Abstract:Interferons (IFNs) can induce the expression of a series of interferon-stimulated genes (ISGs). As direct-acting antiviral factors, ISGs play important roles in host antiviral defense. More and more studies reveal that ISGs directly or indirectly target multiple steps of the virus life cycle. Since diverse ISGs have different structures and locations in the cell, they exert various antiviral actions through different pathways. This review aimed to give a brief introduction to ISGs expression induced by IFNs through the JAK-STAT pathway, and provide an overview of typical antiviral mechanisms of representative ISGs.

Abstract:Exocyst is an octameric protein complex that plays a crucial role in the docking and tethering of post-Golgi secretory vesicles to the plasma membrane in eukaryotic cell. The subunit of exocyst complex directly interacts with small GTPases and provides precise spatiotemporal regulation in polarized exocytosis. The growth of filamentous fungi is accomplished by polarized growth in hypha tip, and it possess strong ability of protein secretion. Filamentous fungi are ideal experimental system in study exocyst complex. Exocyst affects morphogenesis and pathogenesis in filamentous fungi. This review summarized current research on exocyst complex in filamentous fungi, including its composition, localization of subunit, function and regulation.

Abstract:[Objective] We analyzed the Sigma54 mutant regulon data of Bacillus thuringiensis (Bt) HD73 strain and CcpA mutant regulon data of Bacillus cereus ATCC 14579 strain, and verified the binding of promoter and CcpA protein, to determine Sigma54 and CcpA co-regulated genes in Bt HD73 and enrich the understanding of microbial metabolic regulation network. [Methods] Based on the results of transcriptome sequencing, genes regulated by Sigma54 and CcpA were found via the comparison of homology gene in Bt HD73 strain. cre sequence of promoter was found by blast and verified the binding with CcpA protein by electrophoresis mobility shift assays. [Results] Thirty-one genes were co-regulated by Sigma54 and CcpA in Bt HD73 strain. The fourteen promoters of these genes contained the cre sequence, which could bind to the CcpA protein in vitro. [Conclusion] Fourteen genes were directly regulated by CcpA in Bt HD73, while their transcription was controlled by Sigma 54. This finding provides clues to the study of sporulation and crystalformation through the point of view the metabolic regulation, and enriches the Bt metabolic regulation network.

Abstract:[Objective] Endo β-1,4-mannanases play important roles in hydrolysis of the β-1,4 glycosidic linkages in mannans and heteromannans, the second most abundant hemicellulosic polysaccharides in nature. [Methods] In this study, we overexpressed and characterized an endo β-1,4-mannanase from Aspergillus nidulans in Pichia pastoris. [Results] A β-1,4-mannanase was successfully overexpressed in flasks and fermentor, and the yield of overexpressed protein in fermentor reached 3.90 mg/mL. The optimal pH and temperature of the enzyme were 4.0 and 60 respectively, and it was very stable over the pH ranges from 5.0 to 9.0. It was thermally stable below 40, whereas it was inactivated very quickly above 60. Its enzyme activities could be enhanced by Co²⁺ and Zn²⁺, whereas it was inhibited by Pb²⁺, Mn²⁺ and Cu²⁺. [Conclusion] This endo-β-1,4-mannanase could be well produced by Pichia pastoris, and has a potential as commercial enzymes for application.

Abstract:[Objective] We revealed the function of the ArsR (Arsenical resistance regulator) family regulator XanR3 in xantholipin biosynthetic gene cluster. [Methods] Genetically, the xanR3 disrupted mutant CGF03 and complementary strain CGF06 were constructed by conjugation between S. flavogriseus SIIA-A02191 and Escherichia coli ET12567/pUZ8002. The operons within xantholipin biosynthesis cluster were grouped by RT-PCR and the transcriptional level was analyzed in the mutant CGF-3 by quantitative real-time RT-PCR. [Results] Inactivation of the xanR3 gene dramatically reduced xantholipin biosynthesis, and its complementation partially restored xantholipin production. Totally, genes in xantholipin biosynthetic gene cluster were assigned into 18 co-transcription units, and the disruption of xanR3 directly down-regulated the transcription of four operons as xanO1-xanS1, xanB1-xanB3, xanT-xanO9 and xanO10-xanC3.[Conclusion] XanR3, a ArsR family transcriptional regulator, is an activator of xantholipin biosynthesis.

