The 16s rRNA gene sequences of 34 named and unnamed clostridial strains were determined by PCR direct sequencing and were compared with more than 80 previously determined clostridial sequences and the previously published sequences of representative species of other low-G+C-content gram-positive genera, thereby providing an almost complete picture of the genealogical interrelationships of the clostridia. The results of our phylogenetic analysis corroborate and extend previous findings in showing that the genus Clostridium is extremely heterogeneous, with many species phylogenetically intermixed with other sporeforming and non-spore-forming genera. The genus Clostridium is clearly in need of major revision, and the rRNA structures defined in this and previous studies may provide a sound basis for future taxonomic restructuring. The problems and different possibilities for restructuring are discussed in light of the phenotypic and phylogenetic data, and a possible hierarchical structure for the clostridia and their close relatives is presented. On the basis of phenotypic criteria and the results of phylogenetic analyses the following five new genera and 11 new combinations are proposed: Caloramator gen. nov., with Caloramator fervidus comb. nov.; Filifactor gen. nov., with Filifactor villosus comb. nov.; Moorella gen. nov., with Moorella thermoacetica comb. nov. and Moorella thermoautotrophica comb. nov.; Oxobacter gen. nov., with Oxobacter pfennigii comb. nov.; Oxalophagus gen. nov., with Oxalophagus oxalicus comb. nov.; Eubacterium barkeri comb. nov.; Paenibacillus durum comb. nov.; Thermoanaerobacter kivui comb. nov.; Thermoanaerobacter thermocopriae comb. nov.; and Thermoanerobacterium thermosaccharolyticum comb. nov. ~ ~~Great advances, have been made over the past decade in unravelling the phylogenetic complexities within the grampositive endospore-forming bacteria. For example, 16s rRNA oligonucleotide cataloging and, more recently, almost complete rRNA (or gene) sequencing have shown that the aerobic endospore-forming bacilli are phylogenetically very heterogeneous, consisting of at least six highly divergent lines (1,11,51). As a result of these studies, taxonomic reorganization of the genus Bacillus was initiated with the introduction of the genus Alicyclobacillus for some acidophilic species (51) and the genus Paenibacillus (2) for Bacillus polymyxa and its close relatives (rRNA group 3 [l]). Although the remainder of the genus is still in need of taxonomic revision, the phylogenetic groups established by rRNA analysis are already forming the foundation for a new molecular data-based taxonomy for this group of organisms. Knowledge of the natural interrelationships within the anaerobic genus Clostridium is more fragmented than knowledge of the interrelationships among the aerobic bacilli. The earliest, and until recently the most comprehensive, phylogenetic study of the genus Clostridium was published by Johnson and Francis (22), who demonstrated that there is considerable diversity within the genus by u...

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.

Biochemical and chemical studies were performed on some atypical lactobacilli from chicken meat in an attempt to clarify their taxonomy. The present study showed that the majority of the poultry strains could be allocated to the species Brochothrix thermosphacta, Lactobacillus divergens, and Lactobacillus piscicola. The majority of the remaining poultry strains fell into two distinct groups which were worthy of separate species status. Lactobacillus carnis was found to be a member of the species L. piscicola. On the basis of biochemical, physiological, and chemical criteria, we suggest that L. divergens, L. piscicola, and the two unidentified poultry taxa be classified in a new genus, Carnobacterium, as Carnobacterium divergens comb. nov., Carnobacterium piscicola comb. nov., Carnobacterium gallinarum sp. nov., and Carnobacterium mobile sp. nov. The type strains of C . gallinarum and C . mobile are NCFB 2766T and NCFB 2765T, respectively. ~Thornley (22) reported the isolation of some grampositive, catalase-negative, nonsporeforming rods from chicken meat maintained at low temperature. These bacteria resembled lactobacilli in many respects but differed in their inability to grow on acetate media. A subsequent, more comprehensive study indicated that these strains formed three groups, none of which at that time corresponded to any Lactobacillus species or any other recognized taxon (23). A recent numerical phenetic study that included the "atypical lactobacilli" described by Thornley and Sharpe confirmed the presence of three major groups (S. Ferusu and D. Jones, J. Gen. Microbiol., in press). One of these was shown to correspond to Brochothrix thermosphacta, whereas the taxonomic position of the other two groups remained unresolved. In recent years there has been an increasing number of reports of unusual lactobacilli from vacuum-packed meat that are also unable to grow on acetate agar (8,12,18). These studies have resulted in the establishment of two new species, Lactobacillus carnis (19) and Lactobacillus divergens (12). Unfortunately, these two species were not included in the numerical study of Ferusu and Jones, and as far as we are aware there has been no comparative study of the poultry isolates and vacuum-packed meat strains. Therefore, in the present study the biochemical and chemical characteristics of the atypical lactobacilli described by Thornley and Sharpe (23) have been determined in an attempt to clarify their relationship, if any, to the species L. carnis and L . divergens. The recently described salmonid fish pathogen Lactobacillus piscicola was also included in the study because this species shares many properties with the atypical lactobacilli from poultry and meat (9). MATERIALS AND METHODSCultures and cultivation. The test strains used are listed in Table 1. Cells for peptidoglycan and lipid analyses were grown in YGPB broth (5) at 30°C for 3 days. Cultures were checked for purity, harvested by centrifugation, washed with distilled water, and freeze-dried. * Corresponding author.DNA studies. ...

No abstract