Abstract:[Objective] We isolated a novel virulent bacteriophage specifically infecting antibiotics-resistant Enterococcus faecalis from hospital sewage, to study its morphology and to analyze its genome so as to provide basis for treatment and infection control of antibiotics-resistant E. faecalis. [Methods] We used E. faecalis as the host to isolate phage from raw sewage from the sewage treatment center of a hospital. Transmission electromicroscopy, optimal multiplicity of infection (optimal MOI) and one-step growth curve were conducted. The genome DNA was extracted and sequenced by high-throughput sequencing technology. The feature of whole genome, and evolutionary relationship were analyzed. [Results] A lytic bacteriophage designated as vB_E. faecalis_IME196 (IME196) was isolated. The optimal multiplicity of infection (optimal MOI) of IME196 is 0.01. One-step growth curve shows that IME196 has a burst size of 50 PFU and a latent period of 30 min. The genome of IME196 is a 38 895 bp in length, linear, terminally non-redundant double-stranded DNA and has a G+C content of 33.9%. [Conclusion] Isolation and characterization of a lytic Enterococcus faecalis phage will help supply a new way to prevent and control the infection of E. faecalis, and laid a foundation to the treatment of multiple drug resistant bacteria in the future.

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Abstract:[Objective] To determine the role of type VI secretion system 2 (T6SS2) core component VgrG in avian pathogenic Escherichia coli (APEC), we detected the biological characteristics and pathogenicity of APEC vgrG gene mutant strain. [Methods] We constructed the vgrG gene mutant strain and complementary strain of APEC DE719 by the Red recombination system and plasmid pSTV28. Then we analyzed the growth curve, motility, biofilm formation, adhesion and invasion capacity to DF-1 cells and virulence to duck of wild-type strain, mutant strain and complementary strain. [Results] The inactivation of vgrG gene did not affect the growth and motility, nor the biofilm formation of APEC. The vgrG gene mutant showed significantly decreased survival capacity and attenuated virulence in ducks. However, the adhesion capacity to DF-1 cells increased. [Conclusion] T6SS2 core component VgrG was involved in the process of APEC infection, which would help us to comprehensive understand the pathogenicity of APEC.

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Abstract:[Objective] Pseudomonas protegens H78 can secrete three antibiotics including pyoluteorin (Plt), pyrrolnitrin and 2,4-diacetylphloroglucinol. This study focused on regulation of the two component system PhoR/B on phosphorus absorption and utilization and Plt biosynthesis. [Methods] phoR and phoB genes were deleted using non-scar homologous recombination method. The lacZ reporter gene was used to assess the regulation of PhoR/B system on Pst phosphorus transport system. The cell growth of wild-type and mutant was determined, cultured in minimal medium containing different concentrations of phosphate. The production of Plt was determined in KB medium. [Results] The β-galactosidase activity of pstS′-′lacZ fusion in H78 was 15 times that in mutant. The growth of H78 was three times that in mutant when phosphate starvation. The production of Plt in H78 was two times that in mutant in KMB medium. [Conclusion] PhoR/B system positively regulates the expression of the Pst phosphorus transport system. PhoR/B facilitates phosphorus absorption and utilization during phosphate starvation and positively regulates the Plt biosynthesis as well.

Abstract:[Objective] To obtain an engineered strain producing cis-4-L-hydroxyproline and optimize the related transformation conditions. [Methods] The DNA sequence encoding cis-4-proline hydroxylase (cis-P4H) was adjusted based on the codon bias of Escherichia coli and mRNA secondary structure, an engineered strain expressing the optimized cis-P4H gene was constructed. The cis-P4H was purified by Ni-NTA column and its activity and stability were analysed. Whole-cell catalysis was used for the production of cis-4-Hyp, then the related transformation conditions were optimized by single factor experiment and orthogonal experiment. [Results] An engineered strain was obtained to produce cis-4-Hyp. The specific activity of cis-P4H was 2.65 U/mg and the half-life period of cis-P4H was 2.32 h. When the optical density (OD600) of the cells reached up to 0.9, IPTG was added for inducing protein expression, then the cells were used for transformation system. The transformation rate from L-proline to cis-4-Hyp was more than 83.33% at the optimized condition (pH 6.5, 31 °C, 60 h). [Conclusion] The engineered strain and related transformation conditions have a good prospect for industrial application.

