Nocardioides daeguensis sp. nov., a nitrate-reducing bacterium isolated from activated sludge of an industrial wastewater treatment plant.

A Gram-reaction-positive, rod-shaped, non-spore-forming bacterium (strain 2C1-5(T)) was isolated from activated sludge of an industrial wastewater treatment plant in Daegu, South Korea. Its taxonomic position was investigated by using a polyphasic approach. On the basis of 16S rRNA gene sequence similarity, the closest phylogenetic relatives were the type strains of Nocardioides nitrophenolicus (98.6 % similarity), N. kongjuensis (98.5 %), N. caeni (98.4 %), N. simplex (98.3 %), N. aromaticivorans (98.1 %) and N. ginsengisoli (97.5 %); the phylogenetic distance from other species with validly published names within the genus Nocardioides was greater than 3 %. Strain 2C1-5(T) was characterized chemotaxonomically as having ll-2,6-diaminopimelic acid in the cell-wall peptidoglycan, MK-8(H4) as the predominant menaquinone and iso-C16 : 0, C16 : 0 and C17 : 1ω6c as the major fatty acids. The G+C content of the genomic DNA was 74.9 mol%. These chemotaxonomic properties and phenotypic characteristics supported the affiliation of strain 2C1-5(T) to the genus Nocardioides. The results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain 2C1-5(T) from existing species with validly published names. Therefore, strain 2C1-5(T) represents a novel species of the genus Nocardioides, for which the name Nocardioides daeguensis sp. nov. is proposed, with the type strain 2C1-5(T) ( = JCM 17460(T) = KCTC 19799(T)).

Lactobacillus ginsenosidimutans sp. nov., isolated from kimchi with the ability to transform ginsenosides.

Biotransformation of ginsenosides was examined using lactic acid bacteria isolated from several kinds of kimchi. A Gram-positive, facultatively anaerobic, non-motile, non-spore-forming, and rod-shaped lactic acid bacterial strain, designated EMML 3041(T), was determined to have ginsenoside-converting activity and its taxonomic position was investigated using a polyphasic approach. Strain EMML 3041(T) displayed ß-glucosidase activity that was responsible for its ability to transform ginsenoside Rb1 (one of the dominant active components of ginseng) to F2 via gypenoside XVII, ginsenoside Rb2 to compound Y via compound O, ginsenoside Rc to compound Mc via compound Mc1, and ginsenoside Rd to ginsenoside F2. On the basis of the 16S rRNA gene sequence similarity, strain EMML 3041(T) was shown to belong to the genus Lactobacillus and is closely related to Lactobacillus versmoldensis KU-3(T) (98.3 % sequence similarity). Polyphasic taxonomy study confirmed that the strain EMML 3041(T) represents a novel species, for which the name Lactobacillus ginsenosidimutans sp. nov. is proposed, with EMML 3041(T) (=KACC 14527(T) = JCM 16719(T)) as the type strain.

Brevibacterium daeguense sp. nov., a nitrate-reducing bacterium isolated from a 4-chlorophenol enrichment culture.

A Gram-reaction-positive, non-spore-forming, aerobic actinobacterial strain (2C6-41(T)) was isolated from the activated sludge from an industrial wastewater treatment plant in Daegu, South Korea. Its taxonomic position was investigated by using a polyphasic approach. On the basis of 16S rRNA gene sequence similarity, closest phylogenetic relatives to strain 2C6-41(T) were Brevibacterium pityocampae DSM 21720(T) (97.2 %), Brevibacterium salitolerans KCTC 19616(T) (96.7 %), Brevibacterium album KCTC 19173(T) (96.2 %) and Brevibacterium samyangense KCCM 42316(T) (96.2 %). The DNA G+C content of strain 2C6-41(T) was 66.4 mol%. Chemotaxonomic data, which included MK-8(H(2)) as the major menaquinone; meso-diaminopimelic acid, glutamic acid and alanine as cell-wall amino acids; ribose, mannose and glucose as major cell-wall sugars; and anteiso-C(15 : 0), anteiso-C(17 : 0), C(16 : 0) and iso-C(15 : 0) as major fatty acids, supported the affiliation of strain 2C6-41(T) to the genus Brevibacterium. The aromatic ring cleavage enzyme catechol 1,2-dioxygenase was not detected in strain 2C6-41(T), but catechol 2,3-dioxygenase was detected. The results of physiological and biochemical tests, and the low level of DNA-DNA relatedness to the closest phylogenetic relative enabled strain 2C6-41(T) to be differentiated genotypically and phenotypically from recognized species of the genus Brevibacterium. The isolate is therefore considered to represent a novel species in the genus Brevibacterium, for which the name Brevibacterium daeguense sp. nov. is proposed. The type strain is 2C6-41(T) (=KCTC 19800(T) = JCM 17458(T)).

