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).

Advenella faeciporci sp. nov., a nitrite-denitrifying bacterium isolated from nitrifying-denitrifying activated sludge collected from a laboratory-scale bioreactor treating piggery wastewater.

Strain M-07(T) was isolated from nitrifying-denitrifying activated sludge treating piggery wastewater. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that strain M-07(T) belonged to the genus Advenella. 16S rRNA gene sequence similarity between M-07(T) and Advenella incenata CCUG 45225(T), Advenella mimigardefordensis DPN7(T) and Advenella kashmirensis WT001(T) was 96.5, 97.3 and 96.9%, respectively. The DNA G+C content of strain M-07(T) was 49.5 mol%, which was approximately 5 mol% lower than the range for the genus Advenella (53.5-58.0 mol%). The predominant cellular fatty acids of strain M-07(T) were C(16:0), summed feature 3 (comprising C(16:1)ω7c and/or iso-C(15:0) 2-OH), C(17:0) cyclo and summed feature 2 (comprising one or more of C(14:0) 3-OH, iso-C(16:1) I, an unidentified fatty acid with an equivalent chain-length of 10.928 and C(12:0) alde). The isoprenoid quinone was Q-8. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness, strain M-07(T) should be classified as a novel species of the genus Advenella, for which the name Advenella faeciporci sp. nov. is proposed. The type strain is M-07(T) ( = JCM 17746(T)  = KCTC 23732(T)).

Leifsonia lichenia sp. nov., isolated from lichen in Japan.

An actinobacterial strain, 2SbT, isolated from lichen was characterized taxonomically using a polyphasic approach. Strain 2SbT was Gram-positive, strictly aerobic, rod-shaped and non-motile. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain 2SbT was located in the genus Leifsonia. Level of 16S rRNA gene sequence similarity between the isolated strains and the type strains of Leifsonia species were 95.0-99.2%. The value of DNA-DNA relatedness between strain 2SbT and Leifsonia poae, a phylogenetically related and phenotypically similar species, was 39.9/25.2%. The DNA G+C content of the strain 2SbT was 61.7 mol%. Cell wall peptidoglycan type (2,4-diaminobutyric acid), major cellular fatty acids (anteiso-C(15:0), anteiso-C(17:0) and iso-C(16:0)) and quinone type (MK-11 and MK-10) of the isolate support their affiliation to the genus Leifsonia. On the basis of phylogenetic position, physiological and chemotaxonomic properties, strain 2SbT represent a novel species of the genus Leifsonia, for which the name Leifsonia lichenia sp. nov. is proposed. The type strain is 2SbT (=IAM 15426T =JCM 23226T =KCTC 13122T).

Sporosarcina luteola sp. nov. isolated from soy sauce production equipment in Japan.

A Gram-variable, spore-forming, motile rod, designated strain Y1T, was isolated from the hopper surface of equipment used for soy sauce production. Phylogenetic analysis based on 16S rRNA gene sequence revealed that Y1T is affiliated phylogenetically to the genus Sporosarcina, and the strain showed sequence similarities of 95.8-99.2% to those of Sporosarcina species with validly published names. The values of DNA-DNA relatedness between strain Y1T and related type strains of the genus Sporosarcina were below 27%. The major cellular fatty acids were iso-C(15:0) and anteiso-C(15:0). The cell-wall peptidoglycan was of the A4alpha type (Lys-Glu) and the major isoprenoid quinone was MK-7. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The genomic DNA G+C content of the strain was 43.6 mol%. On the basis of phylogenetic analysis and physiological and chemotaxonomic data, the isolate represents a novel species of the genus Sporosarcina, for which the name Sporosarcina luteola sp. nov. is proposed. The type strain is strain Y1T (= JCM 15791T = NRRL B-59180T = NBRC 105378T = CIP 109917T = NCIMB 14541T).

Oceanobacillus soja sp. nov. isolated from soy sauce production equipment in Japan.

A Gram-positive, spore-forming, motile rod-shaped bacterium, designated strain Y27T, was isolated from the bottom of a mold fermenter used in the process of soy sauce production. Phylogenetic analysis of the 16S rRNA gene sequence from this strain placed it within the genus Oceanobacillus, and further sequence analysis revealed that this strain has a sequence similarity of 95.0-98.7% to other known species of Oceanobacillus. The DNA-DNA relatedness between strain Y27T and related type strains of the genus Oceanobacillus is below 43%, indicating that it should be considered a separate species. Characterization of strain Y27T revealed that the major cellular fatty acid is anteiso-C(15:0), the cell wall contains meso-diaminopimelic acid-type peptidoglycans, the major menaquinone is MK-7, and the major polar lipids are diphosphatidylglycerol and phosphatidylglycerol. The genomic DNA G+C content of the strain is 38.0 mol%. On the basis of these phylogenetic, physiological and chemotaxonomic data, we propose that this isolate represents a novel species of the genus Oceanobacillus, and propose the name Oceanobacillus soja sp. nov. The type strain is strain Y27T (= JCM 15792T = NRRL B-59181T = NBRC 105379T = NCIMB 14542T).

Schumannella luteola gen. nov., sp. nov., a novel genus of the family Microbacteriaceae.