Abstract:[Objective] We studied the correlation between low nucleic acid content (LNA) bacteria and their filterability. [Methods] Bacterial community and filterability in three freshwater environments were analyzed by flow cytometry (FCM), denaturing gradient gel electrophoresis (DGGE) and statistical analysis. [Results] The proportion of LNA bacteria and their filterability had a significant correlation (y=0.646x+22.42, R2=0.984, P<0.01), and the filterability of LNA bacteria depended on not only the cell size, but also shape and flexibility. In addition, bacteria community organization positively correlated to the proportion of high nucleic acid content (HNA) bacteria and negative correlated to the proportion of LNA bacteria. [Conclusion] The results indicated that 0.45 μm pore-size-filtration can efficiently screen LNA bacteria from the autochthonous microbial community, and demonstrated that bacterial community organization was closely related to the nucleic acid content measured by flow cytometry.

Abstract:[Objective] Permafrost stores massive amounts of organic carbon, accompanying the thawing permafrost expected to result from the climate change, microbial decomposition of the organic carbon stored in permafrost acts as the positive feedbacks to aggravate the global greenhouse effect. In this study, to understand the distribution and diversity of microbes in the permafrost, the microbial compositions along a stratigraphic permafrost soil profile were studied from the Qinghai-Tibetan Plateau. [Methods] The molecular biology methods were used to amplify the archaeal and bacterial 16S rRNA genes and the fungal ITS sequences, and then constructed their gene clone libraries, respectively. Phylogenetic analyses of the sequences based on those sequence similarity were carried out and the community diversity index was calculated. [Results] Phylogenic analysis of the archaeal 16S rRNA gene clone library revealed that all the archaeal sequences were affiliated to two phyla, Crenarchaeota and Euryarchaeota, which comprise 29.0% and 71.0% of the total clone sequences, respectively; Crenarchaeota was classified into only one lineage, Group1.3b/MCG-A, which accounted for 29.0% of archaeal clone sequences, and Euryarchaeota was classified into four lineages, of which methanomicrobiales (52.0%) was the predominant group in the phylum of Euryarchaeota. In the active layer of permafrost, Group1.3b/MCG-A、Methanomicrobiales and Methanosaetaceae were the predominant groups in the archaeal clore sequences; In the transition layer, The predominant groups belong to Group1.3b/MCG-A and Methanomicrobiales; In the permafrost layer, Methanomicrobiales was the only predominant lineage. The retrieved bacterial 16S rRNA gene sequences were classified into ten lineages, of which Actinobacteria, Firmicutes and Proteobacteria were the predominant groups, accounting for 28.9%, 16.9% and 12.1% of bacterial clone sequences, respectively. In the active layer, the predominant groups of bacterial belong to Proteobacteria and Firmicutes, the transition layer and the permafrost layer are relatively similar on bacterial composition, the predominant groups of bacterial were affiliated to Actinobacteria. All the fungal ITS sequences were affiliated to two phyla, Ascomycota and Basidiomycota, which comprise 75.3% and 24.7% of the total ITS sequences, respectively. Cladosporium sp. and Pseudeurotium bakeri were the predominant lineages affiliated to Ascomycota, while Dioszegia sp. was the predominant lineage affiliated to Basidiomycota, accounting for 35.5%, 34.4% and 22.6% of fungal ITS sequences, respectively. In the active layer of permafrost, Pseudeurotium bakeri was the only predominant lineage in the fungal sequences. in the transition layer and the permafrost layer, the predominant groups belong to Dioszegia sp. and Cladosporium sp.. [Conclusion] The permafrost soil has high diversities of the archaeal, the bacterial and the fungal communities along the vertical soil profile. The microbial composition and distribution between the active layer and the permafrost layer were different significantly.