Sphingomonas ginsengisoli sp. nov. and Sphingomonas sediminicola sp. nov.

Two novel bacteria, designated strains Gsoil 634(T) and Dae 20(T), were isolated in South Korea from soil of a ginseng field and freshwater sediment, respectively and were characterized by a polyphasic approach to clarify their taxonomic positions. Phylogenetic analysis based on 16S rRNA gene sequences indicated that, although they probably represented two distinct species (indicated by a sequence similarity of 96.6 %), both strain Gsoil 634(T) and strain Dae 20(T) belonged to the genus Sphingomonas and were most closely related to 'Sphingomonas humi' PB323 (97.8 % and 96.7 % sequence similarity, respectively), Sphingomonas kaistensis PB56(T) (96.8 % and 96.7 %), Sphingomonas astaxanthinifaciens TDMA-17(T) (96.6 % and 95.4 %) and Sphingomonas jaspsi TDMA-16(T) (95.6 % and 95.8 %). For both novel strains, the major ubiquinone was Q-10, the major polyamine was homospermidine, the major cellular fatty acids included summed feature 7 (C(18 : 1)ω7c, C(18 : 1)ω9t and/or C(18 : 1)ω12t), C(17 : 1)ω6c and C(16 : 0), and the polar lipids included sphingoglycolipid. These chemotaxonomic data supported the affiliation of both strains to the genus Sphingomonas. However, the DNA-DNA relatedness value between strain Gsoil 634(T) and 'Sphingomonas humi' PB323(T) was 31 %. Moreover, the results of physiological and biochemical tests allowed the phenotypic differentiation of strains Gsoil 634(T) and Dae 20(T) from established members of the genus Sphingomonas. Based on these data, the two isolates represent two novel species in the genus Sphingomonas, for which the names Sphingomonas ginsengisoli sp. nov. (type strain Gsoil 634(T) = KCTC 12630(T) = DSM 18094(T) = LMG 23739(T)) and Sphingomonas sediminicola sp. nov. (type strain Dae 20(T)  = KCTC 12629(T) = DSM 18106(T) = LMG 23592(T)) are proposed.

Bioconversion of ginsenoside Rc into Rd by a novel α-L-arabinofuranosidase, Abf22-3 from Leuconostoc sp. 22-3: cloning, expression, and enzyme characterization.

A novel α-L-arabinofuranosidase (Abf22-3) that could biotransform ginsenoside Rc into Rd was obtained from the ginsenoside converting Leuconostoc sp. strain 22-3, isolated from the Korean fermented food kimchi. The gene, termed abf22-3, consisting of 1,527 bp and encoding a protein with a predicted molecular mass of 58,486 Da was cloned into the pMAL-c2x (TEV) vector. A BLAST search using the Abf22-3's amino acid sequence revealed significant homology to that of family 51 glycoside hydrolases. The over-expressed recombinant Abf22-3 in Escherichia coli BL21 (DE3) catalyzed the hydrolysis of the arabinofuranoside moiety attached to the C-20 position of ginsenoside Rc under optimal conditions of pH 6.0 and 30 °C. This result indicated that Abf22-3 selectively converts ginsenoside Rc into Rd, but did not catalyze the hydrolysis of glucopyranosyl groups from Rc or other ginsenosides such as Rb1 and Rb2. Over-expressed recombinant enzymes were purified by two steps with amylose-affinity and DEAE-cellulose chromatography and then characterized. The kinetic parameters for α-L-arabinofuranosidase showed apparent Km and Vmax values of 0.95 ± 0.02 µM and 1.2 ± 0.1 µmol min(-1) mg of protein(-1) against p-nitrophenyl-α-L-arabinofuranoside, respectively. Using a purified MBP-Abf22-3 (10 µg/ml), 0.1 % of ginsenoside Rc was completely converted to ginsenoside Rd within 20 min.