An actinobacterial strain KHIA(T), isolated from lichen in Tokyo, was taxonomically characterized using a polyphasic approach. The isolate is a Gram-positive, anaerobic, non-motile and rodshaped bacterium. Phylogenetic analyses based on 16S rRNA gene sequences revealed that the isolate is represented as an independent lineage distinctive from the Microbacteriaceae genera. The G + C content of DNA was 58.7 mol%. The chemotaxonomic characteristics of the isolate are cell wall peptidoglycan type (2,4-diaminobutyric acid), major cellular fatty acids (anteiso-C(15:0) and iso-C(16:0)) and quinone type (MK-11 and MK-10). On the basis of the phenotypic and phylogenetic distinctness, it is proposed that strain KHIA(T )represents a novel species in a new genus of the family Microbacteriaceae, Schumannella luteola gen. nov., sp. nov. The type strain is KHIA(T )(=JCM 23215(T)=TISTR 1824(T)).

Halobacillus faecis sp. nov., a spore-forming bacterium isolated from a mangrove area on Ishigaki Island, Japan.

A Gram-positive, spore-forming, rod-shaped halophilic bacterial strain, IGA7-4T, was isolated from a mangrove area on Ishigaki Island (Japan), and was characterized taxonomically using a polyphasic approach. Strain IGA7-4T was strictly aerobic and non-motile and formed central endospores. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain IGA7-4T is affiliated to the genus Halobacillus, and exhibits sequence similarities of 99.6-98.0% to the type strains of Halobacillus species. Levels of DNA-DNA relatedness between strain IGA7-4T and the type strains of Halobacillus species were 9.5-46.6%. The DNA G+C content of strain IGA7-4T was 46.5 mol%. The cell-wall peptidoglycan type (Orn-Asp), major cellular fatty acids (anteiso-C15:0, anteiso-C17:0, iso-C15:0 and iso-C16:0) and quinone type (MK-7) of the isolate support its affiliation to the genus Halobacillus. On the basis of phylogenetic analysis, phenotypic characteristics and chemotaxonomic data, the isolate represents a novel species of the genus Halobacillus, for which the name Halobacillus faecis sp. nov. is proposed. The type strain is IGA7-4T (=MBIC08268T=IAM 15427T=KCTC 13121T).

Amphibacillus sediminis sp. nov., an endospore-forming bacterium isolated from lake sediment in Japan.

A facultatively anaerobic, moderately alkaliphilic, Gram-positive, spore-forming and rod-shaped bacterial strain, Shu-P-Ggiii25-2T, was isolated from lake sediment in Shizuoka, Japan, and was characterized taxonomically using a polyphasic approach. Analysis of the 16S rRNA gene sequence indicated that the novel isolate clustered with the type strain of Amphibacillus xylanus and it exhibited sequence similarities of 94.9-95.6% to the type strains of species of the genus Amphibacillus. Isoprenoid quinones and oxidase and catalase activities were not detected for strain Shu-P-Ggiii25-2T. The DNA G+C content was 42.3 mol%, the cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid and the major cellular fatty acids were iso-C14:0, iso-C15:0, anteiso-C15:0, C16:0 and iso-C16:0. On the basis of phylogenetic position and phenotypic properties, strain Shu-P-Ggiii25-2T represents a novel species of the genus Amphibacillus, for which the name Amphibacillus sediminis sp. nov. is proposed. The type strain is Shu-P-Ggiii25-2T (=MBIC08269T=IAM 15428T=KCTC 13120T). An emended description of the genus Amphibacillus is also given.

Sporosarcina saromensis sp. nov., an aerobic endospore-forming bacterium.

Two Gram-positive, endospore-forming, rod-shaped bacterial strains, HG645(T) and HG711, were respectively isolated from surface water of a brackish lake and sediment of a fishery harbour in Japan and were subsequently characterized taxonomically using a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains HG645(T) and HG711 are affiliated phylogenetically to the genus Sporosarcina, and they exhibit sequence similarities of 95.7-97.3 % to the type strains of Sporosarcina species. DNA-DNA relatedness between strain HG645(T) and the type strain of the phylogenetically related species Sporosarcina aquimarina was less than 10 %. The DNA G+C content of strains HG645(T) and HG711 were respectively 46.0 and 45.2 mol%. Major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The cell-wall peptidoglycan type (Lys-Glu), major cellular fatty acids (iso-C(15 : 0) and anteiso-C(15 : 0)) and quinone type (MK-7) of the isolates support their affiliation to the genus Sporosarcina. On the basis of phylogenetic analysis and physiological and chemotaxonomic data, the isolates represent a novel species of the genus Sporosarcina, for which the name Sporosarcina saromensis sp. nov. is proposed. The type strain is strain HG645(T) (=MBIC08270(T)=IAM 15429(T) =KCTC 13119(T)).

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)).

The status of the species Leifsonia rubra Reddy et al. 2003. Request for an opinion.

On the basis of 16S rRNA gene sequence analysis, it is ascertained that the type strain of Leifsonia rubra does not exist in any established culture collection or with the authors who described this species. Therefore, it cannot be included in any scientific study. It is proposed that the Judicial Commission place the name Leifsonia rubra on the list of rejected names if a suitable type strain is not found or a neotype is not proposed within two years following the publication of this Request for an Opinion.

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)).

Cloning, high-level expression and enzymatic properties of an intracellular serine protease from Bacillus sp. WRD-2.

A gene, isp-B, encoding an intracellular serine protease from a newly isolated Bacillus sp. WRD-2 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 960 bp encoding a polypeptide comprised of 319 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other intracellular serine proteases, including active sites, Ser, His and Asp, as well as no signal sequence. The predicted amino acid sequence showed more than 60% homology with the intracellular serine proteases from Bacillus species. When expressed in E. coli, the recombinant enzyme (rISP-B) was overproduced in the cytoplasm as soluble and active form. The purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, EDTA and antipain. The enzyme showed maximum activity at pH 8.0 and 45 degrees C. It was stable atpH from 7.5 to 11.0 and below 50 degrees C.