Abstract:[Objective] We aimed to explore spatial pattern of soil microbial biomass carbon (C) and its driver in temperate grasslands of Inner Mongolia. [Methods] Soil samples were collected from 5 discrete depths (0?10 cm, 10?20 cm, 20?40 cm, 40?60 cm, 60?100 cm) for 17 study sites. The variations of microbial biomass C, major abiotic and biotic factors were analyzed across three community types and five soil depths. Effects of abiotic and biotic factors on microbial biomass C were evaluated. [Results] Soil microbial biomass C in meadow steppe was significantly higher than that of typical steppe and desert steppe. In 0?10 cm soil layer, coefficient of variation among different grassland types was higher than the variation among different sites within meadow steppe and typical steppe and lower than desert steppe. In 0?100 cm soil layer, coefficient of variation among different sites in meadow steppe was lower than typical steppe and desert steppe. Soil microbial biomass C showed significant positive correlation with mean annual precipitation, soil moisture, clay content, soil nutrient content, aboveground biomass and root biomass and negative correlation with mean annual temperature and soil pH value. With the increase of soil depth, soil microbial biomass C, coefficients of variation among different sites in the same grassland type and among different grassland types all decreased significantly. Relationship between soil microbial biomass C and abiotic factors weakened with soil depth increase. Correlation index between soil microbial biomass C in 0?10 cm soil layer and 10?40 cm soil layer was more than 0.5. Nevertheless, correlation index of microbial biomass C between 0?10 cm soil layer and 40?100 cm soil layer was less than 0.3. [Conclusion] Soil microbial biomass C in temperate grasslands of Inner Mongolia showed obvious vertical distributions. Besides, the effects of abiotic factors on soil microbial biomass C dramatically declined with soil depth.

Abstract:[Objective] To study the ammonia nitrogen (NH4+-N) removal characteristic, extraction and activity of its key enzymes in Acinetobacter sp. Y1. [Methods] Sodium citrate and ammonium sulfate were used as the sole carbon and nitrogen sources respectively to investigate NH4+-N removal characteristic of strain Y1. The ultrasonic conditions were optimized using orthogonal experiment, and the crude enzyme obtained from ultrasonic crushing method and osmotic shock method were compared by SDS-PAGE, then the activity of key enzymes——hydroxylamine oxidoreductase (HAO), nitrite reductase (NIR) and nitrate reductase (NAR) were measured. [Results] Bacteria density (OD600) was 1.280, and NH4+-N, total nitrogen (TN), COD removal rate can reach up to 98%, 94% and 92% respectively of strain Y1 in 24 h. Hydroxylamine, nitrite nitrogen and nitrate nitrogen in nitrification process were detected without accumulation, and N2 was produced in the process of denitrification. The optimal ultrasonic conditions were: ultrasonic power 50 W, work/interval time 4/7 s, OD600=1.250 and total working time 20 min, and the corresponding specific activities of HAO, NIR and NAR were 0.011, 0.002, 0.018 U/mg, respectively. Specific activity of HAO obtained by osmotic shock method was 0.067 U/mg. [Conclusion] NH4+-N, TN and COD were removed simultaneously with high efficiency by strain Y1. HAO, NIR and NAR activities were detected after optimizing the ultrasonic conditions. And it was better to use osmotic shock method for HAO extraction than ultrasonic crushing method.

Abstract:[Objective] In order to identify the microbial community structure of the different gymnosperm types. [Methods] Microbial culture and PLFA (Phospholipid fatty-acid analysis) methods were employed to study the microbe quantity, PLFAs types and content of three typical gymnosperms soil in scenic area of Maijishan, gansu Province. [Results] There existed distinct difference on composition and structure of soil microbial. Microbial number was highest in bacteria and lowest in fungi. Total PLFAs content and types were the highest of Taxus chinensis (Pilg.) Rehder and lowest of Larix kaempferi (Lamb.) Carriere. Principal component of PLFA analysis that has high similarity with Taxus and Picea asperata Mast. than Larix kaempferi (Lamb.) Carriere. There was obvious difference with Microbial types and structure form of Larix kaempferi (Lamb.) Carrière. Diversity declined significantly than those in the others rhizosphere soils. [Conclusion] Microbial number and community structure in rhizosphere soil of Larix kaempferi (Lamb.) Carriere have significantly changed comparing with native gymnosperms.

