Abstract:[Objective] Using the 16S rRNA or HSP60 gene sequences to identify the myxobacteria taxa at the levels of species and genus, which were difficult to be classified by their morphological characteristics. [Methods] 15 myxobacterial strains were isolated using the traditional isolation methods, and classified based on their morphological characteristics. The 16S rRNA and HSP60 gene sequences were amplified by PCR methods, and phylogenetically analyzed. [Results] Eleven strains possessed typical mor-phological characteristics, while the other four strains 0085-4, 0121-3, NM03 and Myx9736 were degenerated of fruiting body structures in different extents. The strains were classified into the suborder Cystobacterineae, located in three genera of two families based on their morphological characteristics. The 16S rRNA gene sequences were 95.4% to 99.5% homology, which were in good consistence with the classification of the morphology-based genera; while the HSP60 gene sequences were in longer phylogenetic distances. [Conclusion] The present morphology-based classification of myxobacteria is highly consistent with the phylogenetic results of 16S rRNA gene sequences at the levels of genera or higher taxa; while HSP60 gene sequences provides a more efficient method for identification of closely related myxobacteria species.

Abstract:[Objective] The aim of this study is to analyze the microbial diversity in the sediment of Jingshan hot spring. [Method] We extracted environment DNA from the sediment and then amplified the 16S rRNA genes of prokarotype and internal transcribed spacers(ITS) DNA of eukarotype by PCR with specific conservative primers. The PCR products were ligated to the T vectors and transformed into Escherichia coli to construct libraries. The libraries were analyzed by amplified ribosomal DNA restriction analysis (ARDRA) approach. The clones of dominant ARDRA patterns were selected to sequence. The sequence similarities were analyzed by using the BLAST programs for searching the GenBank DNA data-bases. [Results] We identified 14 dominant operational taxonomic units (OTUs) from prokarotype G. group. The Phy-logenetic tree shows that 7 of them (JS-GU20, JS-GU1, JS-G37, JS-G29, JS-GM2, JS-G42, JS-G41) belong to Bdellovi-brio. They had the highest similarity to Bacteriovorax sp. NE1 (EF092445) and Bdellovibrio sp. JS5 (AF084859), 96% and 99% respectively. Four major OTUs of prokarotype X group belong to Cyanobacterium. One of them, named JS-X2, has a similarity of 95% with an uncultured Cyanobacterium (L35331) which was gained from a hot spring of the Yellow-stone National Park and has a similarity of 89% with Thermosynechococcus elongatus BP-1 (47118315). Most OTUs of eukaryotic group have 90% sequences similarity to that of Penicillium sp.. [Conclusion] The results showed that the mi-crobial diversity in the sediment of Jinshan hot spring is rich. And the result of several clones’ similarity to Bacteriovorax sp. showed that there might be a new group of thermophilic Bacteriovorax sp. in the hot spring sediment.

Abstract:[Objective] To study the diversity and phylogeny of Rhizobia strains from Sesbania cannabina in Jinshajiang arid river valley in Sichuan province, China. [Methods] We used numerical taxonomy, 16S rDNA PCR-Restriction fragment length polymorphism(RFLP), sequences analysis of 16S rDNA and Glutamine synthetaseⅡ(GSⅡ)genes. [Results] Based on the dendrograms generated from numerical taxonomy, the strains were clustered into 6 groups at the similarity of 93%. Four groups were closely related to type strains of R .tropici, R.etli, S. saheli, A .rubi respectively, and two groups were separated with type strains. The results of 16S rDNA PCR-RFLP were in good agreement with that of numerical taxonomy, only two separated groups showed some differences. SCAU176 and SCAU144 representing the strains of two separated groups were selected for sequence analysis .The results of 16S rDNA sequence indicated that SCAU176 and SCAU144 were related to type strains R. huautlense, and the homology coefficient with R. huautlense was 100% and 98.9% respec-tively. GS II sequence analysis revealed that SCAU176 and SCAU144 were clustered together, and the homology coeffi-cient with the nearest type strains R. tropici was below 90%. [Conclusion] Rhizobia of Sesbania cannabina in Jinshajiang arid river valley are highly diverse, they are closely related to Rhizobium, Sinorhizobium and Agrobacterium.

