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.

Aims: To identify Bacillus species and related genera by ®ngerprinting based on ribosomal RNA gene restriction patterns; to compare ribosomal RNA gene restriction patterns-based phylogenetic trees with trees based on 16S rRNA gene sequences; to evaluate the usefulness of ribosomal RNA gene restriction patterns as a taxonomic tool for the classi®cation of Bacillus species and related genera. Methods and Results: Seventy-eight bacterial species which include 42 Bacillus species, 31 species from ®ve newly created Bacillus-related genera, and ®ve species from ®ve phenotypically related genera were tested. A total of 77 distinct 16S rRNA gene hybridization banding patterns were obtained. The dendrogram resulting from UPGMA analysis showed three distinct main genetic clusters at the 75% banding pattern similarity. A total of 77 distinct 23S and 5S rRNA genes hybridization banding patterns were obtained, and the dendrogram showed four distinct genetic clusters at the 75% banding pattern similarity. A third dendrogram was constructed using a combination of the data from the 16S rRNA gene ®ngerprinting and the 23S and 5S rRNA genes ®ngerprinting. It revealed three distinct main phylogenetic clusters at the 75% banding pattern similarity. Conclusions: The Bacillus species along with the species from related genera were identi®ed successfully and differentiated by ribosomal RNA gene restriction patterns, and most were distributed with no apparent order in various clusters on each of the three dendrograms. Signi®cance and Impact of the Study: Our data indicate that ribosomal RNA gene restriction patterns can be used to reconstruct the phylogeny of the Bacillus species and derived-genera that approximates, but does not duplicate, phylogenies based on 16S rRNA gene sequences.

Aims: To assess the congruence between two earlier independent phylogenetic studies on Bacillus thuringiensis strains and on Bacillus-related species and the single, all-encompassing, phylogenetic tree presented here inferred from the combination of the two earlier datasets. Methods and Results: A dendrogram was constructed using a combination of the data from our previous studies on 16S rRNA gene fingerprinting of 86 B. thuringiensis strains and of 77 species of Bacillus and related taxa. It revealed that all B. thuringiensis strains were clustered together in four distinct groups at a DNA similarity rate of 93%, except two serovars, bolivia and finitimus. Four bacilli species, Paenibacillus alvei, P. azotofixans, B. lentus and B. licheniformis, share a DNA similarity rate of 92% with Bt Group IV. Conclusions: Most, but not all, B. thuringiensis strains could be grouped together based on the DNA similarity rate. They were also very close to some other bacilli species. Significance and Impact of the Study: The combined phylogenetic study presented here, inferred from 16S rRNA gene restriction fragment length polymorphism, is congruent with two earlier independent phylogenetic studies, one on B. thuringiensis and the other on bacilli species.

Aims: To determine the 23S and 5S rRNA gene ®ngerprints in order to reveal phylogenetic relationships among Bacillus thuringiensis strains. Methods and Results: Eighty-six B. thuringiensis strains which include 80 serovar type strains, ®ve intraserovar strains and a non-serotypeable strain, wuhanensis, were tested. Total DNA was digested with EcoRI and HindIII. The 23S and 5S rRNA gene restriction fragment length polymorphisms showed 82 distinctive ribopatterns. The dendrogram generated by numerical analysis showed 10 phylogenetic groups and six ungrouped serovars at the 95á5% DNA relatedness rate. A second dendrogram was constructed using a combination of the data from this study and from a previous study on 16S rRNA gene ®ngerprinting. It revealed eight distinct phylogenetic groups and three ungrouped serovars at the 94% DNA relatedness rate. Conclusions: This method permitted the classi®cation and positioning of a wide variety of B. thuringiensis strains on a phylogenetic tree. Bacillus thuringiensis strains appear to be relatively homogeneous and to share a high degree of DNA relatedness. Signi®cance and Impact of the Study: This study contributes a further step to the de®nition of valid taxonomic sublevels for the B. thuringiensis species.

The distribution of IS231 has been analyzed in Bacillus thuringiensis serovars. A 723-bp HaeII conserved fragment from IS231M has been used as a probe against EcoRI-digested B. thuringiensis total DNA to yield serovar-specific hybridization profiles. The approach was useful at revealing the extent of distribution of IS231-like sequences between and within strains. Of the 88 B. thuringiensis strains tested, 70 showed hybridization banding patterns that comprised between one and 20 distinct bands. These 70 B. thuringiensis strains were grouped based on banding pattern similarities. Interestingly, intraserovar strains did not necessarily cluster together.