Abstract:[Objective] To obtain an overview of the diversity of culturable bacteria inside the imago Aspongopus chinesis Dallas. [Methods] Pure culture, reverse transcription factor amplification (BOXA1R-PCR), 16S rRNA gene sequencing and phylogeny analysis were used to analyze the diversity of culturable bacteria inside the Aspongopus chinesis Dallas. Moreover, the antimicrobial activities, indole-3-acetic acid (IAA) production and amylase activities of the isolates were also monitored. [Results] Totally, 52 bacteria strains with different colony phenotype were isolated from six different culture media. Based on the colony phenotype and BOXA1R-PCR patterns, 12 typical strains were selected for 16S rRNA gene sequencing. According to the phylogeny analysis based on the 16S rRNA gene sequence, the isolates were affiliated to 4 genera including Bacillus, Pseudomonas, Stenotrophomonas and Burkholderia. Bacillus comprised the most dominant genus among all the isolates. 84.6% (44/52) of the isolates exhibited antimicrobial activities against the tested pathogens, 94.2% of the isolates showed abilities in producing IAA, and 43 strains (accounting for 82.7%) had amylase activities. [Conclusion] Diverse populations of bacteria were existed in the Aspongopus chinesis Dallas, and showed a potential application prospect.

Abstract:[Objective] A lytic phage of Shewanella was isolated from the sewage and characterized. [Methods] Four strains of Shewanella bacteria were used as the hosts for the isolation of lytic phages from the sewage. A lytic phage against Shewanella oneidensis MR-1, which we named M1, was isolated by the double-layer agar culture method. The plaque morphology was examined. Phage particles of M1 were concentrated by ultracentrifugation and purified by CsCl gradient centrifugation. The morphology of M1 particles was examined by transmission electron microscopy. The genome of M1 was extracted and the nucleic acid was analyzed by enzyme digestion. One-step growth curve was measured. [Results] The plaques of M1 phage were round and transparent with a diameter of about 2.3 mm?2.5 mm. Transmission electron micrographs showed that M1 had an icosahedral head about 55 nm in diameter and a long contractile tail about 170 nm in length. Phage M1 belongs to the Myoviridae family. Its genome is made of linear double-stranded DNA. The infection cycle of M1 was about 15?20 min as indicated by the one-step growth curve. [Conclusion] Bacteriophage M1 belongs to the Myoviridae family. Our results provided materials for the investigation on the roles of bacteriophages in diagenetic process on the earth.

Abstract:[Objective] The objectives were to study and discover thermophilic fungi from soil in western China. [Methods] Different environmental and regional soil samples were collected and cultivated by the enrichment culture using feather. The strains of the genus Myceliophthora were isolated and identified by the classical morphological characters and molecular systematics. [Results] Strain G10 was identified as Myceliophthora heterothallica; EB6301M was M. vellerea and other ten strains were M. thermophile. [Conclusion] M. heterothallica and M. vellerea were two new record species in China.

Abstract:[Objective] To understand the regulatory mechanism of PhoP of Mycobacterium avium, the function of PhoP was analyzed and a Mycobacterium avium strain with defective PhoP gene was constructed. [Methods] DNA fragment of PhoP DNA-binding domain (PhoPC) amplified by PCR was cloned into the pGEX-4T-3 vector, and then the recombinant plasmid was transformed into Escherichia coli BL21(DE3) for expression of GST-PhoPC protein. PhoPC protein was prepared through removal of GST-tag from GST-PhoPC protein by thrombin cleaving. The promotor regions of PhoP, MAV0127, PhoU and Amt were amplified by PCR, respectively. Electrophoretic mobility shift assay (EMSA) was carried out to analyze the binding activity of PhoPC to these promotor fragments. For construction of a Mycobacterium avium strain with defective PhoP gene, PhoP homologous recombinant DNA fragment with defective mutation was obtained by ligasing the upper-stream and down-stream regions of PhoP gene, which were amplified by PCR. The PhoP recombinant DNA fragment was cloned into the suicide plasmid pGMB151. The recombinant plasmid was then transferred into cells of Mycobacterium avium by using electroporation for homologous recombination. The strains harboring defective PhoP gene were selected and identified by PCR. [Results] EMSA results show that PhoPC protein could bind to the promotors of PhoP, MAV0127 and Amt genes, but not bind to the promotor of PhoU. A deletion of 309 bp of the defective PhoP gene was confirmed by PCR and DNA sequencing. [Conclusion] PhoP can regulate the transcription of its downstream genes MAV0127 and Amt, and also regulate the transcription of PhoP gene itself. However, PhoP does not participate in regulating PhoU two-component system. A Mycobacterium avium strain with defective PhoP gene was successfully constructed, which contributes to further study on the role of PhoP in transcription regulation in Mycobacterium avium.

