Anti-miR33 therapy improved hepatopancreatic lipid and immune metabolism disorders in grass carp, Ctenopharyngodon idella.

Lipid metabolism disorders are found ubiquitously in farmed fish and occur as a result of excessive fat accumulation. Previous studies have found that miR-33 is involved in lipid metabolism; however, its role in fish lipid metabolism is unclear. We sought to clarify this relationship in grass carp in vivo and in vitro. Our findings revealed the length of miR-33 to be 65 bp. Phylogenetic tree analysis showed that grass carp miR-33 was most closely related to fish miR-33 (Siganus canaliculatus). Hepatocytes transfected with miR-33 mimic displayed markedly raised TG content (P < 0.05) as well as increased levels of lipid synthesis-related transcription factors (P < 0.05). Compared with blank and saline groups, total serum cholesterol, AST, and LDL levels were suppressed in groups treated with the miR-33 antagomir (P < 0.05). Moreover, the expression levels of PPARγ and SREBP-1c mRNA were significantly decreased in contrast to those found in the control group (P < 0.05). Similar findings were noted in the expression of immune-related proinflammatory molecules (TNFα, IL-1ß, IL-6, and NF-κB), which also demonstrated decreased levels (P < 0.05). Conversely, high expressions of anti-inflammatory factors (TGF-ß1 and IL-10) were noted (P < 0.05). This investigation strongly supports the role of miR-33 in hepatopancreas-based lipid metabolism and immunity. miR-33 may have been highly conserved in early vertebrates in order to facilitate liver-specific metabolic and immunomodulatory functions. Our findings provide a basis for further investigations exploring the mechanisms surrounding fish lipid metabolism and may aid in preventing and treating immunocompromised fish as well as fish with fatty hepatopancreas, and other metabolic diseases.

Azospirillum thermophilum sp. nov., isolated from a hot spring.

A novel Gram-staining negative, aerobic, motile by flagellum, rod-shaped bacterium, designated CFH 70021T was isolated from a hot spring soil sample collected from Tengchong, Yunnan province, PR China. Growth of CFH 70021T occurred at 15-50 °C (optimum 50 °C), pH 5.0-7.0 (optimum pH 7.0) and with 0-3.0 % (w/v) NaCl (optimum 0 %, w/v). The genome of CFH 70021T consisted of four complete circular chromosomes and five plasmids, the genomic DNA G+C content was 69.3 mol%. Comparison of the 16S rRNA gene sequences indicated that CFH 70021T represented a member of the genus Azospirillum and showed close relationship with the type strains of Azospirillum agricola CC-HIH038T (97.8 %), Azospirillum rugosum IMMIB AFH-6T (97.6 %), Azospirillum doebereinerae GSF71T (97.6 %), Azospirillum thiophilum DSM 21654T (97.4 %) and Azospirillum picis IMMIB TAR-3T (97.2 %). The polar lipids of CFH 70021T contained diphosphatidylglycerol, phosphatidylmehtylethanolamine, phosphatidylglycerol, phosphatidylcholine, two aminolipids and an unidentified phospholipid. The predominant cellular fatty acids (>10 %) included C19:0cyclo ω8c (11.4 %), C16 : 0 (27.6 %) and summed feature 8 (C18:1ω7c/C18:1ω6c, 40.9 %). The major isoprenoid quinone was Q-10. On the basis of the low ANIb result (<78 %) and different phenotypic and chemotaxonomic characters, we conclude that strain CFH 70021T represents a novel member of the genus Azospirillum, for which the name Azospirillum thermophilum sp. nov. is proposed. The type strain is CFH 70021T (=KCTC 62259T= CCTCC AB2018121T).

Paracoccus luteus sp. nov., isolated from the intestine of grass carp.