Description of Fimbriimonas ginsengisoli gen. nov., sp. nov. within the Fimbriimonadia class nov., of the phylum Armatimonadetes.

Strain Gsoil 348(T) was isolated from a ginseng field soil sample by selecting micro-colonies from one-fifth strength modified R2A agar medium after a long incubation period. 16S rRNA gene sequence analysis indicated that the strain is related to members of the phylum Armatimonadetes (formerly called candidate phylum OP10). Strain Gsoil 348(T) is mesophilic, strictly aerobic, non-motile and rod-shaped. It only grows in low nutrient media. The major respiratory quinones are menaquinones MK-11 and MK-10, and the main fatty acids are iso-C(15:0), iso-C(17:0), C(16:0) and C(16:1) ω11c. The G+C content is 61.4 mol%. The 16S rRNA gene sequences in public databases belonging to the phylum Armatimonadetes were clustered here into 6 groups. Five of these groups constituted a coherent cluster distinct from the sequences of other phyla in phylogenetic trees that were constructed using multiple-outgroup sequences from 49 different phyla. On the basis of polyphasic taxonomic analyses, it is proposed that strain Gsoil 348(T) (= KACC 14959(T) = JCM 17079(T)) should be placed in Fimbriimonas ginsengisoli gen. nov., sp. nov., as the cultured representative of the Fimbriimonadia class. nov., corresponding with Group 4 of the phylum Armatimonadetes.

Ramlibacter ginsenosidimutans sp. nov., with ginsenoside-converting activity.

A novel beta-proteobacterium, designated BXN5-27(T), was isolated from soil of a ginseng field of Baekdu Mountain in China, and was characterized using a polyphasic approach. The strain was Gram-staining-negative, aerobic, motile, non-spore-forming, and rod shaped. Strain BXN5-27(T) exhibited beta-glucosidase activity that was responsible for its ability to transform ginsenoside Rb1 (one of the dominant active components of ginseng) to compound Rd. Phylogenetic analysis based on 16S rRNA gene sequences showed that this strain belonged to the family Comamonadaceae; it was most closely related to Ramlibacter henchirensis TMB834(T) and Ramlibacter tataouinensis TTB310(T) (96.4% and 96.3% similarity, respectively). The G+C content of the genomic DNA was 68.1%. The major menaquinone was Q-8. The major fatty acids were C16:0, summed feature 4 (comprising C16:1 omega7c and/or iso-C15:0 2OH), and C17:0 cyclo. Genomic and chemotaxonomic data supported the affiliation of strain BXN5-27(T) to the genus Ramlibacter. However, physiological and biochemical tests differentiated it phenotypically from the other established species of Ramlibacter. Therefore, the isolate represents a novel species, for which the name Ramlibacter ginsenosidimutans sp. nov. is proposed, with the type strain being BXN5-27(T) (= DSM 23480(T) = LMG 24525(T) = KCTC 22276(T)).

Streptomyces panacagri sp. nov., isolated from soil of a ginseng field.