Abstract:[Objective] The study was to express and characterize the fusion protein of goose β-defensin (AvBD) 12 in Escherichia coli. [Methods] The cDNA of goose AvBD12 was cloned into EcoR I and Xho I sites of pProEX-HTa vector. The recombinant expression plasmid was translated into E. coli Rosseta and the bacteria were induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant protein was purified. Furthermore, antibacterial activity and salt ionic stability of the recombinant protein were determined by colony-counting assays. [Results] It was demonstrated by Tricine-SDS-PAGE that the recombinant protein (molecular weight, 12 kD) was produced as insoluble bodies in the cells. The recombinant protein showed extensive antibacterial activity against bacteria, including E. coli, Micrococcus tetragenus, Salmonella pullorum, Bacillus subtilis, and Staphylococcus aureus. At high NaCl concentrations, the antibacterial activity of goose AvBD12 decreased significantly. In addition, goose AvBD12 showed little hemolytic activity. [Conclusion] Recombinant goose AvBD12 exhibited extensive antibacterial activity. High salt concentration significantly decreased its antibacterial activity. In addition, AvBD12 showed little hemolytic activity.

Abstract:[Objective] Streptomyces sampsonii KJ07 showed a strongly antifungal activity against Rhizoctonia violacea which caused poplar purple root rot. To investigate its antifungal substances, the main antifungal substance was isolated and purified, and also the partial characteristics were studied. [Methods] Methods of ammonium sulfate precipitation, DEAE Sepharose Fast Flow and Sephadex G-50 were used. [Results] A purified antifungal protein was obtained with a molecular mass of 28.4 kD by SDS-PAGE. It had a wide antifungal spectrum, which made the hyphae deformed, as well as the septa, cell wall and intracellular protoplasm of R. violacea abnormal along with the black substances produced. Stability tests showed that the optimum temperature and pH were 25 °C and 6.0 respectively. The antifungal activity of the protein decreased more than 20% at the higher temperature (≥60 °C), and it declined more than 12% when the pH＜4.0 or ≥8.0. Meanwhile, it was affected by metal cation, but was tolerant of proteinase K. 10 amino acids of the N-terminal sequence were measured by Edman degradation, however no one which had the high homology with the antifungal protein was retrieved through NCBI BLAST. [Conclusion] We speculated that the antifungal protein we obtained maybe a novel protein.

Abstract:[Objective] Oxalic acid (OA) is a virulence factor of several plant pathogens, e.g. Sclerotinia sclerotiorum and Botrytis cinerea. Some strains of Trichoderma spp. have the ability to eliminate OA and thereby reduce disease incidence, while the pathways and mechanism of OA elimination have not been well discovered. [Methods] In this study, T. harzianum LTR-2, performed the best capacity of OA elimination among 42 Trichoderma strains under the stress of 30 mmol/L OA. The developmental features of LTR-2 under OA stresses were analyzed using morphology and in situ observational methods. OA elimination rates, ambient pH and biomass under different OA stresses were analyzed. Then, the growing states of LTR-2 were described under the condition of OA as the sole carbon source. In the end, Real-time PCR was used to analyze expression level of OXDC coding gene LTR_4445. [Results] LTR-2 was cultured on PDA plate containing different concentrations of OA. With 50?80 mmol/L of OA, LTR-2 could survive, but didn’t form normal colon. With 30?50 mmol/L of OA, LTR-2 developed chlamydospores firstly, which then germinated hypha and formed normal colon. LTR-2 could eliminate OA in liquid culture. LTR-2 showed the highest OA elimination rate of 66.50% under 20 mmol/L OA, and the second highest of 55.06% under 10 mmol/L. When OA concentration was enhanced to 50?80 mmol/L, the OA elimination rate was dropped down to 6.75%?38.94%. pH of culture filtrate was enhanced to varying extents along with OA elimination under different OA stresses, and the highest increase occurred under the concentration of 10 and 20 mmol/L. Interestingly, OA showed growth-promoting effect when concentration was under 20 mmol/L. Moreover, LTR-2 could develop macroscopic mycelium pellet when OA (<20 mmol/L) was the sole carbon source in media. Transcriptional level of LTR_4445 was up-regulated under OA stress. [Conclusion] Our study implied that Trichoderma spp. may employ several strategies under OA stress besides degradation, e.g. transformation on stress-resistance mode, converting OA into a precursor of nutrients, etc.

