Methylobacterium persicinum sp. nov., Methylobacterium komagatae sp. nov., Methylobacterium brachiatum sp. nov., Methylobacterium tardum sp. nov. and Methylobacterium gregans sp. nov., isolated from freshwater.

Eight strains, 002-165T, 002-079T, B0021T, Hojyo2, RB603B, RB677T, 002-074T and RB678, isolated from the environment of food-processing factories in Japan, were characterized using a polyphasic approach. The isolates were Gram-negative, strictly aerobic, pink-pigmented, facultatively methylotrophic, non-spore-forming rods. The chemotaxonomic characteristics of these isolates included the presence of C18 : 1omega7c as the major cellular fatty acid and ubiquinone Q-10 as the predominant ubiquinone. The DNA G+C content was 67.1-71.1 mol%. Phylogenetic analyses of 16S rRNA and DNA gyrase B subunit (gyrB) nucleotide sequence confirmed that the eight strains belonged to the Methylobacterium clade. Moreover, a DNA-DNA hybridization analysis showed that the eight isolates represented five novel species. On the basis of their phenotypic and phylogenetic distinctiveness, the isolates represent five novel species within the genus Methylobacterium, for which the names Methylobacterium persicinum sp. nov. (type strain 002-165T =DSM 19562T =NBRC 103628T =NCIMB 14378T), Methylobacterium komagatae sp. nov. (type strain 002-079T =DSM 19563T =NBRC 103627T =NCIMB 14377T), Methylobacterium brachiatum sp. nov. (type strain B0021T =DSM 19569T =NBRC 103629T =NCIMB 14379T), Methylobacterium tardum sp. nov. (type strain RB677T =DSM 19566T =NBRC 103632T =NCIMB 14380T) and Methylobacterium gregans sp. nov. (type strain 002-074T =DSM 19564T =NBRC 103626T =NCIMB 14376T) are proposed.

Virgibacillus halophilus sp. nov., spore-forming bacteria isolated from soil in Japan.

Two Gram-positive, round-spore-forming, rod-shaped, halophilic bacterial strains, 5B73C(T) and 5B133E, were isolated from field soil in Kakegawa, Shizuoka, Japan, and were characterized taxonomically using a polyphasic approach. These two strains were found to comprise strictly aerobic, motile rods that formed subterminal endospores. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains 5B73C(T) and 5B133E are phylogenetically affiliated to the genus Virgibacillus, exhibiting sequence similarities of 94.1-96.4 % with respect to the type strains of Virgibacillus species. The DNA G+C contents of strains 5B73C(T) and 5B133E were 42.6 and 42.3 mol%, respectively. The cell-wall peptidoglycan type (meso-diaminopimelic acid), the major cellular fatty acids (anteiso-C(15 : 0), iso-C(15 : 0), anteiso-C(17 : 0) and iso-C(16 : 0)) and the quinone type (MK-7) of the isolates support their affiliation to the genus Virgibacillus. On the basis of their genotypic and phenotypic characteristics, the isolates represent a novel species of the genus Virgibacillus, for which the name Virgibacillus halophilus sp. nov. is proposed. The type strain is 5B73C(T) (=IAM 15308(T)=KCTC 13935(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)).

Terribacillus saccharophilus gen. nov., sp. nov. and Terribacillus halophilus sp. nov., spore-forming bacteria isolated from field soil in Japan.

Three strains, 002-048(T), RB589 and 002-051(T), isolated from field soil in Japan, were characterized using a polyphasic approach. The isolates were Gram-positive, strictly aerobic, non-motile rods that formed ellipsoidal, subterminal endospores. The chemotaxonomic characteristics of these isolates included the presence of meso-diaminopimelic acid as the cell-wall peptidoglycan, anteiso-C(15 : 0) and anteiso-C(17 : 0) as the major cellular fatty acids and MK-7 as the predominant menaquinone. The DNA G+C content was 44-46 mol%. Phylogenetic analyses based on 16S rRNA gene sequences revealed that the isolates represented an independent lineage that is distinct from related taxa and exhibited less than 94.3 % sequence similarity with respect to those taxa. Moreover, a DNA-DNA hybridization analysis showed that the three isolates represented two species. On the basis of their phenotypic and phylogenetic distinctiveness, the isolates represent two species within a novel genus, for which the names Terribacillus saccharophilus gen. nov., sp. nov. and Terribacillus halophilus sp. nov. are proposed. The type strain of T. saccharophilus is 002-048(T) (=IAM 15309(T)=KCTC 13936(T)) and the type strain of T. halophilus is 002-051(T) (=IAM 15310(T)=KCTC 13937(T)).