A novel Gram-negative bacterium, designated CFH 10530T, was isolated from the intestine of grass carp. The sample was collected from the aquaculture training base at the College of Fisheries, Henan Normal University, Xinxiang, PR China. Cells of strain CFH 10530T were coccoid, ovoid or short-rod-shaped, aerobic, non-spore-forming and non-motile. 16S rRNA gene sequence analysis demonstrated that strain CFH 10530T was closely related to Paracoccus endophyticus SYSUP0003T (97.7 % sequence similarity), Paracoccus halophilus HN-182T (96.5 %) and Paracoccus panacisoli DCY94T (96.1 %). The strain grew optimally at 25-28 °C, at pH 7.0 and with 0-2 % (w/v) NaCl. Cells were positive for catalase and oxidase, nitrate was reduced and H2S was not produced. The isoprenoid quinone was Q-10. Major cellular fatty acids were summed feature 8, C18 : 0 and C18 : 03-OH. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, one unidentified aminolipid and five unidentified polar lipids. The genome size was 3 331 229 bp with a G+C content of 69.6 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between CFH 10530T and the other species of the genus Paracoccus were found to be below the recommended levels for species delineation (ANIm <85, ANIb <80 and dDDH <24 %). Based on its physiological properties, chemotaxonomic characteristics and low ANI and dDDH results, strain CFH 10530T is considered to represent a novel species for which the name Paracoccus luteus sp. nov., is proposed. The type strain is CFH 10530T (=KCTC 62919T=CGMCC 1.16597T).

Grape seed proanthocyanidin extract ameliorates hepatic lipid accumulation and inflammation in grass carp (Ctenopharyngodon idella).

Hepatic lipid metabolism disorder due to excessive fat accumulation in fish is a significant problem in aquaculture. Studies have shown that grape seed procyanidin extract (GSPE) can regulate fish lipid metabolism and improve fish immunity. However, the mechanism is unclear. In this study, we used grass carp that stores excess fat in the liver as a model. In vitro, GSPE treatment of hepatocytes for 3 h significantly decreased TG content, accompanied with decreased expression of SREBP-1c, FAS, and ACC and increased expression of PPARα, ATGL, and LPL. GSPE treatment for 1 h significantly decreased expression of pro-inflammatory cytokines (TNFα, IL-6, IL-1ß, and NF-κB) and increased the expression of anti-inflammatory cytokines (IL-10 and TGF-ß1). In vivo, the administration of GSPE significantly reduced high-fat diet-induced increase of serum CHOL, TG, and HDL, but increased LDL content. GSPE treatment for 3 h increased expression of ATGL and LPL, and significantly decreased the expression of HFD-fed-induced SREBP-1c, ACC, FAS, PPARγ, PPARα, and H-FABP. GSPE treatment for 3 h also significantly decreased the expression of pro-inflammatory cytokines (TNFα, IL-6, and IL-1ß) and increased the expression of the anti-inflammatory cytokine IL-10. The expression levels of the lipogenic miRNAs, miR-33, and miR-122, were suppressed both in vivo and in vitro by GSPE. In summary, GSPE had hypolipidemic and potential anti-inflammatory effects in the liver, potentially mediated by miR-33 and miR-122.

Nocardia yunnanensis sp. nov., an actinomycete isolated from a soil sample.

A novel actinomycete strain, designated CFH S0054T, was isolated from a soil sample collected from Xiaokongshan in Tengchong, Yunnan Province, in south-west PR China. Phylogenetic analysis based on its 16S rRNA gene sequence indicated that strain CFH S0054T belonged to the genus Nocardia. Based on the results of 16S rRNA gene sequence analysis, strain CFH S0054T was most closely related to Nocardia concava IFM 0354T (98.3 %), Nocardia inohanensis NBRC 100128T (97.9 %), Nocardia otitidiscaviarum NBRC 14405T (97.9 %) and Nocardia heshunensis CFH S0067T (97.8 %). Strain CFH S0054T contained C16 : 0, C18 : 1 ω9c, C18 : 0 10-methyl (TBSA) and summed feature 3 as major cellular fatty acids and MK-8 (H4ω-cycl) as the major respiratory quinone. The polar lipid profile included phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and unknown phospholipids. The cell wall contained meso-diaminopimelic acid, and the whole-cell hydrolysates were arabinose, galactose, glucose, fructose, ribose and mannose. The genome of strain CFH S0054T was 7.9 Mbp with a G+C content of 68.4 mol%, the average nucleotide identity (ANI) values between strain CFH S0054T and N. concava IFM0354T were low (ANIm<87.7 % and ANIb=84.1 %). On the basis of phylogenetic, physiological and chemotaxonomic data, strain CFHS0054T is considered to represent a novel species of the genus Nocardia, for which the name Nocardiayunnanensis sp. nov. is proposed, The type strain is CFH S0054T (DSM 46763T=JCM 30082T).