Abstract:[Objective] To obtain the mutants with different toxicity from the wild-type Cry1Ca7. [Methods] Insecticidal crystal protein Cry1Ca7 from Bacillus thuringiensis which is highly toxic to Spodoptera exigua, an important agricultural pest in China, and we mutated this toxin by over-lapping extensive PCR method in different domains to obtain 11 chimeric mutants. [Results] The results of bioassays against Spodoptera exigua neonates showed that several conserved amino acid sites were crucial to insects. The pesticidal activities of most of mutated proteins were decreased, including Glycine138 Serine, Threonine221 Aspartic acid, Threonine221 Argine, Asparagine251 Serine, 439GlycineGlycineThreonine440, Asparagine306 Argine, Tryptophan376 Phenylalanine, Argine522 Glutamic acid and Argine570 Glycine. The activity of those mutated proteins in the DomainⅡ was 439GlycineGlycineThreonine 440 < Asparagine 306 Argine < Tryptophan 376 Phenylalanine. In the DomainⅢ, the mutant Argine522 Glutamic acid < Argine 570 Glycine, their toxicities reduced distinctly compared with Cry1Ca7. The toxicities of the mutant Argine148 Glycine in DomainⅠincreased six-fold, nevertheless the activities of the mutants Glycine138 Serine、Threonine221 Argine and Asparagine251 Serine mutant reduced totally, even the mutant of Threonine221 Aspartic acid was not toxic entirely. In [Conclusion] It is relatively easier to obtain mutant with higher toxicity in DomainⅠof Cry1Ca7 protein than these in both DomainⅡandⅢ. We can use the improved mutant genes as the potential resources to construct novel engineering bacteria and transgenic plant, meanwhile, to perform the study of interaction mechanism between insects and Cry proteins.

Abstract:[Objective] Wheat blue dwarf (WBD) is an important disease in winter wheat district, which causes serious losses in wheat production. Thymidylate kinase (TMK) catalyses the phosphorylation of dTMP to dTDP in the de novo and salvage pathways of dTTP synthesis in both prokaryotes and eukaryotes. In order effectively control this phytoplasma, we isolated the thymidylate kinase gene of WBD phytoplasma, and analyzed the catalytic activity of TMK protein. [Methods] tmk gene was amplified from the phytoplasma of WBD, the amplicons were digested with EcoRⅠand HindⅢ and then inserted into expression vector pET-30a(+). The polyHis-tagged TMK was expressed in E. coli BL21 (DE3) and fusion protein was obtained and purified by Ni-NTA column. The TMK activities were measured by the method of en-zyme-coupled assay involving Mg2+, dTMP and ATP. [Results] Two genes, tmk-1 and tmk-2 were obtained, with the molecular weight of 630 bp and 624 bp. Both of them encoded an amino acid sequence with three conserved functional motifs which related with binding NTP/NMP. The fusion protein, TMK-2 had a higher catalytic activity (112.41 U/mg) than TMK-1 (16.4 U/mg), and its optimum catalytic conditions were 32℃, pH7.3, 1.5 mmol/L Mg2+ and 1 mmol/L ATP. [Conclusion] TMK-1 and TMK-2 had conserved functional motifs in their primary sequence, and suggested that they may function as TMK enzymes. But, the TMK-1-polyHis fusion protein had very low catalytic activity, the possible reason was that two highly conserved regions were absent in TMK-1, and it might function as another type of kinase in WBD phyto-plasma. This experiment lay a foundation for further study of the TMK function in infection and reproduction of WBD phytoplasma.