A Gram-positive, spore-forming, aerobic actinomycete, strain Gsoil 519T, was isolated from soil of a ginseng field of Pocheon province in South Korea. The closest phylogenetic relatives were Streptomyces marinus Sp080513GE-26T (97.94 % 16S rRNA gene sequence similarity), Streptomyces albiaxialis NRRL B-24327T (97.84 %), Streptomyces albus subsp. albus DSM 40313T (97.84 %), Streptomyces almquistii NBRC 13015T (97.81 %), Streptomyces gibsonii NBRC 15415T (97.81 %), Streptomyces rangoonensis NBRC 13078T (97.81 %), Streptomyces sodiiphilus YIM 80305T (97.77 %) and Streptomyces flocculus NBRC 13041T (97.67 %). The G+C content of the genomic DNA was 71.8 mol%. The chemotaxonomic data [MK-9(H6) and MK-9(H8) as the major menaquinones; ll-diaminopimelic acid as a component of the cell-wall peptidoglycan; ribose, xylose, mannose and glucose as the major cell-wall sugars; and anteiso-C15:0, iso-C15:0, iso-C17:0, anteiso-C17:0 and C16:0 as the major fatty acids] supported the affiliation of strain Gsoil 519T to the genus Streptomyces. The physiological and biochemical characteristics and the low level of DNA-DNA relatedness differentiated the isolate genotypically and phenotypically from recognized members of the genus Streptomyces. The isolate, therefore, represents a novel species, for which the name Streptomyces panacagri sp. nov. is proposed, with Gsoil 519T (=KCTC 19139T=DSM 41871T) as the type strain.

Nocardioides ginsengagri sp. nov., isolated from the soil of a ginseng field.

A Gram-reaction-positive, rod-shaped, non-motile, non-spore-forming bacterium (strain BX5-10(T)) was isolated from the soil of a ginseng field on Baekdu Mountain in Jilin district, China. The taxonomic position of this bacterium was determined in an investigation based on a polyphasic approach. On the basis of 16S rRNA gene sequence analysis, strain BX5-10(T) was shown to belong to the family Nocardioidaceae and to be most closely related to Nocardioides plantarum NCIMB 12834(T) (96.5% sequence similarity), Nocardioides dokdonensis KCTC 19309(T) (96.2%) and Nocardioides fonticola NAA-13(T) (95.1%). Strain BX5-10(T) was characterized chemotaxonomically as having ll-2,6-diaminopimelic acid in its cell-wall peptidoglycan, MK-8(H(4)) as the predominant menaquinone and C(18:1)ω9c, C(16:0) and C(17:1)ω8c as its major fatty acids. The G+C content of the genomic DNA was 70.3 mol%. The novel strain could be differentiated genotypically and phenotypically from all recognized species of the genus Nocardioides. Based on the results of the phylogenetic analyses and the genotypic and phenotypic data, a novel species, Nocardioides ginsengagri sp. nov., is proposed. The type strain is BX5-10(T) (=KCTC 19467(T)=DSM 21362(T)).

Paenibacillus sediminis sp. nov., a xylanolytic bacterium isolated from a tidal flat.

A Gram-positive, rod-shaped, xylanolytic, spore-forming bacterium, strain GTH-3(T), was isolated from a tidal flat adjacent to Ganghwa Island, Republic of Korea, and was characterized to determine its taxonomic position. On the basis of 16S rRNA gene sequence similarity, strain GTH-3(T) was shown to belong to the family Paenibacillaceae, being most closely related to the type strains of Paenibacillus ginsengisoli (94.9 %), Paenibacillus anaericanus (94.8 %), Paenibacillus urinalis (94.4 %), Paenibacillus cookii (94.2 %), Paenibacillus alvei (94.1 %) and Paenibacillus chibensis (94.0 %). The G+C content of the genomic DNA of strain GTH-3(T) was 45.9±0.2 mol% (mean±sd). The major menaquinone was MK-7. The major fatty acids were anteiso-C(15:0) and iso-C(16:0). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Phenotypic and chemotaxonomic data supported the affiliation of strain GTH-3(T) to the genus Paenibacillus. The results of physiological and biochemical tests allowed strain GTH-3(T) to be distinguished genotypically and phenotypically from recognized species of the genus Paenibacillus. Strain GTH-3(T) is therefore considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus sediminis sp. nov. is proposed. The type strain is GTH-3(T) ( = DSM 23491(T) = LMG 25635(T)).

