Abstract:Target of rapamycin (TOR) is an evolutionary conservative Ser/Thr kinase that is regarded as a key regulator in cell growth and metabolism responding to environmental stresses in eukaryotic cells. The fungal TOR signaling pathways regulate intracellular homeostasis through ribosome biogenesis, nutrient intake and C or/and N source metabolism under the stimulation of extracellular nutrition and stress. This review summarizes the fungal TOR and its structure, the regulatory mechanism and future perspectives in cell growth, autophagy, metabolism and stress physiological response under different nutritional conditions and stresses, to provide new ideas for the regulating the cell growth and metabolism through the fungal TOR pathway.

Abstract:Carboxyl-terminal repeat domain (CTD) of RNA polymerase Ⅱ largest subunit Rpb1 is essential for transcription regulation. Carboxyl-terminal repeat domain kinase (CTDK-I) is composed of CTK1, CTK2 and CTK3, acting on RNA polymerase Ⅱ carboxyl-terminal repeat domain and phosphorylating CTD heptapeptide repeat (YSPTSPS) for regulating transcription and translation. The specific protein CTK3 binds to cyclin CTK2 to form a heterodimer, controlling CTK1 activity by binding to CTK1. Structural and functional study of CTK1, a homologous protein of cyclin dependent kinase (CDK), may provide a new idea for the research of CDK family, and analysis of the regulatory mechanism of activation of CTK1 by CTK2-CTK3 complex may offer an innovative method for developing cell cycle protein inhibitors. This article reviews the functional characteristics of CTDK-I and the structures and interactions of its subunits and provides a useful guide for the studies of CTDK-I complex in the future.

Abstract:Surfactin, a lipopeptide biosurfactant produced by Bacillus strains, is one of the most surface-active biosurfactant. It is assembled by nonribosomal peptide synthetases (NRPSs) as secondary metabolites in microorganism. Due to its good stability, biodegradability and excellent surface activity in extreme environments, surfactin is gaining increasing attention as a valuable chemical with great potential in medicine, agriculture, food, cosmetics and microbial enhanced oil recovery. However, the low yields and high production costs of natural strain largely limit the large-scale industrial applications of surfactin. This article briefly summarizes the mechanism for surfactin biosynthesis, and reviews the main strategy for improving the yield and changing structure of surfactin including promoter engineering, enhancing efflux system, modifying NRPS domain and engineering fatty acid synthetases. At last, we discussed the prospect of the research on surfactin in the future.

Abstract:A variety of inhibitors are commonly used in related research on ammonia-oxidizing microorganisms, including inhibitors for nitrification and inhibitors against microbial growth. Since the discovery of ammonia-oxidizing archaea, researchers have re-screened different inhibitors to meet the needs of the study of ammonia oxidizing microorganisms. Inhibitors both accelerate the enrichment of archaea, and help researchers to distinguish between archaea and bacteria for their contribution to nitrification and their own anabolic potential. In this paper, the concentrations and inhibitory effects of various inhibitors were reviewed, including traditional inhibitors, like dicyandiamide, 3,4-dimethylpyridine phosphate, and allylthiourea; alkyne inhibitors such as 1-octyne; nitric oxide scavengers and antibiotics. These inhibitors are specific or versatile in their ability to inhibit the activity and growth of ammonia-oxidizing microorganisms. By summarizing these inhibitors, we hope to provide a reference for the choice of inhibitor in the research of ammonia oxidizing microorganisms.

Abstract:Serine/threonine protein kinases play important roles to sense and transduce signal to cope with external environment. However, functions of many serine/threonine protein kinases in cyanobacteria are still unknown. [Objective] This study aims to explore whether SpkC in Synechocystis sp. PCC 6803 responses to high temperature stress. [Methods] We used the homologous recombination method to construct the spkC gene knockout mutant (ΔspkC), and then compared the growth rate, pigment contents of ΔspkC mutant and the wild strain under high temperature stress. In addition, we detected chlorophyll fluorescence under high temperature stress. Recoveries of ΔspkC and wild strain from high-temperature stress were determined through measuring the growth rate under normal temperature. [Results] Compared with wild type strain, ΔspkC mutant grew slower, and the contents of three photosynthetic pigments (chlorophyll, carotenoid and phycobilin) decreased after high temperature stress at 42℃. The activity of photosynthetic system Ⅱ in ΔspkC decreased more rapidly at 45℃. What's more, ΔspkC strain was not able to recover after high-temperature stress for 5 days; their survival rate was significantly lower than wild-type. [Conclusion] The deletion of spkC led to severe defect response to high-temperature stress in Synechocystis sp. PCC 6803, which suggests that serine/threonine protein kinase SpkC is involved in response processes against high temperature stress.

