Alicyclobacillus pomorum sp. nov., a novel thermo-acidophilic, endospore-forming bacterium that does not possess v-alicyclic fatty acids, and emended description of the genus Alicyclobacillus Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1, Yayoi 1-chome, Bunkyo-ku, Tokyo 113-0032, JapanA thermo-acidophilic endospore-forming bacterium was isolated from a mixed fruit juice. The organism, strain 3A T , was rod-shaped, grew aerobically at 30-60 6C (optimum 45-50 6C),

A thermo-acidophilic gram-positive bacterium, strain CP-1T, which grows aerobically at 35-65 degrees C (optimum 55-60 degrees C) and at pH 3.5-6.0 (optimum pH 4.5-5.0), was isolated from a herbal tea made from the dried flowers of hibiscus. Phylogenetic analysis of the 16S rRNA gene sequences showed that this bacterium was clearly distinguishable from previously described species of the genera Alicyclobacillus and Sulfobacillus. Strain CP-1T had unique omega-cycloheptane fatty acids as the major membrane lipid component, a characteristic which is peculiar to Alicyclobacillus cycloheptanicus. However, phenotypic and chemotaxonomic characteristics of strain CP-1T were different from those of the type strain of A. cycloheptanicus. DNA-DNA hybridization between the type strains of Alicyclobacillus species and Sulfobacillus disulfidooxidans was <20%, indicating that strain CP-1T represents a distinct species. On the basis of these results, the name Alicyclobacillus herbarius is proposed for this organism. The type strain is strain CP-1T (= DSM 13609T = IAM 14883T = NRIC 0477T).

Moderately thermophilic, acidophilic, spore-forming bacteria (146 strains) were isolated from various beverages and environments. Based on the results of sequence analysis of the hypervariable region of the 16S rRNA gene, eight of the strains represent novel species of the genus Alicyclobacillus. These strains were designated 3-A191T, 4-A336T, 5-A83JT, 5-A167N, 5-A239-2O-AT, E-8, RB718T and S-TABT. Phylogenetic analyses of 16S rRNA and DNA gyrase B subunit (gyrB) nucleotide sequences confirmed that the eight strains belonged to the Alicyclobacillus clade. Cells of the eight strains were Gram-positive or Gram-variable, strictly aerobic and rod-shaped. The strains grew well under acidic and moderately thermal conditions, produced acid from various sugars, contained menaquinone 7 as the major isoprenoid quinone and did not produce guaiacol. ω-Alicyclic fatty acids were the predominant lipid component of strains 4-A336T, 5-A83JT, 5-A167N, RB718T and S-TABT. No ω-alicyclic fatty acids were detected in strains 3-A191T, 5-A239-2O-AT or E-8, but iso- and anteiso-branched fatty acids and small amounts of straight-chain saturated fatty acids were detected instead. According to the DNA–DNA hybridization data and distinct morphological, physiological, chemotaxonomical and genetic traits, the eight strains represent six novel species within the genus Alicyclobacillus, for which the following names are proposed: Alicyclobacillus contaminans sp. nov. (type strain 3-A191T=DSM 17975T=IAM 15224T), Alicyclobacillus fastidiosus sp. nov. (type strain S-TABT=DSM 17978T=IAM 15229T), Alicyclobacillus kakegawensis sp. nov. (type strain 5-A83JT=DSM 17979T=IAM 15227T), Alicyclobacillus macrosporangiidus sp. nov. (type strain 5-A239-2O-AT=DSM 17980T=IAM 15370T), Alicyclobacillus sacchari sp. nov. (type strain RB718T=DSM 17974T=IAM 15230T) and Alicyclobacillus shizuokensis sp. nov. (type strain 4-A336T=DSM 17981T=IAM 15226T).

Sixty strains of thermoacidophilic bacteria have been isolated from soil and water samples obtained from various acidic environments in Japan. An initial comparative sequence analysis of the hypervariable regions of the 16S rDNA revealed that all strains could be assigned to the Alicyclobacillus acidocaldarius- Alicyclobacillus genomic species 1 group, which could be further subdivided into three clusters (Clusters I-III). On the basis of phenotypic characteristics, chemotaxonomic profiles, and phylogenetic data of six selected strains, five strains were identified as either A. acidocaldarius or Alicyclobacillus genomic species 1; however, one strain (MIH 332) could not be determined to belong to either of these species. 16S rDNA sequence homology values between strain MIH 332 and the reference strains of A. acidocaldarius (ATCC 27009(T)) and Alicyclobacillus genomic species 1 (DSM 11984) were 98.8% and 99.1%, respectively, which were higher than the corresponding similarity between the reference strains (98.4%). On the other hand, DNA-DNA hybridization levels between strain MIH 332 and the reference strains were 39% and 44%, respectively, which were lower than the value between the reference strains (59% or 65%). However, the phenotype of strain MIH 332 was also similar to those of the reference strains, and a typical phenotype could not be found for the strain, thus indicating that the strain may be a new genomic species of A. acidocaldarius, for which the name Alicyclobacillus genomic species 2 is tentatively proposed. The results of this study suggest that A. acidocaldarius and its related species are widely distributed in acidic environments in Japan, with slight regional variations in morphological and genotypic characteristics.

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