by the use of Poull and P s t l . Both endonucleases provided a wider band spectrum (Fig. l(c)). Perhaps the most interesting observation was the lack of noticeable differences in the profiles of our local strain 03 and B. sphaericus 2297. The serotyping of the local isolates was kindly performed by H. de Barjac and there was some uncertainty about the strain 03, which was finely classified into serotype 5a, 5b as the strain

Fig. 1. Hypervariable polymorphic patterns of DNAs from Bacillus sphaericus, digested with different restriction enzymes: (a) EcoRI: Lane 1,2297; lane 2,2362; lane 3, 640. (b) H i n d l l l : Lane 1,099; lane 2,089; lane 3,03; lane 4,2362; lane 5,2297; lane 6,640. (c) Poull: Lane 1,099; lane 2,2362.

M13 D N A Jingerprinting of B. sphaericus 2362. In contrast the results presented here show similarity with strain 2297, belonging to another serotype-25. The apathogenic strain 640 differed significantly from all the other strains studied. Although the detailed analysis of B. sphaericus species is far from complete, the appearance of visible bands in the region of 510 kb, common for the mosquito-pathogenic strains and their absence in the apathogenic one opens the probability that the M13 probe could hybridize to specific alleles related to insect toxicity. The present results show that the DNA fingerprinting with MI3 DNA used as a probe allows one to distinguish between serotypes and phage groups as well as between the pathogenic and apathogenic strains of B. sphaericus. The sensitivity and the potentiality of the method will be clarified in the future but it promises to become an efficient tool in bacterial taxonomy and biotechnology. References BAUMANN, M., OKAMOTO, K., UNTEKMAN, B.M., LYNCH,M.J. & BAUMANN,P. 1984 Phenotypic characterization of Bacillus thuringiensis and Bacillus sphaericus. Journal of Invertebrate Pathology 44, 329-341. DE BARJAC,H., LARGET-THIERY, I., COSMAO DUMAH. 1985 Serological classiNOIR, v. & RIPOUTEAU, fication of Bacillus sphaericus strains on the basis of toxicity to mosquito larvae. Applied Microbiology and Biotechnology 21, 85-90.


GRIGOROVA, R., CHRISTOVA, T., MITEVA,V.. PENEVA, N., TAKOVA, Tz. & ATANASOV,B. 1988 Characterization of isolated local strains of Bacillus sphaericus toxic to mosquitoes. Acta Microhiologica Bulgarica 22, 87-93. HINTERMANN, G., CRAMEKI, R., KIESER,T. & HUTTER, R. 1981 Restriction analysis of the Streptomyces glaucescens genome by agarose gel electrophoresis. Archives of Microbiology 130,218-222. JEFFREYS,A.J., WILSON,V. & THEIN,S.L. 1985 Hypervariable ‘minisatellite’ regions in human DNA. Nature 314,67-73. JINCHARADZE, A.G., IVANOV, P.L. & RYSKOV,A.P. 1987 DNA fingerprinting. Characteristics of cloned sequences of the human genome with M13 as a probe. Dokladi Academii Nauk U S S R 295,23&233. KRYCH,V.K., JOHNSON,J.L. & YOUSTEN, A.A. 1980 Deoxyribonucleic acid homologies among strains of Bacillus sphaericus. International Journal of Systematic Bacteriology 30, 476-484. RYSKOV, A.P., TOKARSKAYA, O.N., VERBOUVAYA,L.V., JINCHARADZE, A.G., GINSBURG, A.L., ZGANGIKOVA, G.B. 1988 DNA finN.A., SHUBIN, F.N. & SMIRNOV, gerprinting in microorganisms the use of M13 DNA as a hybridization probe. Genetica 24, 1310-1313. SINGER, S. 1987 Current status of the microbial larvicide Bacillus sphaericus. In Biotechnology in lnvertebrate Pathology and Cell Culture ed. Maramorosch, K. New York, Academic Press. VASSART, G., GEORGE,M., MONSINEUR, R., BROCAS, D. 1987 A H., LEQUARRE, AS. & CHRISTOPHE, sequence in M13 phage detects hypervariable minisatellites in human and animal DNA. Science 235, 683-684. YOUSTEN, A.A., DE BARJAC, H., HEDRICH, J., COSMAODUMANOIR, V. & MYERS,P. 1980 Comparison between bacteriophage typing and serotyping for the differentiation of Bacillus sphaericus strains. Annales de Microbiologie (Institute Pasteur) 131B, 297-308.

DNA fingerprinting of the mosquito pathogen Bacillus sphaericus with M13 DNA as a probe.

Hypervariable nucleotide sequences were detected in Bacillus sphaericus by hybridization with radioactively labelled M13 DNA. Different serotypes coul...
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