Abstract:[Objective] The aim of this study was to investigate prokaryotic microbial community structures and salt-tolerant genes in hypersaline sediments from Huama salt lake, Northern Shaanxi, China. [Methods] We constructed 16S rRNA library and fosmid library using metagenome of the sediment, and detected prokaryotic microbial distribution and salt-tolerant genes by high-throughput pyrosequencing and bioinformatic analysis. [Results] In total 18978 high-quality reads for 16S rRNA were assigned to 23 phyla, 155 genera and 5221 OTUs. Euryarchaeota and Proteobacteria were the dominant phyla. Sixteen dominant genera such as Halorhabdus, Halorubrum and Pseudomonas and 139 infrequent ones like Halomonas, Psychroflexus and Acinetobacter were explored. A total of 37 salt-tolerant strains were detected from the metagenomic DNA fosmid library containing 4126 strains. The salt-tolerant strains like 5-5, 2E4 and 2F4 exhibited strong tolerance against stresses caused by NaCl, CuSO₄, ZnSO₄ and CdSO₄. High-throughput pyrosequencing of the metagenomic fragment in the strain 5-5 explored 61 unigenes, 23 homogenous genes encoding salt-, or other stress-tolerant proteins in other biological cells, such as inorganic pyrophosphatase, transposase, tellurite resistance protein and calmodulin. [Conclusion] Hypersaline sediments in salt lakes harbor diverse bacterial and arhaeal community and rich salt-tolerant genes.

Abstract:[Objective] To assess the effect of sulfadiazine on gut microbial communities and diversity of Carassius auratus gibelio.[Methods] Fishes were exposed to different concentrations of sulfadiazine (0, 1, 100 and 10000 μg/L) for 14 d. Then, primer sequences were designed according to the conservativeness of bacteria 16S rRNA, and libraries were constructed for high-throughput sequencing. Finally, data were analyzed.[Results] Firmicutes, Proteobacteria and Bacteroidates were the predominant phyla in Carassius auratus gibelio intestine. With different concentrations of sulfadiazine treated, not only the contants of Desulfovlbrlonaceae, Paeudomonas and Hellcobacter in gut decreased observably, but also the numbers of Bifidobacterium, Bacillus and Lactobacillus. The results of Rarefaction Curve, Venn and Diversity index showed that gut microbial diversity decreased siginificantly.[Conclusion] Sulfadiazine led to the microbial diversity decreased significantly and certain pathogenic bacteria could be controlled by sulfadiazine but it also had an nagative impact on some probiotics. This result provided a theoretical support for the rational use of sulfonamides and the healthy growth, disease prevention and control of Carassius auratus gibelio.

Abstract:[Objective] At present, nitrogen pollution has been becoming an important factor in water pollution. In order to unveil the diversity of culturable aerobic denitrifying bacteria in Dianchi Lake and obtain efficient aerobic denitrifying bacteria for providing evidence to bioremediate the polluted water or shallow groundwater. [Methods] Aerobic denitrifying bacteria were obtained through enrichment method and screening processes. The taxonomic positions of these aerobic denitrifying bacteria were analyzed based on 16S rRNA gene sequence comparisons. The efficient aerobic denitrifying bacteria were screened out by detecting nitrogen removal efficiency. [Results] In this study, 260 strains of aerobic denitrifying bacteria were isolated from the sediment and water samples of Dianchi Lake. These bacteria were classified into 59 species, belonging to 14 genera of 13 families of in 2 phyla of bacteria. Pseudomonas composed the major culturable aerobic denitrifying bacteria of Dianchi Lake,followed by Acinetobacter,Aeromonas and Delftia. Twelve strains with perfect nitrogen removal characteristics were screened out. Therein, 8 strains were identified as Pseudomonas spp. and 4 strains were identified as Acinetobacter spp. One efficient denitrifying strain N15-6-1 was screened out by using the quantitative analysis. Its nitrate and total nitrogen removal rates reached 98.81% and 96.27% in 48 h at 30-35℃, C/N=12, repectively. [Conclusion] From all the results, the higher diversity of culturable aerobic denitrifying bacteria inhabited in Dianchi Lake, which enriched the species resources of aerobic denitrifying bacteria, and furthermore, the efficient aerobic denitrifying bacteria provided a significant candidate strains to bioremediate the polluted water or shallow groundwater.

