Proposal of a novel subspecies: Alicyclobacillus hesperidum subspecies aegles.

Alicyclobacillus sp. DSM 11985T was isolated from geothermal soil but had not yet been classified at the species level. The strain produced guaiacol, which is of interest from the viewpoint of food spoilage in the food industry. 16S rRNA gene sequence analysis revealed that strain DSM 11985T was closely related (99.6 % similarity) to Alicyclobacillus hesperidum DSM 12489T. However, strains of A. hesperidum did not produce guaiacol; therefore, we performed the taxonomic characterization of strain DSM 11985T. The results showed that strain DSM 11985T and strains of A. hesperidum showed different phenotypic characteristics in biochemical/physiological tests including guaiacol production. Average nucleotide identity values between strain DSM 11985T and strain DSM 12489T were 95.4-95.9 %, and the in silico DNA-DNA hybridization value using the Genome-to-Genome Distance Calculator between strains DSM 11985T and DSM 12489T was 65.5 %. These values showed that strain DSM 11985T was genetically closely related but separated from strains of A. heparidum. From the above results, a novel subspecies of A. hesperidum, named Alicyclobacillus hesperidum subsp. aegles subsp. nov. is proposed. The type strain is DSM 11985T (=FR-12T=NBRC 113041T).

Pantoea theicola sp. nov., isolated from black tea.

A Gram-negative, facultatively anaerobic strain was isolated from black tea. On the basis of 16S rRNA gene sequence similarity comparisons, strain QC88-366T was grouped into the genus Pantoea, being related most closely to the type strains of Pantoea gaviniae (98.5 %) and Pantoea calida (98.3 %); sequence similarities were ≤ 97.0 % to the type strains of other species of the genus Pantoea. Multilocus sequence analysis based on partial sequences of the gyrB, rpoB, infB and atpD genes also revealed P. gaviniae and P. calida as the closest phylogenetic relatives. The fatty acid profile showed the major fatty acids of strain QC88-366T were C16 : 0, C16 : 1 and C18 : 1, the same as those of its closest related species. However, the ratio of C16 : 1, C17 : 0 cyclo, C18 : 1 and C18 : 2 differed slightly compared with those of the related neighbours. In addition, the results of physiological and biochemical tests also allowed the phenotypic differentiation of strain QC88-366T from its closest phylogenetic neighbours. The G+C content of the DNA was 57.2 mol%. Strain QC88-366T therefore represents a novel species of the genus Pantoea, for which the name Pantoea theicola sp. nov. is proposed. The type strain is QC88-366T ( = DSM 29212T = NBRC 110557T).

Sporolactobacillus putidus sp. nov., an endospore-forming lactic acid bacterium isolated from spoiled orange juice.

A Gram-positive, endospore-forming, lactic acid bacterium was isolated from spoiled orange juice. The organism, strain QC81-06(T), grew microaerobically or anaerobically at 30-45 degrees C (optimum 35 degrees C) and pH 3.5-5.5 (optimum pH 4.5), and produced acid from various sugars. D-Lactic acid was produced. It contained menaquinone-7 as the major isoprenoid quinone. The G+C content of the genomic DNA was 47.5 mol%. The predominant cellular fatty acids of the strain were iso-C(16 : 0), anteiso-C(15 : 0) and anteiso-C(17 : 0). Phylogenetic analyses based on the 16S rRNA gene and gyrB gene (DNA gyrase B subunit gene) revealed that strain QC81-06(T) clustered with Sporolactobacillus species but the strain was clearly distinct from other Sporolactobacillus species with significant bootstrap values. The levels of 16S rRNA gene and gyrB gene sequence similarities between strain QC81-06(T) and the other strains of the cluster were 96.0-97.0 % and 75.1-77.2 %, respectively. On the basis of these results, strain QC81-06(T) should be classified as a novel Sporolactobacillus species for which the name Sporolactobacillus putidus is proposed. The type strain is strain QC81-06(T) (=DSM 21265(T)=JCM 15325(T)).

Proposal of six species of moderately thermophilic, acidophilic, endospore-forming bacteria: Alicyclobacillus contaminans sp. nov., Alicyclobacillus fastidiosus sp. nov., Alicyclobacillus kakegawensis sp. nov., Alicyclobacillus macrosporangiidus sp. nov., Alicyclobacillus sacchari sp. nov. and Alicyclobacillus shizuokensis sp. nov.

