Phylogenetic relationships between Bacillus species and related genera inferred from comparison of 3′ end 16S rDNA and 5′ end 16S–23S ITS nucleotide sequences

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Abstract
The nucleotide sequences of the 3′ end of the 16S rDNA and the 16S–23S internal transcribed spacer (ITS) of 40 Bacillaceae species were determined. These included 21 Bacillus, 9 Paenibacillus, 6 Brevibacillus, 2 Geobacillus, 1 Marinibacillus and 1 Virgibacillus species. Comparative sequence analysis of a 220 bp region covering a highly conserved 150 bp sequence located at the 3′ end of the 16S rRNA coding region and a conserved 70 bp sequence located at the 5′ end of the 16S–23S ITS of the 40 species and six sequences available in GenBank were used to infer the phylogenetic relationships between all 46 taxa. When a maximal distance (D max, where D refers to the number of nucleotide substitutions per site) of 0·31 was introduced as a threshold to determine groupings, 10 phylogenetically distinct clusters were revealed. Twenty-six Bacillus species were separated in seven groups (I, II, III, IV, V, VI and X), but Bacillus circulans remained ungrouped. All six Brevibacillus species under study were in Group VII. The nine Paenibacillus species fell into two distinct groups (VIII and IX). Species with D max values within 0·05 were considered to be very closely related. These were Bacillus psychrophilus and Bacillus psychrosaccharolyticus in Group II; ‘Bacillus maroccanus’ and Bacillus simplex in Group II; Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus mojavensis and Bacillus subtilis in Group VI; Bacillus fusiformis and Bacillus sphaericus in Group VI; Brevibacillus brevis and Brevibacillus formosus in Group VII; Paenibacillus gordonae and Paenibacillus validus in Group VIII; and Bacillus anthracis, Bacillus cereus, Bacillus mycoides and Bacillus thuringiensis in Group X. The phylogenetic classification presented here is, in general, in agreement with current classifications based on phenotypic and molecular data. Our findings suggest, however, that in some cases, further divisions or, conversely, further groupings might be warranted. Should current classifications be re-examined in the light of our results, D max values of 0·31 and 0·05, as exemplified here, may prove useful threshold values for the grouping of Bacillaceae into taxa akin to genera and species, respectively. These D max thresholds may also reveal, in a different way, bacterial species for which further characterization might be warranted for proper classification and/or reassignment.

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