Abstract:

Abstract:[Objective] We studied physiological and biochemical properties of a thermophilic bacterium isolated from hot springs in Changbai Mountain. [Methods] The strain CBS-5 (=JCM 15484) from hot springs in Changbai Mountains was isolated by plating and screening on Olive-rich medium. It was characterized by the physiological and biochemical analysis, 16S rDNA sequencing and determination of (G+C)mol% contents. [Results] The cells were Gram-positive, non-motile rods, spore-forming and generally occurred singly or in pairs. The growth temperature ranges from 48℃ to 70℃, optimum at 65℃; growth pH ranges from 6.5 to 8.5, optimum at 7.7. The strain used glucose, sucrose, maltose, lactose and rhanmose as carbon and energy sources. The G+C content of DNA was 41.9 mol%. The major cellular fatty acids were iso-C15 : 0, C16 : 0 and iso-C17 : 0, respectively representing about 24.20%, 20.45% and 17.42% of total fatty acids. The 16S rDNA sequence analysis showed that the strain belonged to the Anoxybacillus genus, for the sequence similarity was 95.1%-98.5% to other known species of Anoxybacillus. [Conclusion] Strain CBS-5 was a member of Anoxybacillus.

Abstract:[Objective] To identify the distribution of avian influenza virus subtypes among domestic ducks in eastern China. [Methods] One hundred and eighty avian influenza viruses isolated from domestic ducks between 2002 and 2006 were tested for their Hemagglutinin subtypes, and 88 of them were followed by monitoring for Neuraminidase (NA) subtypes. [Results] At least 9 HA subtypes and 6 NA subtypes, which constituted 13 subtypes of avian influenza viruses in domestic ducks, including H1N1, H3N1, H3N2, H3N8, H4N6, H5N1, H5N2, H6N2, H6N8, H8N4, H9N2, H10N3 and H11N2, were circulating in eastern China in recent years. [Conclusion] Multiple subtypes of avian influenza viruses were distributed among domestic ducks in eastern China in recent years. The surveillance and prevention of avian influenza viruses in domestic ducks must be strengthened.

Abstract:[Objective] To obtain the complete nucleotide sequence of Planobispora linear plasmid pPR2, and reveal the novel telomere replication protein and possible replication loci from the centrally located origin. [Methods] We cloned the EcoRI DNA fragments and assembled them to get the whole sequence of pPR2. We analyzed the secondary structure of the telomere DNA and the putative telomere replication protein using software, and detected the possible replication loci by Streptomyces protoplast transformation. [Results] The complete nucleotide sequence of pPR2 consists of 15520 bp in length, with a 68.1% (G+C) content. The 329 bp terminal inverted repeat sequence can’t form the conserved fold-back secondary structure as that of many Streptomyces linear replicons. Lacking of typical Streptomyces tap/tpg locus for te-lomere replication, pPR2.3c encodes a protein with two domains resembling the telomere associated protein of Strepto-myces and helicase of Haemophilus respectively. No typical Streptomyces iteron-rep locus for replication from the cen-trally located origin, two DNA fragments containing almost all pPR2 were cloned and introduced by transformation into S. lividans ZX7, but no transformants were obtained. pPR2 encodes single-strand binding protein which may regulate repli-cation of linear DNA and Tra protein for conjugation transfer in actinomycetes. [Conclusion] pPR2 is the smallest ac-tinomycete linear plasmid beyond Streptomyces. This is the first time to report the complete nucleotide sequence of linear plasmid in the genus Planobispora. pPR2 may have novel mechanism for telomere replication, and pPR2.2c and pPR2.3c encode the possible telomere replication proteins.

Abstract:[Objective] Accumulation of trehalose is critical in improving the stress tolerance of Saccharomyces cerevisiae. Two enzymes are capable of hydrolyzing trehalose: a neutral trehalase (NTH1) and an acidic trehalase (ATH1). We constructed trehalase disruption mutants to provide a basis for future commercial application. [Methods] To retain the accumulation of trehalose in yeast cell, we constructed diploid homozygous neutral trehalase mutants (△nth1), acid trehalase mutants (△ath1) and double mutants (Δath1Δnth1) by using gene disruption. We tested mutants’trehalose content and their tolerance to freezing, heat, high-sugar and ethanol concentrations. [Results] These trehalase disruption mutants were further confirmed by PCR amplification and southern blot. All mutant strains accumulated higher levels of cellular treha-lose and grew to a higher cell density than the isogenic parent strain. In addition, the levels of trehalose in these mutants correlated with increased tolerance to freezing, heat, high-sugar and ethanol concentration. [Conclusion] The improved tolerance of trehalase mutants may make them useful in commercial applications, including baking and brewing. protein.