Abstract:[Objective] To investigate the growth of Staphylococcus aureus in cooked chicken at different incubation temperature and initial inoculating level, three common predictive models were compared and the best-fit one was chosen to build the primary and secondary model, the result of this study could be applied to develop the tertiary model, from which the density of the S. aureus at any time in cooked chicken can be calculated from any combination of temperature and initial inoculating level. [Methods] The S. aureus strain was inoculated into cooked chicken under various initial concentrations of 102, 103, 104 CFU/g and stored at 15, 22, 29, 36 °C. The number of colonies was counted by 3M Petrifilm? Staph Express Count Plate. The modified Gompertz model, modified Logistic model and Baranyi model for describing the growth of S. aureus were established by using Matlab software. The best model was chosen by comparing the residuals and the goodness-of-fit [Residual Sum of Squares (RSS), Akallke Information Crlterlon (AIC), Residual Standard Error (RSE)]. The growth parameter (lag phase, maximum specific growth rate and maximum population density) were then obtained from the best model. The secondary model was set up by using response surface equation. Finally, the reliability of the model was verified by internal and external validation. [results] At 36 and 29 °C, the best choice to describe the growth of S. aureus was modified Gompertz model at all initial inoculating level. At 22 and 15 °C, the most suitable model is modified Gompertz model, modified Logistic model and Baranyi model successively according to the initial inoculation level. By comprehensive considerations, the modified Gompertz model was thought of the optimal primary model. The secondary model was verified by calculating the standard error of prediction (%SEP), Root-Mean-Squares (RMSE), Accuracy factor (Af) and Bias factor (Bf), the results of verification were all within acceptable range. [Conclusion] The modified Gompertz model coupled with response surface model could provide a useful and accurate basis to develop the tertiary model.

Abstract:[Objective] The aim was to analyze the microRNAs expression in PK-15 cells infected with Pseudorabies virus-Fa. [Methods] Illumina deep sequencing was used to identify and analyze the differentially expressed miRNAs in PK-15 cells with or without PRV-Fa infection. The sequencing results for selected miRNAs were confirmed with RT-qPCR. The host target genes were predicted and Gene Ontology (GO) analysis was done. [Results] We identified 384 and 405 miRNAs in the infected and uninfected cells, respectively. Overall, 127 miRNAs were expressed significantly differently after infected with PRV-Fa (60 upregulated and 67 downregulated). RT-qPCR showed that the expressions of miRNAs were consistent with the sequencing results. GO analysis showed that miRNAs were widely involved in signal transduction, cells metabolism, immune response, and gene expression. And miR-10b, miR-16, miR-18a, miR-19b, miR-20a, miR-145-5p, miR-146a, miR-181a and miR-499-5p were associated with immune response. In the regulatory network of target genes, ssc-miR-30d and ssc-miR-30a-5p were in the key positions. Moreover, five miRNAs encoded by PRV-Fa were identified, PRV-miR-LLT2 and PRV-miR-LLT4 were targeted to PRV EPO gene. [Conclusion] The infection of PRV-Fa has significant influence on PK-15 cells encoding miRNAs.

Abstract:[Objective] Sequencing and analysis complete genome of an epidemic foot-and-mouth disease virus of serotype A and construction of its full-length infectious cDNA clone. [Methods] The primers were designed according to the published genome sequence of type A FMDV and a total of four fragments covering the complete genome of A/Sea-97/CHA/2014 were subsequently PCR amplified and sequenced. The four fragments were cloned into pBluescript SKhdv vector in turn with a single restriction sites to construct a full-length cDNA clone of A/Sea-97/CHA/2014 strain. The full-length plasmid linearized with Not Ⅰ and transfected to BSR/T7 cells expressing T7 RNA polymerase to rescue the recombinant virus. [Results] The result of sequence analysis showed that FMDV A/Sea-97/CHA/2014 strain was 8 171 bp in length [except poly(C) and poly(A)], which contains a 5′-UTR with 1 091 bp, a 3′-UTR with 95 bp and a ORF encoding 2 332 amino acids with 6 996 bp. The phylogenetic tree based on the nucleotide sequences of VP1 gene revealed that the A/Sea-97/CHA/2014 strain had a high sequence identity with A/GDMM/CHA/2013 strain (99.1%). The full-length plasmid transfected to BSR/T7 cells, apparent CPE were observed after 68 h incubation. The harvested virus was verified by IFA, RT-PCR and sequencing. The results indicated that infectious FMDV was successfully rescued in vivo. Plaque phenotype and growth curves tests of rescue virus and parental virus showed they have a similar growth phenotype and capacity of proliferation. [Conclusion] This study provided a useful material for studies of the FMD pathogen ecological distribution, molecular epidemiological as well as novel marker vaccines.

