Cohnella panacarvi sp. nov., a xylanolytic bacterium isolated from ginseng cultivating soil

A Gram-positive, aerobic, rod-shaped, nonmotile, endospore-forming bacterium, designated Gsoil 349T, was isolated from soil of a ginseng field and characterized using a polyphasic approach. Comparative analysis of 16S rRNA gene sequences revealed that the strain Gsoil 349T belongs to the family Paenibacillaceae, and the sequence showed closest similarity with Cohnella thermotolerans DSM 17683T (94.1%) and Cohnella hongkongensis DSM 17642T (93.6%). The strain showed less than 91.3% 16S rRNA gene sequence similarity with Paenibacillus species. In addition, the presence of MK-7 as the major menaquinone and anteiso-C(15:0), iso-C(16:0), and C(16:0) as major fatty acids suggested its affiliation to the genus Cohnella. The G+C content of the genomic DNA was 53.4 mol%. On the basis of its phenotypic characteristics and phylogenetic distinctiveness, strain Gsoil 349T should be treated as a novel species within the genus Cohnella for which the name Cohnella panacarvi sp. nov. is proposed. The type strain is Gsoil 349T (=KCTC 13060T = DSM 18696T).     

Chitinophaga soli sp. nov. and Chitinophaga terrae sp. nov., isolated from soil of a ginseng field in Pocheon Province, Korea

Two novel strains of the Cytophaga-Flexibacter-Bacteroides (CFB) group, designated Gsoil 219" and Gsoil 2381, were isolated from soil of a ginseng field of Pocheon Province in Korea. Both strains were Gram-negative, aerobic, nonmotile, nonspore-forming, and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences indicated that both isolates belong to the genus Chitinophaga but were clearly separated from established species of this genus. The sequence similarities between strain Gsoil 219T and type strains of the established species and between strain Gsoil 238T and type strains of the established species ranged from 91.4 to 94.7% and 91.6 to 94.2%, respectively. Phenotypic and chemotaxonomic data (major menaquinone, MK-7; major fatty acids, iso-C15:0 and C(16:1) omega5c; major hydroxy fatty acid, iso-C(17:0) 3-OH; major polyamine, homospermidine) supported the affiliation of both strains Gsoil 219T and Gsoil 238T to the genus Chitinophaga. Furthermore, the results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of both strains from the other validated Chitinophaga species. Therefore, the two isolates represent two novel species, for which the name Chitinophaga soli sp. nov. (type strain, Gsoil 219T=KCTC 12650T=DSM 18093T) and Chitinophaga terrae sp. nov. (type strain, Gsoil 238T=KCTC 12651T=DSM 18078T) are proposed.     

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-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-[α-l-arabinofuranosyl-(1–6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-β-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.     

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 Rb? (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 C??:?, summed feature 4 (comprising C??:? omega7c and/or iso-C??:? 2OH), and C??:? 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)).     

Kinetics of a cloned special ginsenosidase hydrolyzing 3-O-glucoside of multi-protopanaxadiol-type ginsenosides, named ginsenosidase type III

In this paper, the kinetics of a cloned special glucosidase, named ginsenosidase type III hydrolyzing 3-O-glucoside of multi-protopanaxadiol (PPD)-type ginsenosides, were investigated. The gene (bgpA) encoding this enzyme was cloned from a Terrabacter ginsenosidimutans strain and then expressed in E. coli cells. Ginsenosidase type III was able to hydrolyze 3-O-glucoside of multi-PPD-type ginsenosides. For instance, it was able to hydrolyze the 3-O-β-D-(1-->2)-glucopyranosyl of Rb1 to gypenoside XVII, and then to further hydrolyze the 3-O-β-D-glucopyranosyl of gypenoside XVII to gypenoside LXXV. Similarly, the enzyme could hydrolyze the glucopyranosyls linked to the 3-O- position of Rb2, Rc, Rd, Rb3, and Rg3. With a larger enzyme reaction Km value, there was a slower enzyme reaction speed; and the larger the enzyme reaction Vmax value, the faster the enzyme reaction speed was. The Km values from small to large were 3.85 mM for Rc, 4.08 mM for Rb1, 8.85 mM for Rb3, 9.09 mM for Rb2, 9.70 mM for Rg3(S), 11.4 mM for Rd and 12.9 mM for F2; and Vmax value from large to small was 23.2 mM/h for Rc, 16.6 mM/h for Rb1, 14.6 mM/h for Rb3, 14.3 mM/h for Rb2, 1.81mM/h for Rg3(S), 1.40 mM/h for Rd, and 0.41 mM/h for F2. According to the Vmax and Km values of the ginsenosidase type III, the hydrolysis speed of these substrates by the enzyme was Rc>Rb1>Rb3>Rb2>Rg3(S)>Rd>F2 in order.     