Abstract:[Objective] The role of carboxyl terminal activating region of latent membrane protein1 (LMP1) in Epstein-Barr virus infection and oncogenesis is unclear. In this study, we investigated the activating sites and functionary mechanism of LMP1. [Methods] We recombined a deletion mutant type LMP1 (LMP1△232-351), deleted the amino acid residues including 232-351 codons in carboxyl terminal activating region-3 by PCR. Then we compared mutant type LMP1△232-351 with wild type LMP1 (LMP1WT) to alter biological effect in Nasopharyngeal Epithelial Cell line NP69. Moreover, we constructed a Janus Kinase 3 (JKA3) promoter luciferase reporter system (pGL-2/JAK3-LUC). We respectively cotransfected the LMP1△232-351 and LMP1WT with promoter including NF-kB binding sequence or JAK3 promoter luciferase reporter into 293 cells (controlled with pLNSX vector), and compared their actions to activating promoters by results of luciferase activity assay. [Results] (1) The colony forming number (CFN) of NP69-LMP1△232-351 cells significantly decreased to compare with CFN of NP69-LMP1WT (n=3, p<0.01). (2) LMP1WT was able to up-regulate the transcription activity of JAK3 promoter and the level of up-regulation was correlated with its concentration in Human embryonic kidney 293 cell line; while LMP1△232-351 was almost defective ability to activate the promoter. [Conclusion] The carboxyl terminal activating region-3 may be one of the most important function sites of LMP1, which involved in activating the JAK3 promoter and regulating the expression of JAK3 protein.

Abstract:[Objective] To explore the roles of quorum sensing system in establishing symbiosis between bacterium Sinorhizobium sp.1128 and its plant host Melilotus suaveolens Ledeb. [Methods] According to homologous analysis, we designed primers to amplify the autoinducer synthase encoding genes in Sinorhizobium sp.1128 according to Sinorhizobium medicae WSM419 genome sequences. The autoinducer synthase encoding genes were cloned into the expression vector of pYC12 and expressed in E. coli DH5a. Thin-layer chromatography ( TLC) assay was used to study their roles in autoinducer production. A duplicated inactivation of the gene was used to explore its function in plant nodulation. [Re-sults]Homologous analysis showed that at least three annotated acylated homoserine lactone (AHL) synthase genes ex-isted in Sinorhizobium medicae WSM419 genome. We cloned these three autoinducer synthase genes in Sinorhizobium sp.1128. One of these genes named traI2 was over expressed in E. coli DH5α. At least two different AHLs were produced by the recombinant strain. Disruption of traI2 reduced both the autoinducers (AI) activities and AHL production by TLC detection. Furthermore, the complementation of traI2 reverted the phenotype of AI activities. These findings demonstrate that traI2 was responsible for AI synthesis in Sinorhizobium sp.1128. More important, the traI2 deficient strains were defective in nodule formation on their host plant. [Conclusion] The quorum sensing circuits in Sinorhizobium sp.1128 may play an important role in symbiosis between plant and bacterium.

Abstract:[Objective] To achieve the high quality of malo-lactic starter cultures, we investigated the effect of three culture media on the direct inoculation viability, freeze-drying viability and membrane fatty acid composition of Oenococcus Oeni SD-2a. [Methods] We monitored the bacterial growth and change in medium pH when O. oeni SD-2a cells were cultured in ATB, FMATB and MATB media. O. oeni SD-2a cells in early stationary phase were harvested, and subjected to direct inoculation experiments and freeze-drying processes. Then we determined inoculation and freeze-drying viability. Membrane fatty acid composition of those corresponding O. oeni SD-2a cells was determined by GC/MS method. [Results] The results showed ATB medium without sup-plementation of DL-malate had weak pH buffering capability. Compared with FMATB and MATB, O. oeni cells cultured in ATB increased inoculation viability and freeze-drying viability. Concerning the membrane fatty acid composition, it was observed that ATB medium increased distinctly the relative concentration of lactobacillic acid (C19cyc11) and U/S (the unsaturated: saturated fatty acid) ratio in cell membrane lipid composition of O. oeni SD-2a. [Conclusion] The increased resistance to wine stressor and freeze-drying was probably a result of the cross protection conferred by self acid stress response induced in ATB medium, which might be related with changes in membrane fatty acid composition of O.oeniSD-2a. Therefore, ATB medium was more suitable for preparation of O. oeni SD-2a commercial starter cultures.