Abstract:[Objective] Endophytic bacteria of tea leaves may play important roles in tea quality and disease defense. Here we clarify the bacterial communities inside tea leaves, compare the endophytic microbiota with the soil microbial flora, and identify the bacteria that have been selectively enriched in leaves. [Methods] Tea plants and the corresponding soil were sampled from Dengcun, Yichang, Hubei. Genomic DNA was extracted from both leaves and the endophytic bacteria, and the bacterial community was analyzed using Illumina MiSeq sequencing of 16S rRNA gene amplicon (V5-V7). [Results] Camellia sinensis leaf-colonizing endophytic microbiota were mainly composed of five phyla. At the genus level, more than 100 genera were identified among which the genera Methylobacterium, Delftia, Microbacteria, Rhodococcus, Aureimonas and Sphingomonas existed in high abundance. About 40% of the endophytic bacteria were also identified in the soil sample. The endophytic bacteria that did not exist in soil, such as Aureimonas and Delftia, accounted for the rest 60%. [Conclusion] Our findings would provide basis for the bacterium-based strategies in disease control and tea quality.

Abstract:[Objective] This study aimed to investigate the effect of nuclear localization signal (NLS) mutation in the M protein on the virulence and replication of duck-origin Newcastle disease virus (NDV). [Methods] The target fragment owning M/NLS mutation was obtained by overlap PCR method and then used to replace the corresponding region of pNDV/SS1GFP to construct pNDV/SS1GFP-M/NLSm. The M/NLS mutant virus rSS1GFP-M/NLSm was rescued by reverse genetics technology and identified by hemagglutination (HA) assay, fluorescence assay and sequencing analysis of the M gene. The subcellular localization of M protein, biological characteristics, plaque formation ability and proliferation ability of the rescued virus were also detected. [Results] The full-length plasmid pNDV/SS1GFP-M/NLSm was successfully constructed. The allantoic fluid obtained from the first generation had no HA titer, but the HA titer of the rescued virus was detected after three extra chicken egg passages. The results of fluorescence assay and M gene sequencing demonstrated that the rescued virus was rSS1GFP-M/NLSm. In comparison to the parental virus rSS1GFP, the M protein of rSS1GFP-M/NLSm mainly localized in the cytoplasm. In addition, the virulence, the replication ability and the plaque formation capacity of rSS1GFP-M/NLSm were obviously reduced. Moreover, the cytopathic effect and the expression of M protein and green fluorescent protein in rSS1GFP-M/NLSm-infected cells were also markedly decreased, indicating that the in vitro proliferation ability of duck-origin NDV was inhibited by M/NLS mutation. [Conclusion] The disruption of M's nuclear localization caused by M/NLS mutation could obviously attenuate the virulence and replication of duck-origin NDV.

Abstract:[Objective] We studied the regulatory effect of Annexin-1(ANXA1) on type I interferon (I-IFN) production and foot-and-mouth disease virus (FMDV) replication.[Methods] We used overexpression and knockdown assays to determine the role of ANXA1 in FMDV-infected cells.Then we evaluated the influence of ANXA1 on ISRE and IFN-β promoter activation by dual luciferase reporter assays.To confirm the regulatory role of ANXA1 on type I IFN pathway,IFN regulatory factor 3(IRF3) phosphorylation was detected by Western blotting.Expression of interferon-stimulated genes (ISGs) was measured by RT-PCR to investigate the effect of ANXA1 on ISGs expression.[Results] Overexpression of ANXA1 significantly suppressed FMDV replication and knockdown of ANXA1 expression enhanced virus replication,showing an antiviral role of ANXA1 in FMDV-infected cells (P<0.01 or P<0.05).ANXA1 promoted the activation of type I IFN pathway in a dose-dependent manner.Overexpression of ANXA1 enhanced IRF3 phosphorylation and ISGs expression (P<0.01 or P<0.05).[Conclusion] ANXA1 enhances type I IFN production and suppresses FMDV replication.