Characterization of a novel ginsenoside-hydrolyzing α-L-arabinofuranosidase, AbfA, from Rhodanobacter ginsenosidimutans Gsoil 3054T.

The gene encoding an α-L-arabinofuranosidase that could biotransform ginsenoside Rc {3-O-[ß-D-glucopyranosyl-(1-2)-ß-D-glucopyranosyl]-20-O-[α-L-arabinofuranosyl-(1-6)-ß-D-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[ß-D-glucopyranosyl-(1-2)-ß-D-glucopyranosyl]-20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol} was cloned from a soil bacterium, Rhodanobacter ginsenosidimutans strain Gsoil 3054(T), and the recombinant enzyme was characterized. The enzyme (AbfA) hydrolyzed the arabinofuranosyl moiety from ginsenoside Rc and was classified as a family 51 glycoside hydrolase based on amino acid sequence analysis. Recombinant AbfA expressed in Escherichia coli hydrolyzed non-reducing arabinofuranoside moieties with apparent K (m) values of 0.53 ± 0.07 and 0.30 ± 0.07 mM and V (max) values of 27.1 ± 1.7 and 49.6 ± 4.1 µmol min(-1) mg(-1) of protein for p-nitrophenyl-α-L-arabinofuranoside and ginsenoside Rc, respectively. The enzyme exhibited preferential substrate specificity of the exo-type mode of action towards polyarabinosides or oligoarabinosides. AbfA demonstrated substrate-specific activity for the bioconversion of ginsenosides, as it hydrolyzed only arabinofuranoside moieties from ginsenoside Rc and its derivatives, and not other sugar groups. These results are the first report of a glycoside hydrolase family 51 α-L-arabinofuranosidase that can transform ginsenoside Rc to Rd.

Georgenia daeguensis sp. nov., isolated from 4-chlorophenol enrichment culture.

During screening for 4-chlorophenol-degrading micro-organisms in activated sludge from industrial wastewater treatment, a Gram-positive, rod-shaped, aerobic bacterial strain, designated 2C6-43(T), was isolated and characterized taxonomically by using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain 2C6-43(T) belongs to the family Bogoriellaceae, class Actinobacteria, and is related most closely to Georgenia soli CC-NMPT-T3(T) (98.8% sequence similarity), Georgenia muralis 1A-C(T) (97.6%), Georgenia thermotolerans TT02-04(T) (96.8%), Georgenia ruanii YIM 004(T) (96.6%) and Georgenia halophila YIM 93316(T) (96.0%). The G+C content of the genomic DNA of strain 2C6-43(T) was 66.2 mol%. Sugars from whole-cell hydrolysates found in strain 2C6-43(T) were rhamnose, ribose and galactose. The menaquinone MK-8(H(4)) was detected as the predominant quinone. Polar lipid analysis of 2C6-43(T) revealed diphosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylinositol and phosphatidylglycerol. An aromatic compound ring cleavage enzyme of catechol 1,2-dioxygenase was detected but catechol 2,3-dioxygenase was not detected in 2C6-43(T). A fatty acid profile with anteiso-C(15:0), iso-C(15:0) and C(16:0) as the major components supported the affiliation of strain 2C6-43(T) to the genus Georgenia. However, the DNA-DNA relatedness between strain 2C6-43(T) and the type strains of five species of the genus Georgenia ranged from 17 to 40%, clearly showing that the isolate constitutes a new genospecies. Strain 2C6-43(T) could be clearly differentiated from its phylogenetic neighbours on the basis of some phenotypic, genotypic and chemotaxonomic features. Therefore, strain 2C6-43(T) is considered to represent a novel species of the genus Georgenia, for which the name Georgenia daeguensis sp. nov. is proposed; the type strain is 2C6-43(T) (=KCTC 19801(T)=JCM 17459(T)).