Abstract:[Objective] To elucidate the structure and functional mechanism of b subunit of chaperonin from the thermoacidophilic archaeon Sulfolobus solfataricus P2. [Methods] Molecular cloning of the b subunit gene of chaperonin from the thermoacidophilic archaeon Sulfolobus solfataricus P2 was performed by using PCR technique. The gene was expressed in BL21(DE3) of Escherichia coli. After purified and assembled in vitro, the structure of the b subunit homo-oligomer was observed by transmission electron microscope (TEM). The function of this homo-oligomer as a chaperonin was evaluated. [Results] The gene encoding b subunit of chaperonin was amplified by PCR from the genomic DNA of Sulfolobus solfataricus P2 and expressed in BL21(DE3) of E. coli. In vitro, the purified β monomer could assemble to a homo-oligomer in the presence of ATP and Mg2+. As observed by transmission electron microscope(TEM), the b subunit homo-oligomer (b16mer) showed a double-ring structure, which is typical in groupⅡchaperonins. The optimum temperature for ATPase activity of the b16mer was 80℃. The β16mer was able to promote the refolding of denatured GFP and improve the thermostability of xylanase. [Conclusion] According to the prediction and analysis of the chaperonin sequence from thermoacidophilic archaeon Sulfolobus solfataricus P2 genome, we cloned and expressed the b subunit of chaperonin from P2. This subunit formed a homo-oligomer in vitro and showed a typical structure of groupⅡchaperonins. We found that the b16mer was able to function correctly when promoting the refolding and improving the thermostability of some other proteins. Our research has laid a foundation for the further study on the molecular mechanism of ther-moacidophilic archaeon.

Abstract:[Objective] In yeast glycosylphosphatidylinositol (GPI) anchoring is a signal directing localization of GPI proteins to the plasma membrane or cell wall. Some of the cis-requirements for the localization of GPI proteins are now understood, however, little is known the signals directing distribution of the GPI proteins in filamentous fungi. Previously, AfPhoA, a GPI-anchored acid phosphatase in filamentous fungus Aspergillus fumigatus, was first isolated from the cell membrane and latter found to be associated with the cell wall. The actual distribution of the AfPhoA remains unclear. Meanwhile, the signature amino acid motif that determines the distribution of GPI protein in yeast is not found in the C-terminal sequence of the AfPhoA. We aimed to elucidate the cell distribution of the AfPhoA. [Methods] The green fluorescent protein (GFP) was used as reporter to track the localization of the AfPhoA. The C-terminal sequence of the AfPhoA was fused to the C-terminus of the GFP. [Results] We first constructed the expression plasmid pchiGFP, in which the N-terminal signal sequence of the A. fumigatus AfChiB1 was fused to the N-terminus of the GFP. After transformation, a secreted expression of the GFP was achieved in A. fumigatus. Based on this construct, The C-terminal sequence of the AfPhoA was fused to the C-terminus of the GFP to construct a chimeric GFP. After the co-transformation of the fusion construct with plasmid pCDA14, a transformant was confirmed to harbor the chimeric GFP in its genome and could express the chimeric GFP. The transformant cultivated with or without chitin induction could express the chimeric GFP mainly attached to the cell membrane, a prolonged cultivation led to a minor distribution of the chimeric GFP in the cell wall. Although a 30KD of GFP fragment, instead of an intact 43.5KDa chimeric GFP, was also detected in the culture supernatant, which might be released by the cleavage between the fusion protein and its GPI anchor. [Conclusion] Our results suggest that GPI anchoring determines the distribution of the AfPhoA in the cell membrane. In addition to our investigation of the GPI anchoring, an expression vector was also constructed, which would be useful for analyses of the function and regulation of the genes and proteins in A. fumigatus.