Abstract:[Objective] An agarase gene, aga0590, was found in the fine genomics mapping of a deep-sea bacterium Flammeovirga pacifica WPAGA1 using bioinformatics analysis. Recombinant Aga0950 was characterized with heterologous expression in E. coli. [Methods] Genomic sequencing of Flammeovirga pacifica WPAGA1 was performed by Illumina HiSeq2500 and agarase genes were annotated with BLAST. Subsequently, Aga0950 was obtained with heterologous expression in E. coli BL21 (DE3) and the agar-degrading activity was determined by dinitrosalicylic acid method. Enzymatic products of the agarase were analyzed by thin-layer chromatography and ion chromatography. [Results] Thirteen agarase genes with amino acid homologies from 60% to 85% were detected in the genome of Flammeovirga pacifica WPAGA1. The elected gene aga0950, which was aligned with highest identities of 67% in NCBI-nr database, possessed typical sequence characteristics of glucoside hydrolase family 16 (GH16). The specific activity of Aga0950 was 51770 U/mg, and the end-products of agar degradation by Aga0950 were neoagarotetraose (NA4) and neoagarohexaose (NA6). The optimum temperature and pH of Aga0950 was 50℃ and a range of 4.0 to 10.0, respectively. Meanwhile, rAga0950 showed outstanding temperature and pH stability. Activities of Aga0950 was enhanced by Co²⁺ and Mn²⁺ (1 mmol/L), while that was strongly inhibited by Cu²⁺. [Conclusion] Flammeovirga pacifica WPAGA1 has abundant polysaccharide-degrading genes. The agarase Aga0950 was indentified with high agar-degrading activity and excellent stability against acid, alkali and thermo.

Abstract:[Objective] The aim of this research was to express the tyrosinase-coding PPO2 gene from Agaricus bisporus in Saccharomyces cerevisiae and to investigate the characteristics of tyrosinase in vitro and in vivo.[Methods] We cloned PPO2 gene by RT-PCR using the total RNA extracted from Agaricus bisporus. The expression vector pSP-G1-PPO2 was constructed and transformed into yeast. The recombinant protein was purified with Ni-NTA and the tyrosinase enzyme properties were evaluated. [Results] The optimum temperature of tyrosinase in vitro was 45℃. And the optimum pH was 7.0 and 8.0 using tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) as substrate, respectively. In Saccharomyces cerevisiae, the yield of melanin increased with the rise in substrate concentration within 2.5 mg/mL. [Conclusion] We achieved heterologous expression of tyrosinase-coding gene PPO2 from Agaricus bisporus in Saccharomyces cerevisiae and characterized the enzyme properties. The melanin production positively correlates with the concentration of substrate tyrosine which indicates that tyrosinase could be a biosensor to report the content of tyrosine in yeast and could be used for the high throughput screening of high-yield tyrosine strains.

Abstract:[Objective] By testing the internal bacterial growth of SKWP mutants generated in Ralstonia solanacearum strain OE1-1, we evaluated the contribution of these SKWP effectors to bacterial fitness towards host plants. [Methods] The deletion mutant and complementation mutant of R. solanacearum OE1-1 were constructed. A competition assay in mixed infection was adopted to evaluate the contribution of the SKWP to bacterial fitness in tobacco (Nicotiana tabacum cv. Bright Yellow) and eggplant (Solanum melongena cv. Senryo-nigou). [Results] Eggplant was the appropriate host for competitive index (CI) assay of R. solanacearum strain OE1-1. All 6 SKWP mutants affected bacterial fitness in eggplant to some extent while SKWP4 appeared to be most important.[Conclusion] The SKWP effectors were important for bacterial proliferation in eggplant tissues according to the CI analysis, which paving a new way for further identification of the function on whole SKWP family.

Abstract:[Objective] Larix gmelinii in Xinjiashan forest region of Qinling Mountains was taken as the object of this study, and its associated ectomycorrhizal fungi were observed and identified. [Methods] Field investigation was combined with identification methods of morphology and molecular biology to identify ectomycorrhizal fungi. [Results] There were 31 species of ectomycorrhizal fungi, belonging to 2 classes, 4 orders, 11 families and 11 genera. They were Tomentella, Inocybe, Russula, Amphinema, Laccaria, Sebacina, Amanita, Boletus, Cortinarius, Lactarius and Scleroderma; and Inocybe was the predominant group. The diversity of ectomycorrhizal fungi in sunny slope was higher than that in shady slope. The analyses about the chemical properties of mycorrhizosphere soil and non-mycorrhizosphere soil showed that the pH of mycorrhizosphere soil was significantly lower than that of non-mycorrhizosphere soil, while therapidly available phosphorus and potassium contents of mycorrhizosphere soil were significantly higher than those of non-mycorrhizosphere soil. The correlation analyses for chemical properties of mycorrhizosphere soil and ectomycorrhiza infection rate demonstrated that the ectomycorrhiza infection rate of Larix gmelinii was significantly positively correlated with soil pH, and was in extremely significant positive correlation with rapidly available potassium; while the infection rate was significantly negatively correlated with total nitrogen content and available phosphorus content. [Conclusion] The diversity of ectomycorrhizal fungi associated with Larix gmelinii was abundant, and it was affected by the slope aspect. Ectomycorrhizal fungi could reduce the pH of mycorrhizosphere soil, and increase the contents of organic matter, total nitrogen, rapidly available phosphorus, rapidly available potassium, soluble calcium and soluble magnesium of mycorrhizosphere soil. The ectomycorrhiza infection rate was affected by soil pH and contents of total nitrogen, available phosphorus and rapidly available potassium.