Microbacterium ureisolvens sp. nov., isolated from a Yellow River sample.

A Gram-stain positive, aerobic, non-motile and short-rod-shaped strain, CFH S00084T, was isolated from a sediment sample of the Yellow River in Henan Province, China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CFH S00084T clustered within members of Microbacterium and was most closely related to the type strains Microbacterium yannicii JCM 18959T and Microbacterium arthrosphaerae DSM 22421T (98.97 % and 98.36 % similarity, respectively). The strain grew optimally at 25-37 °C, at pH 7.0 and in 0-3 % (w/v) NaCl. The major whole-cell sugars were rhamnose and glucose. The cell-wall peptidoglycan mainly contained glycine, alanine and ornithine. The menaquinones of strain CFH S00084T were MK-13, MK-12 and MK-11. The major fatty acids detected were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The genome of strain CFH S00084T was 4.03 Mbp with a G+C content of 70.5 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between CFH S00084T and the other species of the genus Microbacterium were found to be low (ANIm <85 %, ANIb <75 % and dDDH <24 %). The phylogenomic analysis provided evidence for clear phylogenetic divergence between strain CFH S00084T and its closely related type strains. On the basis of the differential physiological properties, chemotaxonomic characteristics and low ANI and dDDH results, strain CFH S00084T is considered to represent a novel species for which the name Microbacteriumureisolvens sp. nov. is proposed. The type strain is CFH S00084T (=KCTC 39802T=DSM 103157T).

Paracoccus halotolerans sp. nov., isolated from a salt lake.

A novel Gram-stain-negative, aerobic, non-motile, coccus-shaped bacterium, designated CFH 90064T, was isolated from a salt lake sediment sample collected from Yuncheng city, Shanxi province, PR China. 16S rRNA gene sequence comparisons and phylogenetic analyses showed that the strain belonged to the genus Paracoccus and clustered with Paracoccus zeaxanthinifaciens R-1512T (98.2 % similarity), Paracoccus homiensis DD-R11T (97.6 % similarity) and Paracoccus fistulariae 22-5T (96.5 % similarity), respectively. Growth of strain CFH 90064T was observed at 10-37 °C, pH 6.0-9.0 and with NaCl concentrations of up to 6.0 % (w/v). Strain CFH 90064T contained Q-10 as the only isoprenoid quinone, and the major fatty acid was C18 : 1ω7c. Polar lipids of strain CFH 90064T comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified glycolipid, an unidentified aminolipid and an unidentified phospholipid. The genome of strain CFH 90008T was 3.75 Mbp with a DNA G+C content of 65.1 %. Based on the phylogenetic analyses, low average nucleotide identity results, chemotaxonomic characteristics and differential physiological properties, strain CFH 90064T could not be classified into any recognized species of the genus Paracoccus, suggesting that this strain represents a novel species, for which the name Paracoccushalotolerans sp. nov. is proposed. The type strain is CFH 90064T (=CCTCC AB 2016131T=DSM 103234T).

Antribacter gilvus gen. nov., sp. nov., a new member of the family Promicromonosporaceae from a karstic cavern.