Abstract:Heterodimeric beta-galactosidase of Lactobacillus acidophilus belongs to glycoside hydrolase family 2, encoded by two overlapping and translational coupling genes, lacL and lacM. The lacL and lacM genes of the sequenced strain L. acidophilus NCFM encode polypeptides with calculated molecular masses of 73,253 and 35,817 Da, respectively. [objective] To clone, overexpress and characterize the enzyme in Escherichia coli. [Methods] We cloned the fragment (2834 bp) containing ribose-binding site (RBS) and coding regions of the lacLM genes from L. acidophilus ATCC 4356 into expression vector pQE31. RBS and HIS-Tag of pQE31 were substituted by inserted fragment. Recombinant plasmid was electrotransformed into E. coli JM109. Expression product was purified by ammonium sulphate fractionation, an-ion-exchange, affinity chromatography and gel permeation. Native molecular mass of homogenous enzyme was measured by gel permeation, and beta-galactosidase activity was determined by using o-nitrophenyl-b-D-galactopyranoside (ONPG) as the substrate. [Results] Overexpression of the soluble enzyme in E. coli was achieved. Amino acid residue 512 of re-combinant LacL was different from that of L. acidophilus NCFM. Homogenous enzyme was obtained by purification. The homogenous enzyme had a specific activity of 226 U/mg protein, a native molecular mass of 96.3±4.6 kDa, an optimum temperature at 49℃ and an optimum pH of 7. The Km and Vmax values of the enzyme were 2.18±0.12 mmol/L, 273±5 U/mg protein respectively.

Abstract:[Objective] Soil microorganisms play an important role in the vegetable soil ecosystem. Through 16S rRNA gene cloning library technology to analysis the composition of bacteria community structure in the typical vegetable soil. To revealed the microbial diversity in the typical vegetable soil, and laid the foundation for the relationship between land-use changed and the ecological environment. [Methods] Total microbial DNA was directly extracted from a typical vegetable soil of Beijing and Shandong province. The clone library of 16S ribosomal RNA genes was amplified using PCR with universal bacterial primer sets. The PCR products were then subcloned into pGEM-T vector. Each unique restriction fragment polymorphism pattern, created by using two restriction endonucleases (HinfⅠand HaeⅢ), was designated as an operational taxonomic unit (OTU). Amplified DNA was used for diversity analysis. [Results] Constructed bacterial phylogenetic trees of the samples revealed the γ-proteobacteria, b-proteobacteria and α-proteobacteria groups were dominant in both clone libraries. Bacte-rial species composition from Beijing and Shandong province included 124 OTUs and 92 OTUs, respectively. [Conclusion] the dominant species of bacteria populations are proteobacteria in the typical vegetable soil of Beijing and Shandong areas. But bacte-rial diversity in the two typical vegetable soil samples was reduced. This phenomenon may be directly related to Continuous cul-tivation for many years and plant a single vegetable species. At the same time, this phenomenon may also be an important reason to cause vegetable soil diseases widespread occurred and soil degradation.

Abstract:[Objective] The aim of this study was to assess the diversity of arsenite-resistant bacteria in deep sea sediment of the southwest Indian Ocean Ridge. [Method] Arsenite-resistant bacteria in the deep-sea sediment were enriched with 2×10-3 mol/L NaAsO2 and isolated on plates. Their diversity was further examined by Denaturing gradient gel electrophoresis (DGGE) and 16S rDNA library analysis. Furthermore, the gene encoding putative arsenite efflux pump was detected with degenerate primers among these isolates. [Result] Eight arsenite-resistant isolates were obtained; they belonged to five different genera of γ-proteobacteria. One isolate named As-I1-3 can grow in presence of 26×10-3 mol/L NaAsO2, showed highest similarity to Pseudoalteromonas tetraodonis IAM (100%). DGGE result showed that As-I1-3 was dominant in arsenite enriched community, followed by isolate As-I1-5 (Halomonas meridiana,100%) and Mn-I1-6 (Marinobacter vinifirmus, 99%). 16S rDNA library analysis reconfirmed the result of DGGE, which showed that the three bacteria occupied 72.5%, 10% and 7.5% of the total OTUs (operational taxonomic units), respectively. However, only from other two isolates which were not dominant, the gene encoding a putative arsenite efflux pump was obtained. [Conclusion] Different bacteria of arsenite-resistance inhabited in deep sea environment, among which P. tetraodonis (100%) showed highest resistance; they might play a certain role in the geobiocycling of arsenic element in marine environments.

