Application of the hypervariable region of the 16S rDNA sequence as an index for the rapid identification of species in the genus Alicyclobacillus

A comparison of the 16S rRNA gene (rDNA) sequences of seven type strains belonging to different Alicyclobacillus species (i.e., five validated species, one proposed species and one genomic species) suggested that the 5' end hypervariable region (259-273 bases in length) of 16S rDNA was specific for the respective type strains. Further phylogenetic analysis based on DNA sequences of the hypervariable region using 24 Alicyclobacillus strains revealed that the strains could be categorized into five species and the A. acidocaldarius-Alicyclobacillus genomic species 1 group. The hypervariable region was highly conserved among the five species: A. acidiphilus, A. acidoterrestris, A. cycloheptanicus, A. herbarius, and A. hesperidum. The strains in the A. acidocaldarius-Alicyclobacillus genomic species 1 group were subdivided into two clusters (Clusters I and II) based on DNA sequences in the hypervariable region. On the basis of phenotypic characteristics, chemotaxonomic and phylogenetic analyses, and DNA-DNA hybridization data, strains in Cluster I were grouped as Alicyclobacillus genomic species 1 and strains in Cluster II were re-identified as A. acidocaldarius, thereby demonstrating that the hypervariable regions were also highly conserved within these two species. These results suggest that as is the case with Bacillus, the hypervariable region is significantly species-specific in the genus Alicyclobacillus to distinguish Alicyclobacillus species by DNA sequence comparison of the hypervariable region.