Arenimonas daejeonensis sp. nov., isolated from compost.

A Gram-negative, aerobic, motile and rod-shaped bacterium, designated strain T7-07(T), was isolated from compost in Daejeon, Korea. Phylogenetic analysis based on 16S rRNA gene sequencing showed that strain T7-07(T) had 99.0% gene sequence similarity with Arenimonas malthae KACC 14618(T) and 94.7-95.9% with other recognized species of the genus Arenimonas. Cells formed creamy white to yellowish colonies on R2A agar and contained Q-8 as the predominant ubiquinone, C(15:0) iso, C(16:0) iso, C(17:1) iso ω9c and C(11:0) iso 3-OH as the major fatty acids, and diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmethylethanolamine and an unknown aminolipid as the major polar lipids. The DNA G+C content of strain T7-07(T) was 68.3 mol%. DNA-DNA reassociation experiments between T7-07(T) and Arenimonas malthae KACC 14618(T) resulted in a mean relatedness value of 22.2%. Combined genotypic and phenotypic data supported the conclusion that the strain T7-07(T) represents a novel species, for which the name Arenimonas daejeonensis sp. nov. is proposed. The type strain is T7-07(T) (=KCTC 12667(T)=DSM 18060(T)).

Bioconversion of ginsenosides Rb(1), Rb(2), Rc and Rd by novel ß-glucosidase hydrolyzing outer 3-O glycoside from Sphingomonas sp. 2F2: cloning, expression, and enzyme characterization.

A new ß-glucosidase gene (bglSp) was cloned from the ginsenoside converting Sphingomonas sp. strain 2F2 isolated from the ginseng cultivating filed. The bglSp consisted of 1344 bp (447 amino acid residues) with a predicted molecular mass of 49,399 Da. A BLAST search using the bglSp sequence revealed significant homology to that of glycoside hydrolase superfamily 1. This enzyme was overexpressed in Escherichia coli BL21 (DE3) using a pET21-MBP (TEV) vector system. Overexpressed recombinant enzymes which could convert the ginsenosides Rb(1), Rb(2), Rc and Rd to the more pharmacological active rare ginsenosides gypenoside XVII, ginsenoside C-O, ginsenoside C-Mc(1) and ginsenoside F(2), respectively, were purified by two steps with Amylose-affinity and DEAE-Cellulose chromatography and characterized. The kinetic parameters for ß-glucosidase showed the apparent K(m) and V(max) values of 2.9±0.3 mM and 515.4±38.3 µmol min(-1)mg of protein(-1) against p-nitrophenyl-ß-d-glucopyranoside. The enzyme could hydrolyze the outer C3 glucose moieties of ginsenosides Rb(1), Rb(2), Rc and Rd into the rare ginsenosides Gyp XVII, C-O, C-Mc(1) and F(2) quickly at optimal conditions of pH 5.0 and 37°C. A little ginsenoside F(2) production from ginsenosides Gyp XVII, C-O, and C-Mc(1) was observed for the lengthy enzyme reaction caused by the side ability of the enzyme.

Mucilaginibacter composti sp. nov., with ginsenoside converting activity, isolated from compost.

