Assessment of a short phylogenetic marker based on comparisons of 3’ end 16S rDNA and 5’ end 16S-23S ITS nucleotide sequences of the Bacillus cereus group

Abstract

A short phylogenetic marker previously used in the reconstruction of the Order Bacillales and the genus Bacillus was assessed here at a lower taxa level: species in the Bacillus cereus group: B. anthracis, B. cereus, B. thuringiensis and B. weihenstephanensis. This maker is 220 bp in length. It is a combination of 150 bp at the 3’ end of the 16S rDNA and 70 bp at the 5’ end of the 16S-23S ITS sequence. Three additional Bacillus species, B. halodurans, B. licheniformis and B. subtilis, and Clostridium tetani were included for comparison purposes. A total of eight bacterial species and 12 strains were analyzed. A boot- strapped neighbor-joining tree was inferred from comparative analyses of all allelic sequences of the bacterial species and strains under study. Based on its topology, four major Groups were revealed at the 90% nucleotide sequence identities, Group I to IV. Group I contains all al-leles of the Bacillus cereus group. Group II con-tains all alleles of B. halodurans. Group III con-tains all alleles of B. licheniformis and B. subtilis. Group IV contains all alleles of Clostridium tetani. The 220 bp phylogenetic marker used here could resolve different species from different genera. At the genus level, distant species could be dis-tinguished. Very closely-related species, however, were undistinguishable. Species in the B. cereus group, most notably B. cereus, B. anth- racis and B. thuringiensis, could not be distin- guished. After successfully inferring the phylo- genies of the Order Bacillales and the genus Bacillus, we have met the resolving limit of this short phy-logenetic marker: B. cereus, B. anthracis and B. thuringiensis.