Abstract:[Objective] We studied the resistance of Fusarium graminearum against carbendazim, to confirm if the resistance was related to the whole nucleotide sequence of alpha2-tubulin. [Methods] The whole nucleotide sequence of alpha2-tubulin and morphological characteristic were analyzed from different sensitivie strains when treated with carbendazim. [Results] The results indicated that both sensitive strains and moderately resistant strains produced tube with swelled abnormality or germlings with more branches or cells of conidium swelled greatly, when treated with carbendazim at their EC50 or EC90. The full-length nucleotide sequence of α2-tubulin gene from each of 8 F. graminearum strains from China, which had different carbendazim (MBC) sensitivity phenotypes, were separated by using PCR with 4 primer sets designed in accordance with nucleotide sequence of the gene from the genomic sequencing strain, NRRL 31084 (PH-1). The DNA sequence comparison showed no difference in the nucleotide sequence of α2-tubulin gene among 4 sensitive and 4 resistant strains. This result demonstrates that there was no relationship between MBC-resistance and alpha2-tubulin gene. The full-length of the gene spanned 1712 bp, including 4 introns, encoding 453 amino acids. With 100% homology, there were 5 nucleotide differences in alpha2-tubulin gene between PH-1 isolate and the 6 strains from China. The homology of the deduced amino acid sequence of the gene was 100% among the 6 strains and PH-1 isolate, and 64%-89% among other 9 species of fungi. [Conclusion] The resistance of F. graminearum against carbendazim was irrelative to the nucleotide sequence of alpha2-tubulin.

Abstract:[Objective] Toxin produced by phytopathogenic fungi is one of the important microbial herbicides. We found a new compound with herbicidal activity. [Methods] Five different ultraviolet absorption components were isolated from the filtrate of Botrytis cinerea isolate 7–3 culture. [Results] Of the five components, one showed strong inhibitory to Digitaria sanguinalis. The pure fraction with high herbicidal activity was obtained by HPLC purification. Its structure was identified as 10–syn–dihydrobotrydial by Ultraviolet and Visible Spectroscopy, Infrared Spectrum, Nuclear Magnetic Resonance Spectroscopy analysis. [Conclusion] The findings are important for future preparation and application of the herbicide.

Abstract:[Objective] To investigate whether the gene similar with transposase gene (vpiT) from pathogenicity island of Vibrio cholerae exists in V. alginolyticus strains, and to analyze molecular biological characteristic of the gene and its flanking sequences. [Methods] PCR detection of the gene, similar with vpiT in pathogenicity island of V. cholerae was done among 94 strains of V. alginolyticus,. PCR products from positive strains were directly sequenced. Based on acquired partial sequences, we designed primers for reverse PCR, and got the amplification fragment containing complete gene (valT) from V. alginolyticus E0601, which was similar with vpiT gene. The reverse PCR product was cloned and sequenced, and the acquired sequence was analyzed with bioinformatic methods. [Results] We found that among 94 V. alginolyticus strains, only V. alginolyticus E0601 and E0612，from east coastal areas of Guangdong province, produced predicted positive amplification fragments in PCR detection. Sequencing indicated that amplification fragments from V. alginolyti-cus E0601 and E0612 had identical DNA sequence (named valT-S1). Sequence valT-S3 from V. alginolyticus E0601, con-taining complete valT gene and flanking segments, was finally obtained through reverse PCR, cloning, and sequencing. Bioinformatic analysis on valT-S3 suggested that valT was transposase gene, highly similar with vpiT in V. cholerae VPI. [Conclusion] According to above result and related references, we believe that valT and its flanking segments were acquired from heterogenous bacteria, and VPI or its component probably transfers among Vibrio species including V. alginolyticus.

Abstract:[Objective] To isolate bacteriophage of Enterobacter sakazaki from sewage using reference and isolated strains as indicators, and to observe the biological characteristics of the bacteriophage. [Methods] Bacteriophages were isolated from sewage with double layer agar. Specificity and host ranges of the bacteriophages were determined by reference bac-terium from same genus and family. Phage particles were observed by electron microscope and its molecular character was analyzed by Random amplified polymorphic DNA. [Results] Five bacteriophages of E. sakazakii were isolated from sewage and showed relatively narrow host ranges, only E. sakazakii could be lysised. The phage SK2 isolated from ATCC 51329 could form plaques on 24 of all 27 E. sakazakii strains (89%). All five phage particles had the hexagonal heads and tails after observing with negatively stained method by electron microscope. Random amplified polymorphic DNA analy-sis showed the polymorphism of the five phages. [Conclusion] E. sakazakii phages isolated from sewage were only sensi-tive to E. sakazakii, and had potential usage in typing, preventing, treating E. sakazakii and entironment protection.