The Gram-negative, strictly aerobic, non-motile, non-spore-forming, rod shaped bacterial strain designated TR6-03(T) was isolated from compost, and its taxonomic position was investigated by using a polyphasic approach. Strain TR6-03(T) grew at 4-42°C and at pH 6.0-8.0 on R2A and nutrient agar without NaCl supplement. Strain TR6-03(T) had ß-glucosidase activity, which was responsible for its ability to transform ginsenoside Re (one of the dominant active components of ginseng) to Rg(2). On the basis of 16S rRNA gene sequence similarity, strain TR6-03(T) was shown to belong to the family Sphingobacteriaceae and to be related to Mucilaginibacter lappiensis ANJLI2(T) (96.3% sequence similarity), M. dorajii FR-f4(T) (96.1%), and M. rigui WPCB133(T) (94.1%). The G+C content of the genomic DNA was 45.6%. The predominant respiratory quinone was MK-7 and the major fatty acids were summed feature 3 (comprising C(16:1) ω7c and/or iso-C(15:0) 20H), iso-C(16:0) and iso-C(17:0) 3OH. DNA and chemotaxonomic data supported the affiliation of strain TR6-03(T) to the genus Mucilaginibacter. Strain TR6-03(T) could be differentiated genotypically and phenotypically from the recognized species of the genus Mucilaginibacter. The isolate therefore represents a novel species, for which the name Mucilaginibacter composti sp. nov. is proposed, with the type strain TR6-03(T) (=KACC 14956(T) = KCTC 12642(T) =LMG 23497(T)).

A six-well plate method: less laborious and effective method for cultivation of obligate anaerobic microorganisms.

We developed a simple, less laborious method to cultivate and isolate obligate anaerobic microorganisms using a six-well plate together with the AnaeroPack System, designated as the six-well plate method. The cultivation efficiency of this method, based on colony-forming units, colony formation time, and colony size, was evaluated with four authentic obligate anaerobes (two methanogenic archaea and two sulfate-reducing bacteria). The method was found to be comparable to or even better than the roll tube method, a technique that is commonly used at present for the cultivation of obligate anaerobes. Further experiments using 21 representative obligate anaerobes demonstrated that all examined anaerobes (11 methanogens, 5 sulfate- or thiosulfate-reducing bacteria, and 5 syntrophs) could form visible colonies on the six-well plate and that these colonies could be successfully subcultured in fresh liquid media. Using this method, an unidentified sulfate-reducing bacterium was successfully isolated from an environmental sample.

Rhodanobacter panaciterrae sp. nov., a bacterium with ginsenoside-converting activity isolated from soil of a ginseng field.

A novel gammaproteobacterium, designated LnR5-47(T), was isolated from soil of a ginseng field in Liaoning province, China. The isolate was a Gram-negative, aerobic, non-motile, non-spore-forming rod. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain LnR5-47(T) belonged to the genus Rhodanobacter. The isolate was most closely related to Rhodanobacter ginsengisoli GR17-7(T), Rhodanobacter terrae GP18-1(T), Dyella ginsengisoli Gsoil 3046(T), Rhodanobacter soli DCY45(T), Dyella soli JS12-10(T) and Dyella japonica IAM 15069(T) (98.0, 97.9, 97.7, 97.3, 97.2 and 97.1% 16S rRNA gene sequence similarity, respectively). Chemotaxonomic data (Q-8 as the predominant ubiquinone, and iso-C(16:0), iso-C(17:1)ω9c and iso-C(15:0) as the major fatty acids) also supported the affiliation of strain LnR5-47(T) with the genus Rhodanobacter. However, DNA-DNA relatedness between strain LnR5-47(T) and its closest phylogenetic neighbours was <25.8%. Moreover, physiological and biochemical tests phenotypically differentiated the isolate from other members of the genus Rhodanobacter. Therefore, strain LnR5-47(T) represents a novel species, for which the name Rhodanobacter panaciterrae sp. nov. is proposed; the type strain is LnR5-47(T) (=KACC 12826(T)=KCTC 22232(T)=LMG 24460(T)).

Tumebacillus ginsengisoli sp. nov., isolated from soil of a ginseng field.