A novel actinobacterium, designated strain CFH 30434T, was isolated from a soil sample collected from a karst cave in Luoyang, Henan Province, PR China. The taxonomic position of the strain was investigated by using a polyphasic approach. Cells of the strain were aerobic, Gram-stain-positive, non-motile and coccoid or short rods. The strain was found to be oxidase-positive and weakly catalase-positive. Strain CFH 30434T grew optimally at 28 °C, pH 7.0-9.0 and in the presence of up to 0-1.5 % NaCl (w/v). The whole-cell sugars were glucose, mannose and rhamnose. The major isoprenoid quinone was MK-9(H8) and the major fatty acids (>10 % of the total fatty acids compositions) were anteiso-C15 : 0, iso-C15:0 and iso-C14 : 0. The polar lipids detected were diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, an unidentified phosphoglycolipid, an unidentified phospholipid and an unidentified glycolipid. The genomic DNA G+C content was determined to be 72.3 mol%. The results of phylogenetic analysis of 16S rRNA gene sequences indicated that CFH 30434T clustered within the family Promicromonosporaceae, and closely with the type strains of Xylanimicrobium pachnodae DSM 12657T, Myceligenerans crystallogenes DSM 17134T and Promicromonospora xylanilytica CCTCC AA 208046T (97.3 %, 96.2 and 95.9 % sequence similarities, respectively). Phylogenetic analysis showed that strain CFH 30434T formed a separate evolutionary branch, and was parallel to other related genera of Promicromonosporaceae. Its phylogenetic distinctiveness and distinguishing phenotypic characteristics supported that strain CFH 30434T represents a novel genus of the family Promicromonosporaceae, for which the name Antribacter gilvus gen. nov., sp. nov. is proposed. The type strain is CFH 30434T (=CGMCC 1.13856T=KCTC 49093T).

Ornithinimicrobium cavernae sp. nov., an actinobacterium isolated from a karst cave.

A novel actinobacterium, designated strain CFH 30183T, was isolated from a soil sample collected from a karst cave in Luoyang, Henan Province. The taxonomic position of the strain was investigated using a polyphasic approach. Cells of strain CFH 30183T were observed to be Gram-stain positive, motile, asporogenous and coccoid to rod shaped. The strain was found to be aerobic and oxidase positive. On the basis of 16S rRNA gene sequence analysis, strain CFH 30183T was found to be closely related to Ornithinimicrobium murale 01-Gi-040T (97.8% sequence identity). The ANIb/ANIm values between strain CFH 30183T and O. murale DSM 22056T were found to be 80.3%/85.9%. Strain CFH 30183T was found to grow optimally at 28-32 °C, at pH 8.0-9.0 and in the presence of up to 7% NaCl (w/v). Whole cell hydrolysates of strain CFH 30183T contained L-ornithine as the diagnostic diamino acid, and arabinose, glucose, mannose and rhamnose as whole cell sugars. The respiratory quinone was determined to be MK-8(H4), while the major fatty acids were found to consist of iso-C15:0 and iso-C16:0. The polar lipids profile was found to include diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, an unidentified phospholipid, an unidentified phosphoglycolipid and four unidentified lipids. The DNA G + C content of strain CFH 30183T was calculated to be 70.9%. Based on the phenotypic, genotypic and phylogenetic data obtained, strain CFH 30183T is considered to represent a novel species of the genus Ornithinimicrobium, for which the name Ornithinimicrobium cavernae sp. nov. is proposed. The type strain is CFH 30183T (= KCTC 49018T = CGMCC 1.16393T).

Regulation of growth performance and lipid metabolism in juvenile grass carp (Ctenopharyngodon idella) with honeysuckle (Lonicera japonica) extract.

This study investigated the effects of honeysuckle extract (Lonicera japonica, HE) on the growth performance and lipid metabolism of juvenile grass carp (Ctenopharyngodon idella). HE at doses of 10 g kg-1 (LHE), 20 g kg-1 (MHE), and 40 g kg-1 (HHE) were individually mixed with the basal diet and fed to grass carp for 10 weeks, and ginseng extract (20 g kg-1, GSE) was used as a positive control. The results showed that HE administration exerted no effect on growth performance, but the hepatosomatic index (HSI) and muscle and liver lipid contents were significantly decreased in the LHE and MHE groups. The serum levels of LDL-c, total triglyceride (TG) and total cholesterol (TC) also declined in the HE-treated groups. Moreover, the disordered vacuolization and nucleus migration in the liver were alleviated in the MHE and HHE groups, and mRNA expressions of lipogenesis-related genes, such as acc1, fas, srebp1, and pparγ decreased. Similarly, the expression of genes related to lipolysis, such as cpt1, atgl, lpl, and pparα, was found to be significantly increased in the MHE and HHE groups compared with the control. Taken together, HE can effectively improve the lipid metabolism and ameliorate the lipid deposition of grass carp and thus may be a promising feed additive in aquaculture.