Abstract:[Objective] Recently H5N1 subtype avian influenza virus (AIV) is capable of mortality to aquatic bird. The molecular basis for the virulence of this virus is still poorly understood. [Methods] We characterized two H5N1 subtype viruses, A/mallard/Huadong/Y/2003 (Y) is nonpathogenic to mallard whereas A/mallard/Huadong/S/2005 (S) is highly pathogenic to mallard. Using reverse genetics, we constructed a series of single-gene or multiple-gene reassortants from these two viruses. [Results] Substitution of single-gene for PB2, PB1, PA (3P), HA and of combination for 3P gene resulted in complete attenuation of S virus in mallard. However, these corresponding substitutions only slightly increased virulence of Y virus in mallard. Other gene segments had little contribution to the virulence of both viruses. [Conclusion] These results indicate that the pathogenicity of H5N1 AIV to mallard was regulated by multiple gene segments, and these regulations had more sensitive effect on highly pathogenic virus backbone than on low pathogenic virus backbone.

Abstract:[objective] To isolate and identify a new Bacillus strain capable of growing under highly alkaline conditions as alkaline amylase producers and to characterize its enzymatic properties. [Methods] The isolates were sampled from alkaline sewage in Shihezi city, Xinjiang and screened by plating them on the amylase agar medium depending on the halo zone diameter. The alkaline amylase producer with best enzymatic activity was designated as XJU-3. XJU-3 was identified by its physiological and biochemical characteristics, 16S rDNA sequence homol-ogy, and the content of its major cellular fatty acids. [Results] XJU-3 was a Gram-positive, spore-forming, aerobic, motile rod alkaliphilic bacterium. It can grow at a broad range of pH (4.0-12.5) in Luria broth medium and its optimum growth was at pH 10 and 37°C. Its NaCl tolerance was up to 15%. Its major cellular fatty acids were anteiso-C15:0 and iso-C15: 0. Comparative 16S rRNA gene sequence analyses showed that XJU-3 was most closely related to Bacillus flexus, with 99% similarity. The genomic DNA (G+C) content of our isolate was 39.13 mol %. XJU-3 produced extracellular alkaline amylase, and its maximal enzyme activity was observed at 40℃ and pH 10.0. More than 70% of the enzymatic activity was remained at pH 13.0. The enzyme activity was strongly enhanced with the presence of Co2+ and Mg2+. [Conclusion] The strain XJU-3 was confirmed as B. flexus. Owing to its excellent pH tolerance, the kinds of major cellular fatty acids, and several phenotypic characteristics that were different from the description of the reference strain, the strain was further classified as a new variant of the spe-cies B. flexus. The enzymatic properties of XJU-3 alkaline amylase indicated its potential in industrial applications.

Abstract:[Objectives] To optimize the culture conditions of Pseudoalteromonas sp. AJ5 for a higher production of extracellular κ-carrageenase. [Methods] A k-carrageenan-degrading bacterium AJ5, capable of utilizing k- carra-geenan as sole source of carbon and energy, was isolated from the intestine of holothurian Apostichopus japonicus by enrichment culture technique. The strain was identified as the genus Pseudoalteromonas sp. according to its morphological and physiological characterization and 16S rRNA gene analysis. Culture conditions for the bacte-rium were standardized for the maximal productivity of the extracellular k-carrageenase by the single factor and orthogonal tests. [Results] According to the single factor test, the optimal culture conditions were: 75 mL medium in 250 mL Erlenmeyer flask, shaking speed of 150 r/min, inoculum’s volume 7%, and pH 8.0. Based on the single factor and orthogonal tests the optimal medium components were: k-carrageenan (1 g/L), beef extract (2 g/L ), NaCl (20 g/L), K2HPO4·3H2O (1 g/L), MgSO4·7H2O (0.5 g/L), MnCl2·4H2O (0.2 g/L), FePO4·4H2O (0.01 g/L), with the incubation temperature and time of 28°C and 28 h. [Conclusions] Pseudoalteromonas sp. AJ5 secreted an extracellular k-carrageenase. Under the optimal culture conditions, four-fold increase in k-carrageenase activity was achieved as compared to the control.