A gram-reaction-positive, rod-shaped, spore-forming bacterium, designated Gsoil 1105(T), was isolated from soil of a ginseng field in Pocheon Province in South Korea and characterized in order to determine its taxonomic position. Comparative analysis of the 16S rRNA gene sequence showed that the isolate belongs to the order Bacillales, showing the highest level of sequence similarity with respect to Tumebacillus permanentifrigoris Eur1 9.5(T) (94.6 %). The phylogenetic distances from other described species with validly published names within the order Bacillales were greater than 9.0 %. Strain Gsoil 1105(T) had a genomic DNA G+C content of 55.6 mol% and menaquinone 7 (MK-7) as the major respiratory quinone. The major fatty acids were iso-C(15 : 0) and anteiso-C(15 : 0). On the basis of its phenotypic properties and phylogenetic distinctiveness, strain Gsoil 1105(T) represents a novel species of the genus Tumebacillus, for which the name Tumebacillus ginsengisoli sp. nov. is proposed. The type strain is Gsoil 1105(T) ( = KCTC 13942(T)  = DSM 18389(T)).

Inquilinus ginsengisoli sp. nov., isolated from soil of a ginseng field.

A Gram-reaction-negative, chemo-organotrophic, non-motile, non-spore-forming, rod-shaped bacterium (strain Gsoil 080(T)) was isolated from soil collected in a ginseng field in Pocheon Province, South Korea, and was investigated by using a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence analysis showed that strain Gsoil 080(T) was related most closely to Inquilinus limosus strains AU0476(T) and AU1979 (98.9 % similarity to both). Strain Gsoil 080(T) shared ≤91.3 % 16S rRNA gene sequence similarity with the type strains of other recognized species examined. The genus Inquilinus belongs to the family Rhodospirillaceae in the order Rhodospirillales, class Alphaproteobacteria. The predominant ubiquinone was Q-10 and the major fatty acids were summed feature 7 (C(18 : 1)ω9c/ω12t/ω7c) and C(19 : 0) cyclo ω8c. The G+C content of the genomic DNA of strain Gsoil 080(T) was 69.9 mol%. The level of DNA-DNA relatedness between strain Gsoil 080(T) and I. limosus LMG 20952(T) was 12 %. The results of genotypic analyses in combination with chemotaxonomic and physiological data demonstrated that strain Gsoil 080(T) represents a novel species of the genus Inquilinus, for which the name Inquilinus ginsengisoli sp. nov. is proposed. The type strain is Gsoil 080(T) (=KCTC 12574(T) =LMG 23638(T)).

Phycicoccus ginsenosidimutans sp. nov., isolated from soil of a ginseng field.

A Gram-positive, non-motile, non-spore-forming, aerobic, coccoid-shaped bacterium, designated BXN5-13(T), was isolated from the soil of a ginseng field from Baekdu Mountain in Jilin district, China. Strain BXN5-13(T) grew optimally at 30 °C and pH 6.5-7.5 with 0-2  % (w/v) NaCl. Strain BXN5-13(T) had ß-glucosidase activity that was connected with ginsenoside-converting ability, so that it was able to convert ginsenoside Rb(1) to ginsenoside F2. On the basis of 16S rRNA gene sequence analysis, the closest phylogenetic relatives of strain BXN5-13(T) were Phycicoccus aerophilus 5516T-20(T) (98.4  % 16S rRNA gene sequence similarity), P. bigeumensis MSL-03(T) (98.3  %), P. dokdonensis DS-8(T) (97.9  %) and P. jejuensis KSW2-15(T) (96.9  %). Lower sequence similarity (<97.0  %) was found with the type strains of other recognized species of the family Intrasporangiaceae. The predominant quinone was MK-8(H4). The major fatty acids (>10  %) were iso-C15:0, C17:0, anteiso-C15:0 and iso-C16:0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and phosphatidylinositol. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The chemotaxonomic data and the high genomic DNA G+C content of strain BXN5-13(T) (70.8l %) supported its affiliation with the genus Phycicoccus. DNA-DNA relatedness between strain BXN5-13(T) and its closest phylogenetic neighbours was below 16  %. Strain BXN5-13(T) represents a novel species within the genus Phycicoccus, for which the name Phycicoccus ginsenosidimutans sp. nov. is proposed. The type strain is BXN5-13(T) (=KCTC 19419(T)=DSM 21006(T)=LMG 24462(T)).