Comparative analysis of 16S rRNA gene sequences, DNA–DNA hybridization data and phenotypic properties revealed that ‘Sulfobacillus thermosulfidooxidans subsp. thermotolerans’ strain K1 is not a member of the genus Sulfobacillus. Phylogenetically, strain K1 is closely related to unclassified strains of the genus Alicyclobacillus: the 16S rRNA gene sequence of strain K1 is similar to that of Alicyclobacillus sp. AGC-2 (99·6 %), Alicyclobacillus sp. 5C (98·9 %) and Alicyclobacillus sp. CLG (98·6 %) and bacterium GSM (99·1 %). The 16S rRNA gene sequence similarity values for strain K1 and species of the genus Alicyclobacillus with validly published names were in the range 92·1–94·6 %, and for S. thermosulfidooxidans VKM B-1269T the value was 87·7 %. Sulfobacillus disulfidooxidans SD-11T was also phylogenetically related to strain K1 (92·6 % sequence similarity) and thus belonged to the genus Alicyclobacillus. Chemotaxonomic data, such as the major cell-membrane lipid components of strains K1 and SD-11T (ω-alicyclic fatty acids) and the major isoprenoid quinone (menaquinone MK-7) of strain K1, supported the affiliation of strains K1 and SD-11T to the genus Alicyclobacillus. Physiological and molecular biological tests allowed genotypic and phenotypic differentiation of strains K1 and SD-11T from the nine Alicyclobacillus species with validly published names. The G+C content of the DNA of strain K1 was 48·7±0·6 mol%; that of strain SD-11T was 53±1 mol%. DNA–DNA reassociation studies showed low relatedness (22 %) between strains K1 and SD-11T, and even lower relatedness (3–5 %) between these strains and Alicyclobacillus acidocaldarius subsp. acidocaldarius ATCC 27009T, DSM 446T. DNA reassociation of strains K1 and SD-11T with Alicyclobacillus cycloheptanicus DSM 4006T gave values of 15 and 21, respectively. Based on the phenotypic and phylogenetic characteristics of strains K1 and SD-11T, Alicyclobacillus tolerans sp. nov. (type strain, K1T=VKM B-2304T=DSM 16297T) and Alicyclobacillus disulfidooxidans comb. nov. (type strain, SD-11T=ATCC 51911T=DSM 12064T) are proposed.

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