Abstract:[Objective] Fungus Verticillium dahliae caused greensickness of cotton and xylanase is necessary in this pathogenesis. Cloning xylanase gene from V. dahliae and heterologous expression might obtain new xylanase. [Methods] By comparing the amino acid sequences of over 10 xylanases in 11 families from fungi through BLAST, we found 2 highly conserved regions, with a fragment of about 150 amino acids coding sequence in between. Degenerate primers complementary to the ends of these two conserved regions were designed to amplify the in-between sequence from V. dahliae. The whole xylanse gene containing intron was achieved by Genome-walking PCR method. A 63 bp intron was found through BLAST, the whole cDNA xynG was cloned by DpnⅠ-mediated PCR to delete intron. The cDNA was inserted into pHBM905 and expressed in Pichia pastoris GS115, xylanase-secreting transformants were selected on plate containing RBB-xylan. The transformant with the largest halos was selected for study the character of xylanase. [Results] The deduced amino acid sequence showed 72% identity with endo-b-1, 4-xylanase from Cochliobolus carbonum and C. sativus in the GenBank, which means xynG is a new xylanase gene. The optimal pH of the purified recombinant enzyme was pH6. It remains over 50% relative activity at pH5-9. The optimal temperature was 45℃. The most favorable substrate for the xylanase (XYNG) is Beechwood xylan. Mg2+ and Ca2+ improve the enzyme activity by 33.7% and 16.6%, respectively. EDTA, b- Mercap-toethanol and NaN3 don’t affect the enzyme activity. Tween-80 and DMSO activated enzyme activity by 28.4% and 12.8%. Hg+, in concentration of 5 mmol/L, also inhibited the enzyme activity. [Conclusion] The xylanase gene xynG was firstly cloned from the fungi that caused greensickness of cotton. The xylanase genes containing one intron can be efficiently cloned from plant pathogens and white-rot fungi using strategy in this research. It is unnecessary to explore enzyme ex-pression condition and measure enzyme activity of the original strain. Enzyme character analysis showed that the XynG have potential application in the production of xylo-oligosaccharide and the improvement of bread quality.

Abstract:[Objective] To obtain the information of the cultivable microbial population diversity at the rhizosphere of Phyllostachys pubescens. [Methods] We isolated strains from Tianmu Mountain and Jinyun Mountain by diluting plate counting method and analyzed the 16S rDNA sequence of the isolates. [Results] We obtained 51 and 31 strains with different morphological character of colonies from Tianmu Mountain and Jinyun Mountain separately. The 16S rDNA sequence analysis showed that they had similar microbial population diversity. There were 40% and 58% firmicutes, 36.7% and 10.52% actinobacteria, 10% and 5.26% alphaproteobacteria, 10% and 26.32% gammaproteobacteria at the rhizosphere of Phyllostachys pubescens from Tianmu Mountain and Jinyun Mountain separately. The dominant bacteria were the genera Bacillus in both two areas. [Conclusion] The result showed that the cultivable microbial population diversity was abundant and there were some potential novel strains at the rhizosphere of Phyllostachys pubescens. Our research made it is possible to further investigate the function of the rhizosphere microbes and there interaction with the bamboo plant.

Abstract:[Objective] Vibrio is a widely distributed pathogen in aquatic environment. Our study aimed at searching for possible biological control of pathogenic vibrio. [Methods] We collected natural samples from coast and lakes in spring of 2006 and autumn of 2005; and isolated lytic phages by double-layer plate method. We identified the hosts with 16S rDNA sequencing and observed their morphology with phages under electron microscopy. We also tested the physiological characteristics of phages. [Results] We isolated 96 bacteria and 2 phages (Vibio/XM/P1, Vibio/XM/P2). Their hosts be-longed to Vibrio alginolyticus and Vibrio anguillarum. Both phages were hexagonal-headed and one with a tail. Physio-logical tests show that their optimum grow condition were pH7, 25°C and pH8, 30°C. Both phages were sensitive to high temperature and UV light. Vibrio/XM/P2 was sensitive to aether and chloroform whereas Vibrio/XM/P1 not.