Abstract:[Objective] To understand the diversity and antimicrobial activity of intestinal lactic acid bacteria isolated from Gymnocypris przewalskii. [Methods] We identified 47 lactic acid bacteria by the physiological and biochemical properties (pH, temperature and salt resistance) and sequence analysis of 16S rRNA gene. Antibacterial activity of representative strains of lactic acid bacteria were detected. [Results] We identified 23 strains as Lactobacillus fuchuensis (48.94%), 12 strains Lactobacillus curvatus (25.53%), 3 strains Leuconostoc fallax (6.38%), 2 strains Lactobacillus sakei (4.26%), 2 strains Weissella ceti (4.26%); 2 strains Lactococcus cremoris (4.26%), 1 strains Leuconostoc lactis (2.13%), 1 strains Weissella minor (2.13%), and 1 strains Enterococcus devriesei (2.13%). Groups A, B and C could grow between 5 and 50 °C. Groups B and C could grow between pH 3.0 and 10.0. Almost all of the lactic acid bacteria were resistant to 6.5% Nacl concentration. The supernatant could inhibit the growth of indicator strains. By excluding hydrogen peroxide and organic acid, we found that it still has antimicrobial activity. The inhibitive activity decreased after treatment with pepsin, which indicated that this inhibitory material had the features of protein. It could be classified as bacteriocin. [Conclusion] G. przewalskii intestine adhesion diversity of lactic acid bacteria, which will provide quality resources for probiotic lactic acid bacteria and data reference.

Abstract:[Objective] The culture media for the production of pneumocandin B0 by the filamentous fungus Glarea lozoyensis SIIA-F1108 were optimized using the response surface methodology (RSM). The nitrogen source of the culture was optimized to reduce the volume concentration of mycelium and improve the level of dissolved oxygen in fermentation process. [Methods] Through Plackett-Burman design and RSM, the factors that had significant effects on the production of pneumocandin B0 were screened. Afterwards, the path of steepest ascent and the Box-Behnken design were adopted for further optimization, and the optimal concentration levels and the relationships among these factors was found out by quadratic regression model equation with Design-Expert statistic methods. The high-yield and low-viscosity of fermentation medium was obtained by carrying out the whole factor experiment with nitrogen source components. [Results] Mannitol, proline and glucose were found to have greatest influence on the production of pneumocandin B0, and the optimal concentrations were 167.3, 26.1 and 28.5 g/L, respectively. Under the optimal conditions, the production of pneumocandin B0 reached 1 840 mg/L in three baches of shake flask experiments, with an increase of 42% compared with the ordinary culture. The pneumocandin B0 production of 100 L fermentor achieved 1 980 mg/L under the optimal conditions. The viscosity of the fermentation broth was reduced by partly replacing the cottonseed meal with (NH4)2SO4. [Conclusion] The production of pneumocandin B0, which was in agreement with the prediction, was increased by 42% compared with the previous condition. The RSM method was effective for optimizing the composition of medium for the production of pneumocandin B0 by filamentous fungus Glarea lozoyensis SIIA-F1108. After replacing the cottonseed meal with (NH4)2SO4, the viscosity of the fermentation broth was reduced and the level of dissolved oxygen was improved in fermentation process.

Abstract:[Objective] To investigate the role of blood dissemination in the invasion of the central nervous system by the highly pathogenic H5N1 influenza viruses. [Methods] Three H5N1 strains were intranasally inoculated into BALB/c mice to study the replication and pathogenic kinetics of the viruses in lung, brain, and blood. Infection of the brain vascular endothelial cells and nerve tissue around the blood vessels by the virus was investigated by immunohistochemistry and immunofluorescence staining techniques. [Results] Virus replicated efficiently in lungs, leading to viremia quickly after inoculation. At 6 d post inoculation, both the viral titre in lungs and the detection rate of viremia peaked; concomitantly, the viral NP (Nuclear protein) protein began to be detectable in the central nervous system. Viral NP proteins were observed in the brain vascular endothelial cells and in the neurons and astrocytes around blood vessels. [Conclusion] Blood dissemination could be one of the pathways through which the highly pathogenic H5N1 influenza invades the central nervous system.