Paenibacillus esterisolvens sp. nov., isolated from soil.

A Gram-stain-positive, aerobic, motile with peritrichous flagella, rod-shaped bacterium, designated CFH S0170T, was isolated from a soil sample collected from Catba island in Ha Long Bay, Hai Phong City, Vietnam. Comparison of the 16S rRNA gene sequences showed that strain CFH S0170T belonged to the genus Paenibacillus and showed closest relationship with Paenibacillus vulneris CCUG 53270T (98.1 % similarity). Phylogenetic analysis demonstrated that the novel candidate formed a coherent branch with P. vulneris CCUG 53270T and Paenibacillus yunnanensis YN2T. Furthermore, the novel strain shared 87.2 % rpoB gene sequence similarity with P. vulneris CCUG 53270T. Growth of strain CFH S0170T occurred at 10-40 °C, pH 6.0-8.0 and with 0-2.0 % (w/v) NaCl. Strain CFH S0170T contained mannose, glucose and rhamnose as the major whole-cell sugars. The cell-wall peptidoglycan contained meso-diaminopimelic acid, glutamic acid, lysine and aspartic acid. The polar lipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, glycolipids and phospholipids. The dominant cellular fatty acids included anteiso-C15 : 0 and C15 : 0. The genomic DNA G+C content was 50.9 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic analysis, strain CFH S0170T is affiliated to the genus Paenibacillus, but could be distinguished from other valid species of this genus. It is concluded that strain CFH S0170T should be considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus esterisolvens sp. nov. is proposed. The type strain is CFH S0170T (=KCTC 33624T=BCRC 80802T).

Intestinal microbiota and lipid metabolism responses in the common carp (Cyprinus carpio L.) following copper exposure.

The present study was conducted to determine the effects of waterborne copper exposure on the lipid metabolism and intestinal microbiota of juvenile common carp (Cyprinus carpio L.). Common carp were exposed to four waterborne copper (Cu) concentrations (0 (control), 0.07 (low), 0.14 (medium), and 0.28 (high) mg Cu/L) for 8 weeks. Exposure to a high concentration of Cu had a negative effect on growth indices (weight gain rate (WGR) and specific growth rate (SGR)). The biochemical indices measured in serum (low-density lipoprotein (LDL) and triglycerides (TGs)) were significantly affected by exposure to medium concentration levels of Cu. The mRNA levels of lipogenic enzymes (acetyl-CoA carboxylase 1 (ACC-1) and fatty acid synthase (FAS)) and sterol-regulator element-binding protein-1 (SREBP-1) in liver tissue and tight binding protein genes (ZO-1 and occludin) in intestinal epithelial tissue were significantly downregulated in the 0.14 and 0.28 mg/L Cu treatment groups, accompanied by upregulated mRNA levels of lipolysis enzymes (lipoprotein lipase (LPL) and carnitine palmitoyl transferase 1 (CPT-1)) in the liver. The data also showed that the composition of intestinal microbiota was changed following Cu exposure and could alter the α-diversity and ß-diversity. The abundances of few putative short-chain fatty acid (SCFA)-producing bacteria, including Allobaculum, Blautia, Coprococcus, Faecalibacterium, Roseburia, and Ruminococcus, decreased significantly. More specifically, Roseburia sequences were positively associated with lipogenic enzymes, total protein (TP), and TGs and negatively associated with lipolysis enzymes. Other sequences related to probiotics (Lactobacillus, Bacillus and Akkermansia) were also found to decrease, accompanied by an increase in sequences related to pathogens (Pseudomonas and Acinetobacter). To the best of our knowledge, the present study provides the first evidence that waterborne, chronic Cu exposure can disturb the composition of intestinal microbiota related to lipid metabolism and immunity in freshwater fish, thereby increasing the risk of pathogen invasion.