Abstract:[Objective] To study whether the antiviral specificity of chicken Mx protein is determined by an amino acid substitution at position 631. [Methods] We used PCR site-directed mutagenesis technique by which a single amino acid was reciprocally substituted G with A at position 2032bp of chicken Mx cDNA. Sequence analysis confirmed successful mutation from G to A at 2032bp of chicken Mx cDNA. The fragments amplified by PCR containing the mutation site were subcloned into a enteukaryotic expression vector. Then the recombinant vector was transfected into COS-Ⅰcell, Mx gene and Mx protein in the transfected COS-Ⅰcell were detected by RT-PCR and indirect fluorescence assay. [Results] The results showed that COS-Ⅰcell transfected the recombinant plasmid could stably express the Mx protein. The antivirus assay showed that Mx protein had characteristics of resistance to infection of Newcastle Disease Virus. [Conclusion] This study may provide a basis of the virus-resistant mechanism of Mx protein and production of virus-resistant transgenic chicken.

Abstract:[Objective] To purify and to detect reactivity of non-structural proteins 3A, 3B and 2C expressed in the Escherichia coli. [Methods] FMDV NSP 3A, 3B and 2C containing the major B-cell antigenic sites were expressed in E. coli. We got renatured 2C protein by lysing of isolated inclusion body using high concentration of urea, and then diluted in a buffer system containing oxidized/reduced glutathione. Purified 3A, 3B and 2C were obtained by Ni-NTA His Bind Resin affinity chromatography. The reactivity of three NSPs with sera of different origin was measured using an indirect ELISA and Western-blot. The reactivity of three proteins was compared with 3ABC and 3D by detecting sera of clinically healthy sheep that were collected from epidemic region of AsiaⅠFMD. [Results] Proteins 3A and 3B were solubly expressed in bacteria, and 2C was expressed to form inclusion body. All three products could react specifically with sera from FMDV infected animal by western-blot and ELISA. The high coincident rates were observed between 3A, 3B, 2C and 3ABC. [Conclusion] The results would provide useful materials for establishment of immunoelectro-transfer blot (EITB) diagnostic method, which could be used for differentiation of the FMDV infected animals from the vaccinated animals．

Abstract:[Objective]: To assess the public health risk, we studied the prevalence of enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing E. coli (STEC) among pig, cattle and human in Guizhou Province. [Methods]: E. coli isolates from fecal samples were investigated for their virulence markers by polymerase chain reaction (PCR) assays. [Results]: Of 333 E. coli isolates, ETEC was predominant and detected in 73 of 112 isolates from patients, 82 of 106 isolates from pigs, and 18 of 115 isolates from cattle. The distribution of genes st, lt, and st/lt was equivalent in ETEC isolates. The detection rate of STEC from pig isolates was higher than that from patient and cattle isolates, most of which carried genes for st or lt or both. Furthermore, we analyzed the presence of the fedA gene encoding the major subunit of F18 fimbriae in E. coli isolates. Although most isolates were negative in the PCR, the presence of F18 fimbriae in the E. coli isolates was always associated with enterotoxin genes. In 25 stx-positive STEC isolates, however, only 4 STEC from pigs with diarrhea detected fedA. [Conclusion]: These results indicate that ETEC, coexisting with F18 fimbriae, is common in patients, cattle, and pigs, while STEC is dominant in pigs in Guizhou Province, China.

Abstract:[Objective] The attenuated vaccine of equine infectious anemia virus (EIAV) is the first lentiviral vaccine that provides solid protection against the infection of EIAV virulent strains. Study of the immune response induced by EIAV vaccine is an important approach to understand the immunity to other lentiviruses. IFN-g expressed by specifically stimulated lymphocytes is an important indicator for the evaluation of T cell-mediated immunity. A flow cytometry based assay was established in this study to accurately and effectively detect IFN-g expression in different subtypes of T lymphocytes in EIAV-infected horses. [Methods] Peripheral blood mononuclear cells (PBMC) were prepared from horses inoculated with EIAV vaccine stain FDDV (fetal donkey dermal-attenuated virus vaccine), virulent strain LV or saline (health control), were stimulated in vitro with FDDV or PMA + Inomycin. Brefeldin A was added into the cell culture to block protein secretions. Stimulated cells were then labeled with monoclonal antibodies specific for equine CD4 and CD8 surface markers, as well as an IFN-g-specific monoclonal antibody. Flow cytometry was applied to detect CD4+ and CD8+ lymphocytes expressing IFN-g. [Results] IFN-g positive cell population isolated from FDDV-immunized horses was 1.7±0.9% in CD4+ T cells and 6.1±1.2% in CD8+ T cells (n=4). In contrast, only 0.6±0.1% of IFN-g positive cells in CD4+ subset and 2.4±0.9% of IFN-g positive cells in CD8+ subset of T cells were detected for PBMC isolated from LV-infected horses (n=4). [Conclusion] The multi-fluorescence cell flowmetry detecting both the IFN-g expressing cells and subsets of T lymphocytes simultaneously, is specific, stable and reproducible in evaluating antigen-specific response of IFN-g- producing lymphocytes. This is an essential approach to study the protective immunity induced by EIAV vaccine.

