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.

Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals. It was thought to differ from Bacillus cereus, an opportunistic pathogen and cause of food poisoning, by the presence of plasmids pXO1 and pXO2, which encode the lethal toxin complex and the poly-␥-D-glutamic acid capsule, respectively. This work describes a non-B. anthracis isolate that possesses the anthrax toxin genes and is capable of causing a severe inhalation anthrax-like illness. Although initial phenotypic and 16S rRNA analysis identified this isolate as B. cereus, the rapid generation and analysis of a high-coverage draft genome sequence revealed the presence of a circular plasmid, named pBCXO1, with 99.6% similarity with the B. anthracis toxin-encoding plasmid, pXO1. Although homologues of the pXO2 encoded capsule genes were not found, a polysaccharide capsule cluster is encoded on a second, previously unidentified plasmid, pBC218. A͞J mice challenged with B. cereus G9241 confirmed the virulence of this strain. These findings represent an example of how genomics could rapidly assist public health experts responding not only to clearly identified select agents but also to novel agents with similar pathogenic potentials. In this study, we combined a public health approach with genome analysis to provide insight into the correlation of phenotypic characteristics and their genetic basis.

Representative strains of the Bacillus cereus group of bacteria, including Bacillus anthracis (11 isolates), B. cereus (38 isolates), Bacillus mycoides (1 isolate), Bacillus thuringiensis (53 isolates from 17 serovars), and Bacillus weihenstephanensis (2 isolates) were assigned to 59 sequence types (STs) derived from the nucleotide sequences of seven alleles, glpF, gmk, ilvD, pta, pur, pycA, and tpi. Comparisons of the maximum likelihood (ML) tree of the concatenated sequences with individual gene trees showed more congruence than expected by chance, indicating a generally clonal structure to the population. The STs followed two major lines of descent. Clade 1 comprised B. anthracis strains, numerous B. cereus strains, and rare B. thuringiensis strains, while clade 2 included the majority of the B. thuringiensis strains together with some B. cereus strains. Other species were allocated to a third, heterogeneous clade. The ML trees and split decomposition analysis were used to assign STs to eight lineages within clades 1 and 2. These lineages were defined by bootstrap analysis and by a preponderance of fixed differences over shared polymorphisms among the STs. Lineages were named with reference to existing designations: Anthracis, Cereus I, Cereus II, Cereus III, Kurstaki, Sotto, Thuringiensis, and Tolworthi. Strains from some B. thuringiensis serovars were wholly or largely assigned to a single ST, for example, serovar aizawai isolates were assigned to ST-15, serovar kenyae isolates were assigned to ST-13, and serovar tolworthi isolates were assigned to ST-23, while other serovars, such as serovar canadensis, were genetically heterogeneous. We suggest a revision of the nomenclature in which the lineage and clone are recognized through name and ST designations in accordance with the clonal structure of the population.

One hundred and nineteen strains of alkaliphilic and alkalitolerant, aerobic endospore-forming bacteria were examined for 47 physiological and biochemical characters, and DNA base composition. Numerical analysis (S, and SS&JPGMA clustering) revealed 11 clusters that comprised three or more strains. Most of the phena were further characterized by analysis of carbohydrate utilization profiles using the API 50CH system, but strains of two taxa could not be cultured by this method. DNA reassociation studies showed that nine of the phena were homogeneous, but strains of phenon 4 and phenon 8 were each subdivided into two DNA hydridization groups. The strains could therefore be classified into 13 taxa plus a number of unassigned single-membered clusters. Two taxa were equated with Bacillus cohnii and B. alcalophilus and nine of the remainder are proposed as new species with the following names: B. agaradhaemns sp. nov., B. clarkii sp. nov., B. clausii sp. nov., B. gibsonii sp. nov., B. halmapalus sp. nov, B. halodurans comb. nov., B. horikoshii sp. nov., B. pseudalcalophilus sp. nov. and B. pseudofirmus sp. nov. Two taxa were insufficiently distinct to allow confident identification and these have therefore not been proposed as new species.

Three hundred and sixty-eight strains of aerobic, endospore-forming bacteria which included type and reference cultures of Bacillus and environmental isolates were studied. Overall similarities of these strains for 118 unit characters were determined by the SSM, S, and D p coefficients and clustering achieved using the UPGMA algorithm. Test error was within acceptable limits. Six cluster-groups were defined at 70% SSM, which corresponded to 69% Sp and 48-57% SJ. Groupings obtained with the three coefficients were generally similar but there were some changes in the definition and membership of cluster-groups and clusters, particularly with the SJ coefficient.The Bacillus strains were distributed among 31 major (4 or more strains), 18 minor (2 or 3 strains) and 30 single-member clusters at the 83% SsM level. Most of these clusters can be regarded as taxospecies. The heterogeneity of several species, including Bacillus breuis, B. circulans, B. coagulans, B. megateriun, B . sphaericus and B . stearothermophilus, has been indicated and the species status of several taxa of hitherto uncertain validity confirmed. Thus on the basis of the numerical phenetic and appropriate (published) molecular genetic data, it is proposed that the following names be recognized; BacillusJlexus (Batchelor) nom. rev., Bacillus fusiformis (Smith et al.) comb. nov., Bacillus kaustophilus (Prickett) nom. rev., Bacilluspsychrosaccharolyticus (Larkin & Stokes) nom. rev. and Bacillus simplex (Gottheil) nom. rev. Other phenetically well-defined taxospecies included ' B. aneurinolyticus', 'B. apiarius', 'B. cascainensis', 'B. thiaminolyticus' and three clusters of environmental isolates related to B . firmus and previously described as 'B. firmus-B. lentus intermediates'. Future developments in the light of the numerical phenetic data are discussed.

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