Abstract:[Objective] To elucidate differences of biological characteristics and cytopathogenicities between T3SS1 and T3SS2 in Vibrio parahaemolyticus. [Methods] With the main structural protein genes vcrD1 and vcrD2 as the research object, we used the homologous recombination technique to construct a series of deletion mutants ΔvcrD1, ΔvcrD2, ΔvcrD1-vcrD2, and complementary strains CΔvcrD1, CΔvcrD2 of V. parahaemolyticus SH112 strain, then analyzed the differences of growth, biofilm, motility, cytotoxicity to RAW264.7 macrophages and Caco-2 cells, and transcriptional regulation of pro-inflammatory cytokine IL-1β, IL-6 in RAW264.7 macrophages between mutant strain and wild strain. [Results] Compared to the wild strain, the mutant strains had no difference on growth rate, however, the vcrD1 mutant obviously weakened biofilm formation, motility, cytotoxicity of V. parahaemolyticus; the vcrD2 mutant positively upregulated the transcriptional levels of pro-inflammatory cytokines, and significantly attenuated cytotoxicity to cells. The ΔvcrD1-vcrD2 double deletion mutant showed weaker biofilm, motility, cell toxicity than vcrD1 mutant strain, and as similar to the vcrD1 mutant, in the mRNA levels of inflammatory cytokines when compared with the wild strain. [Conclusion] Contributions of two T3SSs to biological characteristics and cytopathogenicities of V. parahaemolyticus are different. The T3SS1 was mainly involved in biofilm formation and motility of V. parahaemolyticus, but also has a significant cytotoxic effect; T3SS2 did not affect bacterial biofilm formation and motility, but played a negative regulation on cell inflammatory reaction, potentially contributing to bacterial immune evasion in host. T3SS1 contributed to bacterial survival in the environment; T3SS2 may help V. parahaemolyticus to evade the host immune response. Moreover, T3SS1 and T3SS2 may play related functions on biological characteristics and cytopathogenicities of V. parahaemolyticus, but the specific mechanism still needs further study.

Abstract:[Objective] To sequence and analyze plasmid p1173-CTXM from the clinical multidrug-resistant Shigella sonnei isolate 1173 with the aim of investigating the mechanism of bla_CTX-M-64resistance gene transfer. [Methods] A double-disk synergy test was conducted to determine if strain 1173 produced extended-spectrum beta-lactamase (ESBL), then antibiotic resistance genes were PCR-amplified and sequenced. Conjugation experiments were used to verify the transferability of plasmids carrying ESBL genes. The transconjugant was detected to certify the production of ESBL and the existence of corresponding resistance genes. Strains 1173 and 1173-CTXM-EC600 underwent testing for antimicrobial drug susceptibility. Plasmid p1173-CTXM-EC600 was sequenced by high-throughput genomic sequencing to illustrate the mechanism of resistance gene transfer. [Results] The multidrug-resistant isolate Shigella sonnei 1173 was shown to be an ESBL-producing strain and to carry the antibiotic resistance genes bla_CTX-M-64 and bla_TEM; however, only bla_CTX-M-64 was horizontally transferred to the recipient strain EC600 which endowed the corresponding antibiotic resistant profiles to EC600. Sequencing and bioinformatics analysis revealed that the resistance gene bla_CTX-M-64 was carried by the ISEcp1-bla_CTX-M-64-Δorf477 transposition unit. [Conclusion] The resistance gene bla_CTX-M-64 carried by plasmid p1173-CTXM mediated the resistance of Shigella sonnei isolate 1173 to multiple antibacterial agents. Horizontal transfer of bla_CTX-M-64 was mediated by the ISEcp1-bla_CTX-M-64-Δorf477 transposition unit.