<Emphasis Type="Italic">Terrabacter ginsengisoli</Emphasis> sp. nov., isolated from ginseng cultivating soil

A Gram-positive, strictly aerobic, nonmotile, yellowish, coccus-rod-shaped bacterium (designated Gsoil 653^T) isolated from ginseng cultivating soil was characterized using a polyphasic approach to clarify its taxonomic position. The strain Gsoil 653^T exhibited optimal growth at pH 7.0 on R2A agar medium at 30°C. Phylogenetic analysis based on 16S rRNA gene sequence similarities, indicated that Gsoil 653^T belongs to the genus Terrabacter of the family Humibacillus, and was closely related to Terrabacter tumescens DSM 20308^T (98.9%), Terrabacter carboxydivorans PY2^T (98.9%), Terrabacter terrigena ON10^T (98.8%), Terrabacter terrae PPLB^T (98.6%), and Terrabacter lapilli LR-26^T (98.6%). The DNA G + C content was 70.5 mol%. The major quinone was MK-8(H₄). The primary polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidyl-ethanolamine. The predominant fatty acids were iso-C_15:0, iso-C_16:0, iso-C_14:0, and anteiso-C_15:0, as in the case of genus Terrabacter, thereby supporting the categorization of strain Gsoil 653^T. However, the DNA-DNA relatedness between Gsoil 653^T and closely related strains of Terrabacter species was low at less than 31%. Moreover, strain Gsoil 653^T could be both genotypically and phenotypically distinguished from the recognized species of the genus Terrabacter. This isolate, therefore, represents a novel species, for which the name Terrabacter ginsengisoli sp. nov. is proposed with the type strain Gsoil 653^T (= KACC 19444^T = LMG 30325^T).     

Characterization of diverse heterocyclic amine-degrading denitrifying bacteria from various environments

Although, there have been many published bacterial strains aerobically degrading the heterocyclic amine compounds, only one strain to date has been reported to degrade pyrrolidine under denitrifying conditions. In this study, denitrifying bacteria degrading pyrrolidine and piperidine were isolated from diverse geological and ecological origins through selective enrichment procedures. Based on the comparative sequence results of 16S rRNA genes, 30 heterocyclic amine-degrading isolates were grouped into ten distinct phylotypes belonging to the genera Thauera, Castellaniella, Rhizobium, or Paracoccus of the phylum Proteobacteria. The representative isolates of individual phylotypes were characterized by phylogenetic, phenotypic and chemotaxonomical traits, and dissimilatory nitrite reductase gene (nirK and nirS). All isolates completely degraded pyrrolidine and piperidine under both aerobic and anaerobic conditions. The anaerobic degradations were coupled to nitrate reduction. A metabolic pathway for the anaerobic degradation of pyrrolidine was proposed on the basis of enzyme activities implicated in pyrrolidine metabolism from three isolates. The three key pyrrolidine-metabolizing enzymes pyrrolidine dehydrogenase, γ-aminobutyrate/α-ketoglutarate aminotransferase, and succinic semialdehyde dehydrogenase, were induced by heterocyclic amines under denitrifying conditions. They were also induced in cells grown aerobically on heterocyclic amines, suggesting that the anaerobic degradation of pyrrolidine shares the pathway with aerobic degradation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and Characterization of a Novel Terrabacter ginsenosidimutans sp. nov. β-Glucosidase That Transforms Ginsenoside Rb1 into the Rare Gypenosides XVII and LXXV