Abstract:[Objective] To study the photodynamic inactivation of Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli by hematoporyrin monomethyl ether. [Methods] Bacteria incubated with different concentrations of hematoporyrin monomethyl ether (HMME) and then irradiated with visual light for 30minutes, bacteria inactivation efficiency was detected with the reduction of colony unit, and morphological changes were observed with atomic force microscope (AFM). [Results] Results indicated that 90% of Staphylococcus aureus was photoinactivated by illumination with visible light for 30 min (power density 200 mW/cm2) in the presence of 50 mg/mL HMME. The bacteria killing efficiency to Staphylococcus aureus with light irradiation was much obvious than that in dark at the same concentration of HMME, although without noticeable damage to E. coli with illumination or in dark. AFM ultrastructure images showed that the cells surface of photodynamic inactivated bacteria was all damaged seriously without the leakage of cell contents. [Conclusion] We concluded that the attacked sites to bacteria cells by hematoporyrin monomethyl ether were bacteria membrane structure. Atomic force microscopy provides us a visual technique to study the mechanism of bacteria reacted with photosensitizers.

Abstract:[Objective] To express phosphoenolpyruvate carboxykinase (PEPCK) of tuberculosis and evaluate its immune diagnostic ability. [Methods] PEPCK was cloned and expressed in prokaryotic expression system. We analyzed immune response of recombinant PEPCK with Western blot and studied the immunity of it through mice immunization. We also investigated the sensitivity and specificity of immunodiagnosis of PEPCK via detecting the sera from tubercular patients by using ELISA. The results were evaluated by comparing with a Mycobacterium tuberculosis antibody Colloidal Gold Diagnostic Kit. [Results] The recombinant protein was 72 kDa and specifically reacted with anti-BCG antibody. Specific humoral immune response was elicited after mouse immunization with PEPCK protein and the high titer specific antibody was generated (1:1280). Antibodies were detected against M. tuberculosis 17.3% in all tuberculosis patients, 32.5% in patients harboring Mycobacterium currently and 12.9% in patients without M. tuberculosis. The sensitivity and specificity of ELISA were 51.0% and 96.7%. [Conclusion] These results indicated that the recombinant PEPCK might be one of the suitable antigens for serodiagnosis of tuberculosis.

Abstract:[Objective] Thrombolytic therapy is a safe and effective treatment for thrombotic diseases. Microorganisms are possible sources of thrombolytic drugs. We purified and characterized fibrinolytic enzyme produced by Bacillus subtilis strain BS-26. [Methods] We examined the fibrinolytic enzyme activity by fibrin plate and purified fibrinolytic enzyme by ammonium sulfate fractional precipitation, anion-exchange chromatography on DEAE-Sepharose Fast Flow and prepara-tive PAGE. [Results]The fibrinolytic enzyme of the strain BS-26 was stable blow 50℃ and pH5.0-11.0, the optimal temperature was 42℃ and optimal pH was 9.0. Ca2+ and Mg2+ ions enhanced the fibrinolytic activity, whereas Cu2+ com-pletely inhibited the enzyme. Phenylmethylsulfonyl fluoride (174.2 mg/mL), chicken ovomucoid (1000 mg/mL) and soy-bean trypsin inhibitor (1000 mg/mL) could inhibit enzyme activity, which indicated that the enzyme belonged to serine protease group. On plasminogen-free fibrin plates and plasminogen fibrin plates, the fibrinolytic activity had no obvious difference, indicating that the enzyme was a fibrinolytic enzyme which degraded fibrin directly, but not a plasminogen activator which degraded fibrin by activating plasminogen. A fibrinolytic enzyme was purified from the fermentation broth with recovery yield of 3.2%, purification factor of 41.0 fold and the specific activity 8750.0 U/mg. SDS-PAGE analysis of the purified protein showed only one band with molecular mass of 32 kDa. [Conclusion]A single fibrinolytic enzyme was purified, which provided the basis for large-scale production of fibrinolytic enzyme.