Abstract:[Objective] We studied the formation of a mixed biofilm formed by Vibrio parahaemolyticus and Vibrio cholerae. [Methods] Biofilm formation was quantified using crystal violet staining, abundance of extracellular polysaccharide and extracellular protein, fluorescence in situ hybridization and colony plate count. Biofilms formed by individual and mixed strains were determined from the start and the following intervals at 4, 8, 12, 24, 36, 48, 60 and 72 h. [Results] The amount of biofilm produced by the mixed strains was less than the total amount of biofilm produced by the individual strains, indicating there was no synergism between the strains. However, in the mixed biofilm the time of maturation was increased, the stability of the biofilm to external perturbation was enhanced and the cell density was higher. [Conclusion] The biofilm formed by a mixed population of V. parahaemolyticus and V. cholera is therefore more persistent and can potentially pose a greater risk for recontamination and subsequent development of disease.

Abstract:[Objective] This study aims to explore the inhibitory effect of alkannin on Candida albicans and the mechanism. [Methods] The minimal inhibitory concentration and minimal fungicidal concentration of alkannin on C. albicans were measured by microdilution method. The effect of alkannin on the membrane permeability of C. albicans was determined by ultraviolet spectrophotometry. The effect of alkannin on the morphology of C. albicans was observed by scanning electron microscope and the effect of alkannin on intracellular calcium ion concentration of C. albicans was determined by laser scanning confocal microscope. The effect of alkannin on membrane phospholipid enzyme activity of C. albicans was determined by egg-yolk agar plate medium method. The effect of alkannin on amount of gene expression of PLB1 and PLB2 was determined by RT-PCR. [Results] Alkannin had potent inhibitory effect on C. albicans, the minimal inhibitory concentration and minimal fungicidal concentration of alkannin on C. albicans were 16 mg/mL and 32 mg/mL respectively. Alkannin could damage the cytomembrane integrity of C. albicans and increased the permeability of cytomembrane, leading to the leakage of macromolecular substances such as DNA and RNA in cells and the loss of intracellular calcium ions. The concentration of macromolecular substances such as DNA and RNA in the supernatant increased by 117.32% (P<0.01) after alkannin acted on C. albicans for 16 h and the intracellular[Ca²⁺] decreased by 72.02% (P<0.01). The scanning result of electron microscopy also proved the destructive effect of alkannin on C. albicans cytomembrane. Alkannin could inhibit secretion of phospholipase by C. albicans and showed the dose-dependence. Alkannin in minimal inhibitory concentration decreased phospholipase secretion of C. albicans by 56.3% (P<0.01) compared with the control group. RT-PCR results demonstrated that alkannin could inhibit the expression of PLB1 and PLB2, which were a pair of gene encoding phospholipase B. After alkannin in 1/2 minimal inhibitory concentration acted on C. albicans for 16 h, the expression of PLB1 and PLB2 decreased by 56.4% and 61.4% respectively (P<0.01) compared with the control group. [Conclusion] Alkannin had a strong inhibitory effect on C. albicans by damaging cytomembrane integrity of C. albicans and increasing the permeability, leading to leak of intracellular macromolecule such as DNA and RNA and loss of[Ca²⁺], eventually causing cell death. The inhibition of alkannin on phospholipase secretion of C. albicans prevented cells from maintaining and repairing the damage of cytomembrane caused by alkannin, was also one of reasons of cell death.

Abstract:[Objective] This study aims to classify Tetragenococcus halophilus strains from soy sauce moromi mash using taxonomy analysis, and to characterize these strains. [Methods] T. halophilus strains were classified using multilocus sequence typing combined with cluster analysis of their carbohydrates metabolisms. Physiological characteristics of T. halophilus strains were studied by analyzing their tolerance to stress.[Results] Twelve T. halophilus strains of soy sauce mash origin were classified into two groups. The cluster or group type of individual strain is correlated with the environment where it derived from. T. halophilus strains C25, C33, C3 and R55 tolerated osmotic stress (high concentrations of sugar and salt), whereas strains R44, C1 and R23 exhibited better resistance to high concentrations of salt. Growth of stains C33 and R44 in acidic medium (pH 5.0) was not significantly inhibited though T. halophilus generally grows poorly under acidic conditions. Growth of T. halophilus strains was significantly inhibited when grown at low temperatures (15℃ and 25℃). [Conclusion] T. halophilus strains from soy sauce moromi mash origin were classified into two groups, using the methods based on genotype and phenotype categorizing. Strains from different groups exhibit differences in physiological characteristics.