A fluorescence method for determination of glucose transport by intestinal BBMV of common carp.

Epithelial brush-border membrane vesicles (BBMVs) were isolated from the intestine of common carp and studied systematically by enzyme activity, transmission electron microscopy and immunoblotting. The uptake time course and the substrate concentration effect were assessed, and then, the ability of phlorizin and cytochalasin B to inhibit uptake was analyzed. The results show that sucrase, alkaline phosphatase and Na+-K+-ATPase activities in these vesicles were enriched 7.94-, 6.74- and 0.42-fold, respectively, indicating a relatively pure preparation of apical membrane with little basolateral contamination. The vesicular structure was in complete closure, as confirmed by electron microscopy. The presence of SGLT1 on the BBMVs was confirmed by Western blot analysis. In the time course experiment, the glucose uptake by BBMVs in Na+ medium displayed an initial accumulation (overshoot) at 5 min followed by a rapid return to equilibrium values at 60 min. Over the 2-NBDG concentration range selected, the external 2-NBDG concentration in NaSCN medium graphed as a curved line. Phlorizin and cytochalasin B had an obvious inhibitory effect on 2-NBDG transport in carp BBMVs, and the detected fluorescence intensity decreased. The inhibition rate in the 1000 µM group was the strongest at 64.18% and 63.61% of phlorizin and cytochalasin B, respectively, indicating the presence of carriers other than SGLT1. This study is the first to demonstrate that 2-NBDG can be used as a convenient and sensitive probe to detect glucose uptake in fish BBMVs. This technology will provide a convenient method to discover new effects and factors in glucose metabolism.

Laceyella thermophila sp. nov., a thermophilic bacterium isolated from a hot spring.

A novel themophilic bacterium, designated YIM 79486T, was isolated from a sediment sample collected from Jinze hot spring in Tengchong county, Yunnan province, south-west China. Phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that strain YIM 79486T should be assigned to the genus Laceyella and formed a monophyletic clade with the type strain Laceyella putida KCTC 3666T (98.7 % similarity). Strain YIM 79486T formed white aerial mycelium and brown substrate mycelium. Abundant endospores were produced on short sporophores. Cell-wall peptidoglycan contained meso-diaminopimelic acid. The predominant menaquinones were MK-9 and MK-8. The genomic DNA G+C content observed for strain YIM 79486T was 47.8 mol%. Based on low DNA-DNA hybridization data, chemotaxonomic characteristics and differential physiological properties, strain YIM 79486T is considered to represent a novel species within the genus Laceyella, for which the name Laceyella thermophila sp. nov. is proposed. The type strain is YIM 79486T (=CCTC AB 2015040T=NBRC 110772T).

Nocardia heshunensis sp. nov., an actinomycete isolated from soil.

A novel Gram-stain-positive, aerobic, non-motile and acid-fast actinomycete strain, designated CFH S0067T, was isolated from a soil sample collected from Heshun old town in Tengchong, Yunnan province, in south-west PR China. The taxonomic position of strain CFH S0067T was studied in detail using a polyphasic approach. Phylogenetic analysis, based on 16S rRNA gene sequences, revealed that strain CFH S0067T belongs to the genus Nocardia and is closely related to Nocardia concava JCM 12351T (99.3 % similarity), forming a separated branch with this type strain. However, the strain shared 96.0 % gyrB gene sequence similarity with N. concava JCM 12351T. Furthermore, DNA-DNA hybridization showed 56.5±0.6 % DNA relatedness between the novel strain and N. concava JCM 12351T. The whole-cell hydrolysates contained meso-diaminopimelic acid (type IV) and arabinose, galactose, fructose and mannose. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and one unidentified lipid. Strain CFH S0067T contained MK-8 (H4ω-cycl) as the predominant menaquinone. C16 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C18 : 1ω9c and C18 : 0 10-methyl (TBSA) were the major cellular fatty acids. Mycolic acids were also detected. The G+C content of the genomic DNA was determined to be 66.9 mol%. A combination of the low DNA-DNA hybridization values and phenotypic properties demonstrated that strain CFH S0067Tis clearly distinguishable from its most closely related strain, N. concava JCM 12351T. On the basis of this polyphasic study, it is concluded that strain CFH S0067T should be considered to represent a novel species of the genus Nocardia, for which the name Nocardia heshunensis sp. nov. is proposed. The type strain is CFH S0067T (=DSM 46764T=JCM 30085T).