A new β-glucosidase from a novel strain of Terrabacter ginsenosidimutans (Gsoil 3082^T) obtained from the soil of a ginseng farm was characterized, and the gene, bgpA (1,947 bp), was cloned in Escherichia coli. The enzyme catalyzed the conversion of ginsenoside Rb1 {3-O-[β-d-glucopyranosyl-(1-2)-β-d-glucopyranosyl]-20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to the more pharmacologically active rare ginsenosides gypenoside XVII {3-O-β-d-glucopyranosyl-20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol}, gypenoside LXXV {20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol}, and C-K [20-O-(β-d-glucopyranosyl)-20(S)-protopanaxadiol]. A BLAST search of the bgpA sequence revealed significant homology to family 3 glycoside hydrolases. Expressed in E. coli, β-glucosidase had apparent K_m values of 4.2 ± 0.8 and 0.14 ± 0.05 mM and V_max values of 100.6 ± 17.1 and 329 ± 31 μmol·min⁻¹·mg of protein⁻¹ against p-nitrophenyl-β-d-glucopyranoside and Rb1, respectively. The enzyme catalyzed the hydrolysis of the two glucose moieties attached to the C-3 position of ginsenoside Rb1, and the outer glucose attached to the C-20 position at pH 7.0 and 37°C. These cleavages occurred in a defined order, with the outer glucose of C-3 cleaved first, followed by the inner glucose of C-3, and finally the outer glucose of C-20. These results indicated that BgpA selectively and sequentially converts ginsenoside Rb1 to the rare ginsenosides gypenoside XVII, gypenoside LXXV, and then C-K. Herein is the first report of the cloning and characterization of a novel ginsenoside-transforming β-glucosidase of the glycoside hydrolase family 3.Ginseng refers to the roots of members of the plant genus Panax, which have been used as a traditional medicine in Asian countries for over 2,000 years due to their observed beneficial effects on human health. Ginseng saponins, also referred to as ginsenosides, are the major active components of ginseng (27). Various biological activities have been ascribed to ginseng saponins, including anti-inflammatory activity (43), antitumor effects (23, 39), and neuroprotective and immunoprotective (15, 31) effects.Ginsenosides can be categorized as protopanaxadiol (PPD), protopanaxatriol, and oleanane saponins, based on the structure of the aglycon, with a dammarane skeleton (29). The PPD-type ginsenosides are further classified into subgroups based on the position and number of sugar moieties attached to the aglycon at positions C-3 and C-20. For example, one of the largest PPD-type ginsenosides, Rb1 {3-O-[β-d-glucopyranosyl-(1-2)-β-d-glucopyranosyl]-20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol}, contains 4 glucose moieties, two each attached via glycosidic linkages to the C-3 and C-20 positions of the aglycon (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Chemical structures of protopanaxadiol and protopanaxatriol ginsenosides (5). The ginsenosides represented here are all (S)-type ginsenosides. glc, β-d-glucopyranosyl; arap, α-l-arabinopyranosyl; araf, α-l-arabinofuranosyl; rha, α-l-rhamnopyranosyl; Gyp, gypenoside; C, compound.Because of their size, low solubility, and poor permeability across the cell membrane, it is difficult for human body to directly absorb large ginsenosides (44), although these components constitute the major portion of the total ginsenoside in raw ginseng (30). Moreover, the lack of the availability of the rare ginsensoides limits the research on their biological and medicinal properties. Therefore, transformation of these major ginsenosides into smaller deglycosylated ginsenosides, which are more effective in in vivo physiological action, is required (1, 37).The production of large amounts of rare ginsenosides from the major ginsenosides can be accomplished through a number of physiochemical methods such as heating (17), acid treatment (2), and alkali treatment (48). However, these approaches produce nonspecific racemic mixtures of rare ginsenosides. As an alternative, enzymatic methods have been explored as a way to convert the major ginsenosides into more pharmacologically active rare ginsenosides in a more specific manner (14, 20).To date, three types of glycoside hydrolases, β-d-glucosidase, α-l-arabinopyranosidase, and α-l-arabinofuranosidase, have been found to be involved in the biotransformation of PPD-type ginsenosides. For example, a β-glucosidase isolated from a fungus converts Rb1 to C-K [20-O-(β-d-glucopyranosyl)-20(S)-protopanaxadiol] (45), and an α-l-arabinopyranosidase and α-l-arabinofuranosidase have been isolated from an intestinal bacterium that hydrolyze, respectively, Rb2 {3-O-[β-d-glucopyranosyl-(1-2)-β-d-glucopyranosyl]-20-O-[α-l-arabinopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to Rd {3-O-[β-d-glucopyranosyl-(1-2)-β-d-glucopyranosyl]-20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol} and Rc {3-O-[β-d-glucopyranosyl-(1-2)-β-d-glucopyranosyl]-20-O- [α-l-arabinofuranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to Rd (34). Two recombinant enzymes that convert major ginsenosides into rare ginsenosides have been cloned and expressed in Escherichia coli: Solfolobus solfataricus β-glycosidase, which transforms Rb1 or Rc to C-K (28), and β-glucosidase from a soil metagenome, which transforms Rb1 to Rd (16). Both of these glycoside hydrolases are family 1 glycoside hydrolases.Here, we report the cloning and expression in E. coli of a gene (bgpA) encoding a new ginsenoside-hydrolyzing β-glucosidase from a novel bacterial strain, Terrabacter ginsenosidimutans sp. nov. Gsoil 3082, isolated from a ginseng farm in Korea. BgpA is a family 3 glycoside hydrolase, and the recombinant enzyme employs a different enzymatic pathway from ginsenoside-hydrolyzing family 1 glycoside hydrolases. BgpA preferentially and sequentially hydrolyzed the terminal and inner glucoses at the C-3 position of ginsenoside Rb1 and then the outer glucose at the C-20 position. Thus, BgpA could be effective in the biotransformation of ginsenoside Rb1 to gypenoside (Gyp) XVII {3-O-β-d-glucopyranosyl-20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol}, Gyp LXXV {20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol}, and C-K.     