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-A191(T), 4-A336(T), 5-A83J(T), 5-A167N, 5-A239-2O-A(T), E-8, RB718(T) and S-TAB(T). 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. omega-Alicyclic fatty acids were the predominant lipid component of strains 4-A336(T), 5-A83J(T), 5-A167N, RB718(T) and S-TAB(T). No omega-alicyclic fatty acids were detected in strains 3-A191(T), 5-A239-2O-A(T) 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-A191(T)=DSM 17975(T)=IAM 15224(T)), Alicyclobacillus fastidiosus sp. nov. (type strain S-TAB(T)=DSM 17978(T)=IAM 15229(T)), Alicyclobacillus kakegawensis sp. nov. (type strain 5-A83J(T)=DSM 17979(T)=IAM 15227(T)), Alicyclobacillus macrosporangiidus sp. nov. (type strain 5-A239-2O-A(T)=DSM 17980(T)=IAM 15370(T)), Alicyclobacillus sacchari sp. nov. (type strain RB718(T)=DSM 17974(T)=IAM 15230(T)) and Alicyclobacillus shizuokensis sp. nov. (type strain 4-A336(T)=DSM 17981(T)=IAM 15226(T)).

Alicyclobacillus pomorum sp. nov., a novel thermo-acidophilic, endospore-forming bacterium that does not possess omega-alicyclic fatty acids, and emended description of the genus Alicyclobacillus.

A 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 degrees C (optimum 45-50 degrees C), pH 3.0-6.0 (optimum pH 4.0-4.5) and produced acid from various sugars. It contained menaquinone-7 as the major isoprenoid quinone. The G+C content of the DNA was 53.1 mol%. The predominant cellular fatty acids of the strain were iso-C(15 : 0), anteiso-C(15 : 0), iso-C(16 : 0), iso-C(17 : 0) and anteiso-C(17 : 0), but omega-alicyclic fatty acids, which are characteristic of the genus Alicyclobacillus, were not found in the strain. Phylogenetic analyses based on both 16S rRNA and gyrB (DNA gyrase B subunit gene) gene sequences showed that strain 3A(T) falls into the Alicyclobacillus cluster, validated by significant bootstrap values. However, strain 3A(T) did not show a close relationship to the other species of the cluster. The level of 16S rDNA similarity between strain 3A(T) and other strains of the cluster was between 92.5 and 95.5 %. The level of gyrB sequence similarity between strain 3A(T) and other strains of the cluster was between 68.5 and 74.4 %. DNA-DNA hybridization values between strain 3A(T) and phylogenetically related strains of the genera Alicyclobacillus, Bacillus and Sulfobacillus were under 13 %, indicating that strain 3A(T) represents a distinct species. On the basis of these results, strain 3A(T) should be classified as a novel Alicyclobacillus species. The name Alicyclobacillus pomorum is proposed for this organism. The type strain of Alicyclobacillus pomorum is strain 3A(T) (=DSM 14955(T)=IAM 14988(T)).

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.

Alicyclobacillus acidiphilus sp. nov., a novel thermo-acidophilic, omega-alicyclic fatty acid-containing bacterium isolated from acidic beverages.

A novel thermo-acidophilic bacterium was isolated from an acidic beverage that had the odour of guaiacol. The cells are aerobic, gram-positive, spore-forming rods. The organism, strain TA-67T, grows at temperatures from 20 to 55 degrees C (optimum, 50 degrees C) and at pH values from 2.5 to 5.5 (optimum, pH 3.0). It possesses omega-cyclohexane fatty acid as a major cellular fatty acid. The G+C content of the DNA is 54.1 mol%. Phylogenetic analysis of the 16S rRNA gene sequences indicated that strain TA-67T constituted a distinct lineage in the Alicyclobacillus cluster, with Alicyclobacillus acidoterrestris as the closest neighbour (96.6% homology). Phenotypically, it is similar to, but can be distinguished from, omega-cyclohexane fatty acid-possessing alicyclobacilIi (A. acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus hesperidum and 'Alicyclobacillus mali') by the morphology of spores and sporangia, by the growth response to different temperatures, and by the profiles for acid production from carbon sources. It is the alicyclobacillus that produces guaiacol, a causative substance for an 'off' flavour of orange juice. On the basis of the phenotypic and phylogenetic evidence, it is concluded that strain TA-67T represents a new species of the genus Alicyclobacillus, for which the name Alicyclobacillus acidiphilus is proposed. The type strain is TA-67T (= DSM 14558T = IAM 14935T = NRIC 6496T).

Identification of thermoacidophilic bacteria and a new Alicyclobacillus genomic species isolated from acidic environments in Japan.

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.

Alicyclobacillus herbarius sp. nov., a novel bacterium containing omega-cycloheptane fatty acids, isolated from herbal tea.

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).