Gene cloning, expression, and characterization of a novel ß-mannanase from the endophyte Paenibacillus sp. CH-3.

An endophyte with high yield of ß-mannanase, designated as Paenibacillus sp. CH-3, was isolated from black locust (Robinia pseudoacacia) seeds. The ß-mannanase gene (manB) has been cloned, expressed, purified, and characterized. With an open reading frame of 984 bp, the manB gene encodes 327 amino acid residues, which belongs to GH family 5. The recombinant ß-mannanase (ManB) has an apparent molecular weight of 50.4 kDa with an optimal activity level at pH 7.0 and 45 °C, and it is stable in the range of pH 4-9. By optimizing, the temperature, isopropyl ß-d-thiogalactoside concentration, and induction time were 28 °C, 0.05 mM, and 12 H, respectively. The highest activity reached at 1,054.17 U/mL. In addition, ManB had Km values of 7.30, 8.69, and 27.17 mg/mL for locust bean gum (LBG), guar gum, and konjac glucomannan (KGM), respectively. Its catalytic efficiency (kcat /Km ) was 176.31 mL/(mg·Sec) for LBG, 116.69 mL/(mg·Sec) for guar gum, and 115.49 mL/(mg·Sec) for KGM. The products of glucomannans, which were hydrolyzed by ManB, mainly contain mannobiose, mannotetrose, and a higher manno-oligosaccharide. The characteristics of ManB revealed that it could be exploited as an effective additive in the animal feed industry.

Nocardia tengchongensis sp. nov., isolated from a soil sample.

The taxonomic status was determined of two actinomycetes, designated CFH S0057T and CFH S0065, that were isolated from soil samples collected from an extinct volcano in Tengchong county, Yunnan province, south-west China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains CFH S0057T and CFH S0065 belong to the genus Nocardia and formed a single clade within this genus. The two isolates were able to grow at 4-45 °C, pH 5.0-7.0 and with a NaCl tolerance up to 5.0% (w/v). The whole-cell hydrolysates were rich in meso-diaminopimelic acid, galactose, arabinose and fructose. Mycolic acids were present. Strains CFH S0057T and CFH S0065 exhibited a menaquinone system with MK-8 (H4, ω cyclo), and the major polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and one unidentified phospholipid. Major fatty acids were C16:0, Summed features 3, C18:1 ω9c and C18:0 10-methyl (TBSA). The genomic DNA G + C contents of strains CFH S0057T and CFH S0065 were 65.7 and 66.1 mol%, respectively. The combined genotypic, phenotypic and chemotaxonomic results indicated the isolates are considered to represent a novel species of the genus Nocardia, for which the name Nocardia tengchongensis sp. nov. is proposed. The type strain is CFH S0057T (= KCTC 29485T = JCM 30083T).

Agromyces insulae sp. nov., an actinobacterium isolated from a soil sample.