Isolation and Characterization of Bacteria Capable of Degrading Phenol and Reducing Nitrate Under Low-Oxygen Conditions

Nitrate-reducing bacteria capable of degrading phenol were isolated from natural and contaminated environments under low-oxygen conditions with nitrate-containing media, using phenol as a sole carbon and energy source. A total of 27 bacteria able to degrade phenol and reduce nitrate under low-oxygen conditions were isolated from all of the inoculum samples, regardless of previous phenol contamination. For all of these bacteria, oxygen was an essential requirement for phenol degradation. Nitrate reduction by 19 of the strains was insensitive to 10 mM sodium azide, and these strains were placed into the - and -subclasses of Proteobacteria and two were Gram-positive bacteria. To date, the order of Rhizobiales has hardly been reported to have an ability to degrade aromatic compounds. Interestingly, our study showed that all isolates that were placed into the -subclass of Proteobacteria are in the order of Rhizobiales. Furthermore, the genus Agrobacterium was isolated from most inoculum samples and one genus of Gram-positive bacteria, Staphylococcus, was also isolated. In the case of the remaining eight strains, nitrate reduction was inhibited by 10 mM sodium azide. Of these strains, seven were placed into the -subclass of Proteobacteria.     

Roseomonas sediminicola sp. nov., isolated from fresh water

A Gram-stain negative, strictly aerobic, non-motile, non-spore-forming, and rod-shaped bacterial strain designated FW-3^T was isolated from fresh water and its taxonomic position was investigated by using a polyphasic approach. Strain FW-3^T was found to grow at 10–37 °C and at pH 7.0 in the absence of NaCl on nutrient agar. On the basis of 16S rRNA gene sequence similarity, strain FW-3^T was shown to belong to the family Acetobacteraceae and to be related to Roseomonas lacus TH-G33^T (97.2 % sequence similarity) and Roseomonas terrae DS-48^T (96.4 %). The G+C content of the genomic DNA was determined to be 68.0 %. The major menaquinone was determined to be Q-10 and the major fatty acids were identified as summed feature 7 (comprising C_18:1 ω9c/ω12t/ω7c as defined by the MIDI system; 55.4 %), and C_18:1 2OH (29.8 %). DNA and chemotaxonomic data supported the affiliation of strain FW-3^T to the genus Roseomonas. Strain FW-3^T could be differentiated genotypically and phenotypically from the recognized species of the genus Roseomonas. The novel isolate therefore represents a novel species, for which the name Roseomonas sediminicola sp. nov. is proposed, with the type strain FW-3^T (=KACC 16616^T = JCM 18210^T).