Phylogenetic analysis ofBacillus thuringiensisserovars based on 16S rRNA gene restriction fragment length polymorphisms

Abstract

Aims: To determine the 16S rRNA gene ®ngerprints of Bacillus thuringiensis strains to reveal phylogenetic relationships among them. Methods and Results: Using 16S rRNA gene restriction fragment length polymorphisms generated by HindIII and EcoRI, 86 Bacillus thuringiensis strains were classi®ed. This includes 80 B. thuringiensis serovars and ®ve more strains, kurstaki HD-1, subtoxicus, dendrolimus, tenebrionis and sandiego, to assess not only interserovar DNA relatedness but also intraserovar DNA relatedness, and the non-motile strain, hence non-serotypeable, B. thuringiensis var. wuhanensis. All 86 B. thuringiensis strains tested showed distinct ribotypes. The dendrogram resulting from the numerical analysis of the distance matrix shows four distinct phylogenetic groups and two ungrouped serovars, ®nitimus and bolivia, at the 92á5% DNA relatedness rate. Conclusions: 16S rRNA gene ®ngerprinting cannot only be used for the classi®cation of B. thuringiensis strains amenable or not to serotyping, but can also reveal phylogenetic relationships between strains. Signi®cance and Impact of the Study: In future screening programmes, 16S rRNA gene restriction pattern analysis could be determined for novel B. thuringiensis strains, allowing them not only to be grouped but also to be positioned on the phylogenetic tree.

Fingerprinting of Bacillus thuringiensis Type Strains and Isolates by Using Bacillus cereus Group-Specific Repetitive Extragenic Palindromic Sequence-Based PCR Analysis

“…Other serovars that shared the same Bc-REP-PCR pattern were serovars amagiensis and seoulensis. This is in agreement with a previous random amplified polymorphic DNA analysis of these strains (16); ribotyping also connects both strains in the same group (27).…”

“…Likewise, such segregation should be reviewed for the highly related serovars sumiyoshiensis and fukuokaensis, indiana and thompsoni; amagiensis, seoulensis, and kyushuensis; thuringiensis and sooncheon; azorensis and vazensis; and monterrey and oswaldocruzi. All these serovars appear closely related among each other in the phylogenetic dendrogram generated by the Bc-REP-PCR fingerprinting and by previously reported ribotyping (27). Other serovars highly related by their Bc-REP-PCR pattern, such as serovars silo and ostriniae, palmanyolensis and darmstadiensis, japonensis and kenyae, and colmeri and mexicanensis show less of a relationship by ribotyping analysis (27).…”

“…All these serovars appear closely related among each other in the phylogenetic dendrogram generated by the Bc-REP-PCR fingerprinting and by previously reported ribotyping (27). Other serovars highly related by their Bc-REP-PCR pattern, such as serovars silo and ostriniae, palmanyolensis and darmstadiensis, japonensis and kenyae, and colmeri and mexicanensis show less of a relationship by ribotyping analysis (27).…”

“…On the other hand, Bc-REP-PCR patterns from the serovar sotto and its biovar dendrolimus slightly differ from each other, with the serovar sotto pattern identical to that of serovar dakota and the serovar leesis pattern the closest to that of biovar dendrolimus. Interestingly, ribotyping of these strains (27) indicated that while biovar dendrolimus and serovars leesis and dakota were phylogenetically related, serovar sotto was located in a separate group. Other serovars that shared the same Bc-REP-PCR pattern were serovars amagiensis and seoulensis.…”

“…Diversity of rRNA intergenic spacer sequences of 31 strains proved insufficient to discriminate between isolates (97 to 99% similarity) (6). On the other hand, ribotyping (16S, 23S, and 5S rRNA gene RFLP) of 80 serovars of B. thuringiensis showed a great diversity of patterns (27,28), similar to the diversity found with fluorescent AFLP, when 34 B. thuringiensis serovars were analyzed along with strains of B. cereus and B. anthracis (21).…”