Bacillus strains with >99.7% 16S rRNA gene sequence similarity were characterized with DNA:DNA hybridization, cellular fatty acid (CFA) analysis, and testing of 100 phenotypic traits. When paired with the most closely related type strain, percent DNA:DNA similarities (% ) for six Bacillus strains were all far below the recommended 70% threshold value for species circumscription with Bacillus nealsonii. An apparent genomic group of four Bacillus strain pairings with 94%–70% was contradicted by the failure of the strains to cluster in CFA- and phenotype-based dendrograms as well as by their differentiation with 9–13 species level discriminators such as nitrate reduction, temperature range, and acid production from carbohydrates. The novel Bacillus strains were monophyletic and very closely related based on 16S rRNA gene sequence. Coherent genomic groups were not however supported by similarly organized phenotypic clusters. Therefore, the strains were not effectively circumscribed within the taxonomic species definition. 1. Introduction CBD 118 was one of the two first Bacillus strains not related to the B. cereus group reported to harbor the capsule genes carried on pXO2 by Bacillus anthracis (USF Center for Biological Defense (CBD)) [1, 2]. Luna et al. isolated and sequenced the capsule operon (capA, capB, capC, capD, and promoter), repA, capR, acpA, IS1627, ORF43, ORF48, and ORF61 on a large plasmid in CBD 118 [1]. Its status as a carrier of B. anthracis capsule genes spurred research into determining its closest relatives, to aid in circumscribing the reservoir of genes essential for virulence in B. anthracis. When near full length 16S rRNA gene sequences were compared, the most similar type strains to strain CBD 118 were Bacillus circulans ATCC (98.9%) and Bacillus nealsonii DSM (99.3%). Strain CBD 118 differed from B. circulans ATCC and B. nealsonii DSM for 10 and 12 of 100 phenotypic traits evaluated, respectively. The percentages of DNA:DNA binding in two pairings each of strain CBD 118 to B. circulans ATCC and B. nealsonii DSM were 12.5 and 10.2% and 10.8 and 8.3%, respectively. Thus, strain CBD 118 is differentiated by phenotypic and genome-based methods from the only validly named species with greater than 98.7% 16S rRNA gene sequence similarity [3–5]. Strain CBD 118 was the sole exemplar of a novel species. Prior to the proposal of novel species, studies of ten or more strains are recommended in order to detail intraspecies diversity and to foster appropriate type strain assignment [6–8]. To identify the requisite closely related strains, the
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