Abstract:[Objective] The targeted type I polyketide-derived compounds was explored to discover diverse compounds with good biological activity from Streptomyces albus DSM 41398. The biosynthetic pathways of the isolates were further elucidated based on the structure determination and bioinformatics analysis. [Methods] The target compounds were discovered based on comparative HPLC analysis of the fermented broths of wild type and type I PKS gene cluster inactivated mutants. Their chemical structures were elucidated based on ¹H-,¹³C-NMR and HR-ESI-MS. Additionally, their biosynthetic pathways were illuminated by bioinformatics analysis. [Results] Two type I polyketide-originated compounds with antitumor activity, elaiophilin and actinopyranone, were isolated from 5 L fermented broth of S. albus DSM 41398. Their gene clusters were located, and the biosynthetic pathways were proposed, respectively. Notably, the biosynthesis gene cluster of actinopyranone was reported for the first time in this study. [Conclusion] The investigation of elaiophilin and actinopyranone not only offered a strategy to discover type I polyketide compounds through genome mining, but also paved ways for further compound structural modifications.

Abstract:[Objective] The nitrogen fixation (nif) gene operon (nifBHDKEfNXhesAnifV) of Paenibacillus polymyxa WLY78 encoding the nitrogenase enabled Escherichia coli to synthesize functional nitrogenase. The genome-wide transcriptional profiling of the recombinant E. coli 78-7 was examined for improving its nitrogenase activity. [Methods] The transcriptomic analysis of the recombinant E. coli 78-7 cultured under non-N₂-fixing (air and 100 mmol/L NH₄⁺) and N₂-fixing (without O₂ and NH₄⁺) conditions was implemented. [Results] These results reveal that nif genes were significantly transcribed under both conditions, indicating that the negative regulation of nif gene transcription by O₂ and NH₄⁺ is bypassed in heterogeneous E. coli. The non-nif genes specifically required for nitrogen fixation, such as mod, cys and feoAB encoding transporters of Mo, S, Fe and electron transporters, respectively, were transcribed at different levels in both conditions. The transcription levels of suf operon and isc system specific for the synthesis of the Fe-S cluster varied greatly. The genes involved in nitrogen metabolism were notably up-regulated in N₂-fixing conditions.[Conclusion] These data suggest that the non-nif genes specifically required for nitrogen fixation in recombinant E. coli had obvious effects on its expression of nitrogenase. Our results will provide valuable exploration regarding the improvement for nitrogenase activity of heterogeneous hosts.

Abstract:[Objective] To study the role of response regulator rstA of TCS RstA/RstB in pathogenesis of uropathogenic Escherichia coli (UPEC). [Methods] By using λ Red recombination system, we generated the rstA knockout mutant U17ΔrstA and the complementation strain ReU17ΔrstA. We then compared and analyzed the characterization of the mutant strain and the wild type strain in vivo and in vitro.[Results] The growth curves in LB showed that the deletion of rstA did not affect growth kinetics of mutant, whereas in LB-Fe medium, the growth rate of U17ΔrstA was lower than that of the wild-type strain U17. Under the selected environmental stress conditions in vitro, the bacterial survival experimental results showed that the mutant was not sensitive to acid, alkali, high osmotic pressure, urea and oxidants. Strain biofilm assay showed that the biofilm formation ability of the mutant was similar to that of the wild-type strain. qRT-PCR results showed that the rstA gene was significantly upregulated in LB-Fe medium, indicating that the iron-deficiency environment may be the stimulus signal evoking the RstA regulator. The mouse model of ascending urinary tract infection demonstrated that the deletion of rstA led to attenuation of virulence, because the mutant showed significantly decreased colonization compared with the wild type strain in urine, bladder and kidney, whereas the complementation strain restored the virulence to resemble that of wild-type strain. [Conclusion] The rstA gene was a potential virulence factor and associated with the pathogenesis of UPEC.

Abstract:[Objective] To investigate the inhibitory effect of honokiol on Candida albicans in vitro. [Methods] The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of magnolol for C. albicans were determined by microdilution method. Transmission electron microscopy was used to examine the effect of different concentrations of honokiol on the ultrastructure of C. albicans. Apoptosis of C. albicans caused by honokiol was analyzed by Annexin V-FITC/propidium iodide (PI) double staining and the accumulation of reactive oxygen species (ROS) in C. albicans cells was determined by DCFH-DA staining. The effects of honokiol on mitochondrial membrane potential and cell membrane permeability of C. albicans were analyzed by JC-1 staining and PI staining respectively. [Results] Honokiol had strong inhibitory effect on C. albicans, with MIC₉₀ of 16 µg/mL and MFC of 32 µg/mL. Honokiol affected cell wall, cell membrane, and cytoplasm of C. albicans, induced both apoptosis and necrosis in C. albicans, probably through ROS production and disruption of mitochondrial function. It also changed the permeability of cell membrane, leaded to the cell wall damage and the binding of ergosterol. [Conclusion] Inhibition of C. albicans by honikiol involves multiple mechanisms including ROS production accompanied by a series of cellular damages. Honikiol appears to be a potential antifungal drug candidate.