Comparative Analysis of Bacillus anthracis, Bacillus cereus, and Related Species on the Basis of Reverse Transcriptase Sequencing of 16S rRNA

Abstract

The primary structures of the 16s rRNAs of Bacillus anthracis, Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis were determined by using the reverse transcription-dideoxy sequencing method. All of the strains exhibited very high levels of sequence similarity (>9%) that were consistent with the close relationships shown by previous DNA hybridization studies. The species Bacillus anthracis, Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis were originally described on the basis of their habitats, their pathogenicity for mammals or insects, and their morphological and physiological characteristics. However, the taxonomic interrelationships of these species are equivocal. All four species share many phenotypic properties, and several workers have questioned their status as separate species (6, 11, 12). DNA-DNA hybridization studies on strains of B. anthracis, B. cereus, and B. thuringiensis have also shown that these organisms share relatively high levels of chromosomal base sequence similarity (7, 10, 13). However, inconsistencies in reported levels of DNA relatedness make it difficult to draw firm conclusions regarding species differentiation within this group of organisms.Small-subunit rRNA is now recognized as a powerful molecular chronometer (16). Degrees of sequence conservation, ranging from highly variable to highly conserved regions, enable systematists to measure small as well as great genealogical distances. In this study we determined partial primary 16s rRNA sequences of B. anthracis, B. cereus, B. mycoides, and B. thuringiensis in order to investigate the genealogical interrelationships of these organisms. MATERIALS AND METHODSCultures and cultivation. Details concerning the test strains which we examined are shown in Table 1. Strains were grown in shake flasks containing nutrient broth no. 2 (Oxoid) to late exponential phase at 30°C.Extraction and sequence determination of 16s rRNA. Total cellular rRNA was extracted from ca. 2 g of wet cells by mechanical disruption, using glass beads and a Braun homogenizer, and was purified as described by Embley et al. (5). Nucleotide sequences were determined by the Sanger dideoxynucleotide method (9) directly from cellular rRNA, using avian myeloblastosis virus reverse transcriptase (8). The sequences of the oligonucleotide primers were the same as those described by Embley et al. (5). In addition, the * Corresponding author.following primer was used: S'TCACCAACTAGCTAATG, which is complementary to positions 258 to 242 (Escherichia coli nomenclature). This primer was included to enable determination of the sequence between positions 100 and 150, which could not always be fully established by using the primer at position 357 described by Lane et al. (8).Nucleotide sequence accession numbers. The 16s rRNA sequences have been deposited in the EMBL Data Library (accession numbers X55059 to X55063). RESULTS AND DISCUSSIONThe 16s rRNA sequences of B. anthracis Sterne, B. cereus NCDO 1771T (T = type strain), B. cereus NCTC 11143 (emetic strain)...

How Close Is Close: 16S rRNA Sequence Identity May Not Be Sufficient To Guarantee Species Identity

“…In such cases the 16s rRNA data would be sufficient to identify any strains that were clear outsiders but could never convincingly establish that the remaining genetic diversity was consistent with the existence of only one species. This interpretation has already been recognized and implemented by Collins and coworkers (1,6,27). In recent studies of Lactobacillus lactis (6), Streptococcus parasanguis (27), and Mycobacterium intracellulare (4) strains, less than 10 sequence differences were encountered in each case.…”

Bosea eneae sp. nov., Bosea massiliensis sp. nov. and Bosea vestrisii sp. nov., isolated from hospital water supplies, and emendation of the genus Bosea (Das et al. 1996)

“…As described previously for several species, including Bacillus spp. (Ash et al, 1991;Fox et al, 1992), the study of 16S rDNA sequences alone may not be sensitive enough to discriminate confidently between groups of bacteria; therefore, these data must be accompanied by DNA-DNA hybridization data for accurate analyses to be made (Stackebrandt & Goebel, 1994). Indeed, species definition must be based on the results of DNA-DNA hybridization and phenotypic data (Wayne et al, 1987;Grimont, 1998).…”

Description of Afipia birgiae sp. nov. and Afipia massiliensis sp. nov. and recognition of Afipia felis genospecies A.

“…It should be noted that genomic data are increasingly available for these species (up to 40 16S rDNA sequences for the Bradyrhizobium species) (Willems et al, 2001), but phenotypic data that could help in the polyphasic taxonomy of these bacteria are still lacking. As observed among Bradyrhizobium species and for the species described in this study or as described previously for Bacillus species (Ash et al, 1991 ;Fox et al, 1992), over-reliance on 16S rDNA sequence comparisons may be foolhardy and should therefore be associated with DNA-DNA hybridization data . In this study, A. felis and A. felis genospecies A represent two distinct genospecies on the basis of DNA-DNA hybridization and phenotypic data such as susceptibility to antibiotics, SDS-PAGE profile and whole-cell fatty acid composition (Wayne et al, 1987 ;Grimont et al, 1998), whereas they exhibit levels of 16S rDNA sequence similarity of 99n9%.…”