Xiao-Lin Meng

De Novo Characterization of Japanese Scallop Mizuhopecten yessoensis Transcriptome and Analysis of Its Gene Expression following Cadmium Exposure

Background Japanese scallop has been cultured on a large-scale in China for many years. However, serious marine pollution in recent years has resulted in considerable loss to this industry. Moreover, due to the lack of genomic resources, limited research has been carried out on this species. To facilitate the understanding at molecular level immune and stress response mechanism, an extensive transcriptomic profiling and digital gene expression (DGE) database of Japanese scallop upon cadmium exposure was carried out using the Illumina sequencing platform. Results RNA-seq produced about 112 million sequencing reads from the tissues of adult Japanese scallops. These reads were assembled into 194,839 non-redundant sequences with open reading frame (ORF), of which 14,240 putative amino acid sequences were assigned biological function annotation and were annotated with gene ontology and eukaryotic orthologous group terms. In addition, we identified 720 genes involved in response to stimulus and 302 genes involved in immune-response pathways. Furthermore, we investigated the transcriptomic changes in the gill and digestive gland of Japanese scallops following cadmium exposure using a tag-based DGE system. A total of 7,556 and 3,002 differentially expressed genes were detected, respectively, and functionally annotated with KEGG pathway annotations. Conclusion This study provides a comprehensive transcripts sequence resource for the Japanese scallop and presents a survey of gene expression in response to heavy metal exposure in a non-model marine invertebrate via the Illumina sequencing platform. These results may contribute to the in-depth elucidation of the molecular mechanisms involved in bivalve responses to marine pollutants.

The transcriptomic response to copper exposure in the digestive gland of Japanese scallops (Mizuhopecten yessoensis)

The present study was conducted to elucidate the effects of copper exposure on the immune system and lipid metabolism of the Japanese scallop, Mizuhopecten yessoensis. Transcriptional levels of differentially expressed genes (DEGs)in M. yessoensis digestive gland tissue were analyzed using the deep-sequencing platform Illumina HiSeq™ 2000. In total, 841 and 877 genes were identified as significantly up- or down-regulated, respectively. In addition, significant enrichment analysis identified 3 gene ontology terms and 15 pathways involved in the response to copper exposure. Analysis of transcripts related to the immune response revealed a complex pattern of innate recognition receptors, including toll-like receptors, NOD-like receptors and downstream pathway effectors, including those involved in apoptosis. Furthermore, genomic analysis revealed that genes involved in extracellular matrix (ECM)-receptor interactions were enriched in Cu-exposed scallop glands. These results will provide a resource for subsequent gene expression studies regarding heavy metal exposure and the identification of copper-sensitive biomarkers for the aquaculture of M. yessoensis.

Molecular cloning, mRNA expression and tissue distribution analysis of Slc7a11 gene in alpaca (Lama paco) skins associated with different coat colors

Slc7a11 encoding solute carrier family 7 member 11 (amionic amino acid transporter light chain, xCT), has been identified to be a critical genetic regulator of pheomelanin synthesis in hair and melanocytes. To better understand the molecular characterization of Slc7a11 and the expression patterns in skin of white versus brown alpaca (lama paco), we cloned the full length coding sequence (CDS) of alpaca Slc7a11 gene and analyzed the expression patterns using Real Time PCR, Western blotting and immunohistochemistry. The full length CDS of 1512bp encodes a 503 amino acid polypeptide. Sequence analysis showed that alpaca xCT contains 12 transmembrane regions consistent with the highly conserved amino acid permease (AA_permease_2) domain similar to other vertebrates. Sequence alignment and phylogenetic analysis revealed that alpaca xCT had the highest identity and shared the same branch with Camelus ferus. Real Time PCR and Western blotting suggested that xCT was expressed at significantly high levels in brown alpaca skin, and transcripts and protein possessed the same expression pattern in white and brown alpaca skins. Additionally, immunohistochemical analysis further demonstrated that xCT staining was robustly increased in the matrix and root sheath of brown alpaca skin compared with that of white. These results suggest that Slc7a11 functions in alpaca coat color regulation and offer essential information for further exploration on the role of Slc7a11 in melanogenesis.

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).

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).

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 β-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.

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).

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

An endophyte with high yield of β‐mannanase, designated as Paenibacillus sp. CH‐3, was isolated from black locust (Robinia pseudoacacia) seeds. The β‐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 β‐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 β‐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.

Symbiotic bacteria associated with puffer fish Gastrophysus spadiceus and evaluation of their antimicrobial activities.

The present study reports the diversity of culturable bacteria associated with the puffer fish Gastrophysus spadiceus. During the study, a total of 31 strains affiliated to the genera Pseudomonas, Janthinobacterium, Rahnella, and Psychrobacter were isolated from liver, intestines, and flesh of G. spadiceus. These strains exhibited a diverse range of metabolites as indicated by the HPLC and TLC profiles of the chemical extracts of their fermentation products. Some of these crude extracts showed strong antimicrobial activities against pathogenic bacterial strains. In addition, few crude extracts exhibit insecticidal activity against Artemia salina.