A novel Gram-reaction-positive, non-motile, aerobic bacterium, designated CFH S0483T, was isolated from a soil sample collected from Catba island in Halong Bay, Vietnam. 16S rRNA gene sequence analysis showed that the strain is a member of the genus Agromyces and has highest 16S rRNA gene sequence similarities with Agromyces humatus DSM 16389T (97.3 %) and Agromyces ramosus DSM 43045T (97.1 %), and similarities < 97.0 % with type strains of other species of the genus Agromyces. Strain CFH S0483T was able to grow at 10-37 °C, at pH 7.0-9.0 and tolerated NaCl up to 2.0 % (w/v). The whole-cell sugars were mannose, galactose, glucose and ribose. The isolate contained l-2,4-diaminobutyric acid, d-alanine, d-glutamic acid and glycine in the cell-wall peptidoglycan. Strain CFH S0483T exhibited a menaquinone system with MK-12, and the major fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The genomic DNA G+C content of strain CFH S0483T was 71.6 mol%. Based on the phylogenetic and phenotypic analysis, and low DNA-DNA hybridization values, strain CFH S0483T could not be classified into any recognized species of the genus Agromyces. Strain CFH S0483T is therefore considered to represent a novel species of the genus Agromyces, for which the name Agromyces insulae sp. nov. is proposed. The type strain is CFH S0483T ( = KCTC 39117T = CCTCC AB 2014301T).

Crenalkalicoccus roseus gen. nov., sp. nov., a thermophilic bacterium isolated from alkaline hot springs.

Two closely related thermophilic bacterial strains, designated YIM 78023T and YIM 78058, were isolated from samples collected from two alkaline hot springs in Tengchong county, Yunnan province, south-west China. The novel isolates were Gram-stain-negative, non-motile, aerobic ovoid- to coccoid-shaped and non-spore-forming. Strain YIM 78023T grew at 20-60 ºC and pH 6.0-9.0 with optimal growth observed at 40-50 ºC and pH 8.0, while strain YIM 78058 grew at 25-60 ºC and pH 6.0-10.0 with optimal growth at 45-50 ºC and pH 8.0. Phylogenetic analysis based on 16S rRNA gene sequences affiliated these two isolates within the family Acetobacteraceae with high sequence similarities to members of the genera Roseomonas and Belnapia (all sequence similarities <94.5 %). In addition to the above two genera, these strains also clustered with the genera Craurococcus and Paracraurococcus (having sequence similarities <93.3 %) in the phylogenetic tree, but with a distinct lineage within the family Acetobacteraceae. The major ubiquinone was Q-10 and the major fatty acids observed were C18:1ω7c, summed feature 4 and C16:0. The genomic DNA G+C contents observed for strains YIM 78023T and YIM 78058 were 74.3 and 74.0 mol%, respectively. Morphological, phylogenetic and chemotaxonomic results suggest that strains YIM 78023T and YIM 78058 are representatives of a novel species of a new genus within the family Acetobacteraceae, for which the name Crenalkalicoccus roseus gen. nov., sp. nov. is proposed. The type strain of Crenalkalicoccus roseus is YIM 78023T (=JCM 19657T=KACC 17825T).

Amycolatopsis xuchangensis sp. nov. and Amycolatopsis jiguanensis sp. nov., isolated from soil.

Two novel actinomycetes, designated strains CFH S0322T and CFH S01580T, were isolated from soil samples collected from Xuchang city and Ji Guan cave in Henan province, China, respectively. Their taxonomic positions were investigated by a polyphasic approach. The two isolates were Gram-reaction positive, non-motile, aerobic and catalase-positive actinomycetes. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that the two isolates are closely related to the members of the genus Amycolatopsis, sharing high similarities with Amycolatopsis magusensis KCTC 29056T (99.1 %, CFH S0322T) and Amycolatopsis nigrescens DSM 44992T (98.1 %, CFH S01580T), respectively. The major whole cell sugars of strains CFH S0322T and CFH S01580T were arabinose and galactose and the predominant menaquinone was MK-9(H4). The genomic DNA G+C contents of strains CFH S0322T and CFH S01580T were 70.6 and 68.6 mol%, respectively. Morphological, phylogenetic and chemotaxonomic results suggest that strains CFH S0322T and CFH S01580T are representatives of two new species, for which the names Amycolatopsis xuchangensis sp. nov. and Amycolatopsis jiguanensis sp. nov. are proposed. The type strains are CFH S0322T (=NBRC 110769T =KCTC 39516T) and CFH S01580T (=DSM 101526T =CCTCC AA 2015032T).