Abstract:[Objective] A gene encoding family 3 β-glucosidase from Streptomyces sp. GXT6 was cloned and expressed. The enzymatic properties of recombinant protein were studied in detail. And the related amino acid residues were modified to improve glucose-tolerance. [Methods] Based on the analysis of the genome sequence of Streptomyces sp. GXT6, one of the genes annotated as glycosyl hydrolase family 3 was cloned into expression vector pSE380. The recombinant plasmid pSE-bgl3 was constructed and transformed into Escherichia coli XL1-blue for induction of expression. The recombinant protein was purified with Ni-NTA. The features of the recombinant protein BGL3-GXT6 were characterized. The recombinant enzyme was modified through site-directed saturation mutation. [Results] A new gene encoding β-glucosidase of glycosyl hydrolase family 3 was cloned from Streptomyces sp. GXT6. The properties of BGL3-GXT6 were identified. Its optimal pH and temperature were 6.0 and 40 μmol/(min·mg), and K_i value was (1.8880±0.1307) mmol/L. BGL3-GXT6 was able to hydrolyze daidzin, genistin, polydatin and icariin. Furthermore, the possible amino acids related to glucose tolerance of BGL3-GXT6 (81-Trp and 233-Trp) were substituted. Then, twenty-five mutants with activity were obtained and further characterized. As a result, it was found K_m and K_i values of mutants of W233 were significantly changed compared with the wild-type BGL3-GXT6. And the glucose tolerance of these mutants was improved to some extent, with the highest increase of 209 times. [Conclusion] The β-glucosidase BGL3-GXT6 in this study has the ability to hydrolyze rubusoside, daidzin, genistin, polydatin and icariin. These characteristics indicate that BGL3-GXT6 has important applications in theoretical research and in industry.

Abstract:[Objective] In order to systematically investigate the richness of endophytic fungi derived from the medicinal plant Nicotiana tabacum L., and to reveal their biodiversity and community structures, the resource library of the tobacco endophytic fungi has been established, to provide a foundation for the development and effective use of the fungal endophytes. [Methods] The tobacco endophytic fungi were isolated by tissue separation method and identified with morphology and molecular biology methods. The Shannon index (H') and relative frequency (RF) were used to reflect the biodiversity and distribution of the tobacco endophytic fungi. [Results] A total of 539 strains were isolated under different tissues, developmental periods, and altitudes. According to morphological characteristics and rDNA-ITS sequences, they were identified as 73 taxa in 31 genera, of which the genera Aspergillus sp. and Fusarium sp. were confirmed as the dominant flora, with the relative frequency values of 22.63% and 12.99%, respectively. Meanwhile, the Shannon diversity index was 2.78, indicating considerable richness of the fungal endophytes. The diversity of the endophytic fungi in stems was higher compared with those of in roots and leaves. With the extension of the developmental period, the diversity of the fungal endophytes increased gradually, whereas the trend of the species diversity index of the fungal endophytes was downward with altitude. [Conclusion] Tobacco endophytic fungi have abundant biodiversity, and their distribution shows the specialization of tissues, developmental periods, and altitudes, respectively. To illustrate the distribution of endophytic fungi in tobacco will provide scientific basis for the development and application of the fungal endophytes in agricultural industry.

Abstract:[Objective] The study is aimed to reveal the diversity of culturable actinobacteria in Xiaoerkule salt lake, to lay a foundation for further development. [Methods] Actinobacterial diversity of Xiaoerkule lake was studied by culturable method and phylogenetic analysis based on 16S rRNA gene sequences. Component factors of samples were tested by conventional methods. Activity for protease, amylase and asterase was tested by inoculating single colony method. Antibacterial activity of novel actinomycetes was detected by bacteriostasis ring method. [Results] In total 51 OTUs isolated from the salt lake belonged to 24 different genera, and 15 strains are novel actinomycetes. Streptomyces is predominated microorganisms for 16.25% of the total isolated amounts. Sample environmental factors affected the population of actinomycetes in this lake. Tested actinomycetes showed excellent functional enzyme activity and antibacterial activity. Strain XHU 5011, a novel actinomycete, showed great potential for anti-Staphylococcus aureus, anti-Mycobacterium smegmatis and anti-Pseudomonas fluoressens. [Conclusion] There is abundant actinobacterial diversity in the sediment of Xiaoerkule lake, and the result implies that there are large numbers of unknown actionobacterial groups here. These actinomycetes have excellent functional enzymes and provide the basis for further study on secondary metabolites.