Reclassification of Methylobacterium chloromethanicum and Methylobacterium dichloromethanicum as later subjective synonyms of Methylobacterium extorquens and of Methylobacterium lusitanum as a later subjective synonym of Methylobacterium rhodesianum.

Phylogenetic analysis based on 16S rDNA sequences was performed on all type strains of the 14 validly described Methylobacterium species to ascertain the genealogic relationships among these species. The results showed that type strains of Methylobacterium were divided into two monophyletic groups whose members were distinct species with sequence similarity values greater than 97.0% between any two of the members in the same group. Only M. organophilum JCM 2833(T) and ATCC 27886(T) were not divided into those two groups. In particular, strains of M. dichloromethanicum and M. chloromethanicum exhibited extremely high similarity values (99.9 and 100%, respectively) with the type strain of M. extorquens. To clarify the relationships among Methylobacterium species in more detail, phylogenetic analysis based on the 5' end hyper-variable region of 16S rDNA (HV region), ribotyping analysis, fatty acid analysis, G+C content analysis and DNA-DNA hybridization experiments was performed on 58 strains of Methylobacterium species. Results of the ribotyping analysis and the phylogenetic analysis based on HV region sequences indicated that many Methylobacterium strains, including M. 'organophilum' DSM 760(T), have been erroneously identified. The DNA G+C content of Methylobacterium strains were between 68.1 and 71.3%. Results of whole-cell fatty-acid profiles showed that all strains contained 18 : 1omega7c as the primary fatty acid component (82.8-90.1%), with 16 : 0 and 18 : 0 as minor components. M. dichloromethanicum DSM 6343(T), M. chloromethanicum NCIMB 13688(T), and M. extorquens IAM 12631(T) exhibited high DNA-DNA relatedness values between each other (69-80%). M. lusitanum NCIMB 13779(T) also showed a close relationship with M. rhodesianum DSM 5687(T) at DNA-DNA relatedness levels of 89-92%. According to these results, many Methylobacterium strains should be reclassified, with M. dichloromethanicum and M. chloromethanicum regarded as a synonym of M. extorquens, and M. lusitanum a synonym for M. rhodesianum.

Reclassification of Brevibacillus brevis strains NCIMB 13288 and DSM 6472 (=NRRL NRS-887) as Aneurinibacillus danicus sp. nov. and Brevibacillus limnophilus sp. nov.

Comparison of the hypervariable region (269-279 bases in length) at the 5' end of the 16S rDNA sequences of 29 bacterial strains that were identified previously as Brevibacillus brevis showed that 13 strains clustered with Aneurinibacillus species, eight strains clustered with Bacillus species and eight strains clustered with Brevibacillus species. Based on DNA-DNA hybridization results, 27 strains, not including [Brevibacillus brevis] NCIMB 13288 and [Brevibacillus brevis] DSM 6472, were reidentified as Aneurinibacillus migulanus, Aneurinibacillus thermoaerophilus, Bacillus methanolicus, Bacillus oleronius, Brevibacillus agri, Brevibacillus brevis and Brevibacillus parabrevis. [Brevibacillus brevis] NCIMB 13288, which was located in the Aneurinibacillus cluster, showed low DNA-DNA relatedness (<14 %) and low 16S rDNA sequence similarity (96.8-97.9 %) to other Aneurinibacillus species. [Brevibacillus brevis] DSM 6472, which was located in the Brevibacillus cluster, also showed low DNA-DNA relatedness (<12 %) and low 16S rDNA sequence similarity (95.4-98.8 %) to other Brevibacillus species. These genotypic and phylogenetic data, plus phenotypic and chemotaxonomic characteristics, suggest that [Brevibacillus brevis] NCIMB 13288 (=IAM 15048) and [Brevibacillus brevis] DSM 6472 (=NRRL NRS-887) represent novel species of the genera Aneurinibacillus and Brevibacillus, respectively, for which the names Aneurinibacillus danicus sp. nov. and Brevibacillus limnophilus sp. nov. are proposed.

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.

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.