Abstract:[Objective] Bermuda grass white leaf is an important disease on Bermuda grass all over the world. The aim of this research is to identify the pathogen which leads to Bermuda grass white leaf occurring on the Chinese mainland. [Methods] PCR amplification technique, sequence analysis and Southern hybridization were used. [Results] A 1.3 kb fragment was amplified by PCR phytoplasma universal primers and total DNA sample extracted from ill Bermuda grass as the amplified template. Sequence analysis of the amplified fragment indicated it clustered into Candidatus Phytoplasm Cynodontis. Southern hybridization analysis showed differential cingulums. [Conclusion] The pathogen of Bermuda grass white leaf on the Chinese mainland contains phytoplasma, which provides a scientific basis for further identification, prevention and control of the disease.

Abstract:[Objective] To study fermentative production of exopolysaccharide (EPS) from a Libertella sp. and to evaluate its antioxidant activity. [Methods] We studied the effect of carbon source, nitrogen source, growth factors, pH and culture time on the production of Libertella exopolysaccharide. By determining the reduction capability, the decoloration of 1,1-diphenyl-2- picrylhydrazyl (DPPH) and the elimination of hydroxyl radicals, we evaluated the antioxidant activity of the Libertella exopolysaccharide. [Results] The optimum conditions for Libertella exopolysaccharide production were in a medium containing potato juice 20%, maltose 20 g/L, (NH4)2SO4 5 g/L and L-cystine 0.01 g/L and at pH 5.0 and 25℃ fermented for 10 d. Under this condition, exopolysaccharide concentration in the fermentation broth reached 37.52 mg/L, 84.10 % higher than that of the control. The scavenging effects of the Libertella exopolysaccharide on 1,1-diphenyl-2-picrylhydrazyl radical reached 14.21% with 20 mg/L exopolysaccharide, and the clearance rate of hydroxyl radicals reached 50.81% with 50 mg/L exopolysaccharide. [Conclusion] The ferment conditions have significant influence on the production of Libertella exopolysaccharide. The exopolysaccharide showed obvious antioxidant activity.

Abstract:[Objective] In order to monitor the present situation of Avian influenza virus(AIV) and Newcastle disease virus(NDV) in migratory waterfowls effectively, 158 tracheal and cloacal swab samples for wild birds were collected from Sanjiang natural reserve during migratory seasons in October 2005, April 2006 and October 2006. [Methods] Serial passages in specific pathogen free embryonated chicken eggs, haemagglutination activity (HA) text, hemagglutination inhibition (HI) text and RT-PCR detection were used to isolate and identify AIV and NDV. [Results] Twenty AIV isolates and 13 NDV isolates were collected in the test. Twenty AIV isolates were all from aquatic birds in October 2006, and among these isolates, 12 AIV subtypes were identified definitely, 11 subtypes were found in mallards-H2N2 (2/20), H2N6 (2/20), H3N4 (1/20), H3N6 (2/20), H3N7 (2/20), H3N8 (2/20), H6N2 (2/20), H11N2 (1/20), H11N3(1/20), H11N5 (2/20), H11N6 (1/20), and 1 subtype was found in garganey-H5N2 (1/20). Thirteen NDV isolates were collected in all three migratory seasons from 5 different species of waterfowls, including mallard (8/13), bean goose (1/13), white-fronted goose (1/13), common teal (1/13) and mandarin duck (2/13). [Conclusion]The results indicated that mallard, which possesses huge population size and world wide distribution, could be considered one of the most important natural carrier of AIV and NDV and may have more important ecological significance on viruses transmission than other species of wild birds.

Abstract:b-Proteorhizobia and nonrhizobial species—— A review

Abstract:Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen. Successful injection of virulence factors into host cells, evasion of phagocytosis and promotion of pathogenesis depend primarily on the function of type III secretion system (T3SS). A complex set of signaling pathways have been shown to modulate T3SS expression. In this review, a brief introduction is given on the composition, function, and molecular determinants that regulate P. aeruginosa T3SS gene expression.

Abstract:Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) have recently been identified as cytoplasmic sensors for RNA viruses. The RLR signaling cascades induce the production of type I interferons and pro-inflammatory cytokines, which are rigorously regulated by the host. On other side, RNA viruses have evolved strategies to evade RLR signaling. In this review, we present our current understanding of RLR signaling in RNA virus recognition and antiviral innate immunity.