Abstract:[Objective] In this experiment we explored the expression, purification condition and tertiary structure of the Curli family protein CsgF of Escherichia coli CFT073, to provide a theoretical basis for studying the biosynthetic mechanism of Curli. [Methods] The csgF gene was amplified by PCR from E. coli CFT073 genomic DNA, and the recombinant plasmids such as pET28a-csgF(nsp)-N-6His, pET28a-csgF(20?129)-N-6His, pET28a-csgF-C-6His, and pET28a-csgF(nsp)-C-6His, were constructed. These were then transformed into E. coli strain DH5α, and the expression of the csgF gene in E. coli BL21(DE3) was induced by isopropyl β-D-1-Thiogalactopyranoside (IPTG). Next, CsgF was separated and purified by Ni-chelating affinity chromatograph and gel exclusion chromatography, and was determined by SDS-PAGE and Western blotting assay. The protein interaction of CsgF and CsgG was studied by pull down experiments. The tertiary structure of CsgF was constructed based on homology modeling. [Results] The purified CsgF protein of high stability was obtained in condition of 50 mmol/L sodium acetate (pH 5.0), 150 mmol/L NaCl and 5% glycerol. Pull down experiments showed there was interaction between CsgF and CsgG. Homology modeling demonstrated that the tertiary structure of CsgF was of (β/α) model. [Conclusion] Our findings can lay a foundation for studying the structure and function of CsgF.

Abstract:[Objective] In order to improve the yield and quality of the Metarhizium conidia spores, response surface design was used to optimize the medium components for solid fermentation of the Metarhizium anisopliae CY-1. [Methods] On the basis of one-factor experiment, medium components were optimized by the response surface design. [Results] The carbon and nitrogen sources were added to the best solid fermentation medium, which also contained cornmeal and rice husk (8:2, W/W), water ratio 1:0.8, 0.8% glucose, 2.5% ammonium sulfate, 0.8% KH2PO4. In the optimized solid fermentation medium, the theory of spores production was 7.45×109 CFU/g, and the validation was actually 6.94×109 CFU/g. [Conclusion] the response surface method was applied to optimize the components of solid fermentation medium of the M. anisopliae, and the spores powders were obtained, which laid a foundation for the research of the control the underground soil borne pathogens and insect pests and the research of the powder preparation and processing.

Abstract:Bacillus as novel micro-ecological agent has been widely used in livestock and poultry industry due to its robust resistance to extreme environment and the advantages of some probiotic effects, such as regulating intestinal microbial balance, promoting the digestion and absorption, enhancing animal immune function, and improving animal production. Our review focuses on effects of probiotic Bacillus on animal immune function covering animal immune organs, specific immune function, non-specific immune function and erythrocyte immune function. In addition, it shows the mechanism of probiotic Bacillus on animal immune function including immune regulation and synthesis of antimicrobial compounds. Furthermore, future research needs are proposed.

Abstract:Soil microbial community composition was the hot issue of soil science, microbiology and ecology. This research conducted in China was in the international forefront and more and more relevant research has been published in high influential international journals. The phospholipid fatty acid (PLFA) method plays a significant role in the analysis of soil microbial communities, both domestic and foreign. But it also has some shortages, which should arouse the concern of research scholars. This paper reviewed the related research focusing on the development history and the application of PLFA method. This review highlights the caution when using and interpreting PLFA data, summarizes the PLFA biomarker and provides some suggestions towards the combination with the new approach in order to facilitate future studies.

Abstract:[Objective] To shorten the process of screening excellent hybridized strains in Hypsizygus marmoreus. [Methods] We used the allochroic method in this study. [Results] The results showed that the color change of the allochroic medium was concordant with decolorization ratio (D value), of which the medium was yellow and the D value was positive value, and the medium was blue and the D value was negative value. Furthermore, we found that the D value was related to the main agronomic characters closely, and the mycelial growth of the strains with positive D value was faster than the strains with negative D value, the output per bottle and the number of normal fruit bodies had positive relationship with D value, and the number of abnormal fruit bodies had negative relationship with D value. [Conclusion] Application the allochroic method could screen the excellent hybridized strains quickly and easily, and could shorten the breeding process of H. marmoreus.