Comparative 16S rRNA sequence analysis has demonstrated that the genus Bacillus consists of at least five phyletic lines. rRNA group 3 bacilli of Ash, Farrow, Wallbanks and Collins (1991) comprising Bacillus polymyxa and close relatives is phylogenetically so removed from Bacillus subtilis, the type species of the genus and other aerobic, endospore-forming bacilli that they warrant reclassification in a new genus Paenibacillus. The genus Paenibacillus can be readily distinguished from other Bacillus groups using a battery of phenotypic characters and a highly specific gene probe based on 16S rRNA.

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)...

The small‐subunit rRNA sequences of 51 species of Bacillus were determined by reverse transcription to elucidate the phylogenetic structure of the genus. Comparative analysis of the sequence data revealed five phylogenetically distinct clusters. Group 1 (Bacillus sensu stricto) included B. subtilis the type species of the genus and 27 other species. Group 2 consisted of B. sphaericus and five other bacilli and Sporosarcina ureae clustered within the confines of this group. Group 3 consisted of a phylogenetically coherent group of 10 species whereas groups 4 and 5 comprised two and three species respectively. It is evident that the genus Bacillus in genetically extremely heterogeneous and requires extensive taxonomic revision. The rRNA structures defined in the present study will provide a firm basis for the division of Bacillus into several phylogenetically distinct genera.

The phylogenetic status of members of the genus Lactococcus and some motile strains which react with Lancefield group N antiserum was examined by reverse transcriptase sequencing of 16S ribosomal ribonucleic acid. In agreement with earlier nucleic acid hybridization and immunological studies of superoxide dismutase the 16S sequence data clearly demonstrate that the lactococci represent a distinct phylogenetic group equivalent in rank to the genera Enterococcus and Streptococcus. The motile group N strains from chicken faeces and river water, however, were found to be phylogenetically unrelated to lactococci but displayed a closer, albeit loose, association with members of the genus Enterococcus. On the basis of the present sequence data and earlier chemotaxonomic studies it is proposed that the motile group N strains be classified in a new genus Vagococcus, as Vagococcus fluvialis sp. nov. The type strain of V. fluvialis is NCDO 2497.

The primary structures of the 23S ribosomal RNA genes of Bacillus anthracis and an emetic strain of Bacillus cereus were determined by direct sequencing of enzymatically amplified chromosomal DNA. The 23S rRNA gene sequences of B. anthracis and B. cereus were found to be almost identical and showed only two differences (a single nucleotide change, and a single base insertion in B. cereus). The feasibility of using PCR-direct sequencing for the rapid sequence determination of large-subunit rRNA genes is demonstrated.

Summary The small subunit rRNA sequences of 55 species of the genus Lactobacillus were determined by reverse transcription in an attempt to elucidate their natural interrelationships. Comparative analysis of the sequence data revealed the presence of 3 phylogenetically distinct clusters. Cluster 1 (designated) the L. delbrueckii group) included L. delbrueckii the type species of the genus and 11 other obligately homofermentative species. Cluster 2 (designated the L. casei group) comprised 32 Lactobacillus species and 5 Pediococcus species (including P. damnosus, the type species of the genus). With the exception of 5 obligately homofermentative species (L. animalis, L. ruminis, L. salivarius, L. sharpeae, L. yamanashiensis) most members of cluster 2 were heterofermentative. Cluster 3 (designated the Leuconostoc paramesenteroides group) included the atypical heterofermentative lactobacilli, L. confusus L. kandleri, L. minor, L. viridescens and Leu. paramesenteroides. The sequence data clearly demonstrate that the genus Lactobacillus as presently constituted is phylogenetically very heterogeneous. Further, the rRNA groups identified in the present study do not correspond to the classical division of Lactobacillus into the subgenera Thermobacterium, Streptobacterium and Betabacterium established on the basis of phenotype.

A phylogenetic analysis based on 16S rRNA was performed on the genera Planococcus, Marinococcus, Sporosarcina and endospore-forming rods. In agreement with earlier 16S rRNA cataloguing data, Planococcus citreus and Sporosarcina ureae clustered with Bacillus pasteurii and other bacilli containing lysine in their cell walls. Sporosarcina halophila was shown to be genetically distinct from S. ureae and formed a loose association with the main Bacillus subtilis grouping. Marinococcus halophilus (formerly Planococcus halophilus) exhibited low levels of relatedness to all reference species examined and formed a distinct line of descent.

The primary structures of the 23S ribosomal RNA genes of Bacillus anthracis and an emetic strain of Bacillus cereus were determined by direct sequencing of enzymatically amplified chromosomal DNA. The 23S rRNA gene sequences of B. anthracis and B. cereus were found to be almost identical and showed only two differences (a single nucleotide change, and a single base insertion in B. cereus). The feasibility of using PCR‐direct sequencing for the rapid sequence determination of large‐subunit rRNA genes is demonstrated.