Abstract:[Objective] We constructed cDNA library of Magnaporthe grisea. The good quality cDNA library could facilitate finding proteinaceous elicitors of M. grisea, and elucidating the mechanisms of the M. grisea--rice interaction. [Methods] The Oligo(dT) combined with the magnetic bead was used to extract mRNA from total RNA of Magnaporthe grisea and as primers to synthesize the first-strand cDNA. Terminal transferase introduced PolyA into 3’terminal of the first cDNA strand, then the PolyA was used for amplifying the second-strand cDNA. Restriction enzyme and adapter were avoided in this research, which could solve technical limitation of the traditional method. Because all reactions were done in one centrifuge tube, this process could reduce the risk of cDNA loss and cross-contamination. The primers designed in this research could clone the amplified cDNAs into expression vector in a desirable orientation. [Results]The cDNA library constructed had a high titer of 8.9×106 cfu/mL, and contained a total clones of 8.9×107 cfu, with an average inserts size of about 1380 bp. [Conclusion]Constructing cDNA library with magnetic bead was a highly efficient method useing only small amount of experimental materials within a short period.

Abstract:Herpesvirus of turkey (HVT) is an alpherpesvirus and widely used as a live vaccine against Marek’s disease (MD) because of its antigenic relationship with Marek’s disease virus (MDV). [Objective] The aim of this study was to construct Herpesvirus of turkey Fc126 strain as an infectious bacterial artificial chromosome (BAC). [Methods] Using the selection marker Eco-gpt (Xanthine-guanine phosphoribosyl transferase)(1.3 kb) and BAC vector pBeloBAC11(7.4 kb), we constructed the transfer plasmid pGAB-gpt-BAC11. Then, the transfer plasmid and HVT-infected cells’ total DNA were cotransfected into primary chicken embryo fibroblasts (CEFs). After six rounds of selection in medium containing mycophenolic acid, xanthine and hypoxanthine, we obtained purified recombinant viruses. Genomic DNA was extracted and electroporated into Escherichia coli DH10B competent cells. BAC clones were identified by restriction enzyme digestion and PCR analysis, and then tested for infectivity after transfection into CEFs using calcium phosphate. [Results] We obtained 25 BAC clones, and reconstituted recombinant viruses by transfection of HVT-BAC6 DNA, HVT-BAC8 DNA and HVT-BAC10 DNA into CEFs respectively. [Conclusion] In this study, we cloned the complete genome of HVT Fc126 strain as an infectious bacterial artificial chromosome.

Abstract:[Objective] To enhance the production of plantaricin by Lactobacillus plantarum ZJ316 isolated from infant feces. [Methods] We analyzed fermentation parameters influencing cell growth and plantaricin production with different medium composition and under different cultivation conditions. [Results] MRS (DE MAN, ROGOSA, SHARPE) medium was more suitable for producing bacteriocin than other media. The maximum plantaricin production was obtained in modified MRS medium containing 10 g/L maltose and 10 g/L glucose, 10 g/L yeast extract, 10 g/L tryptone and 2g/L tri-ammonium citrate, 1 mL/L Tween80, 6 g/L K2HPO4 ? 3H2O, 5 g/L sodium acetate, 0.2 g/L magnesium sulfate and 0.05 g/L manganese sulfate. The optimal initial pH and temperature for plantaricin production were 6.5 and 30℃ for 24 h. [Conclusion] After optimizing, the production of plantaricin was increased 2.3-fold using the optimized medium, compared with in the basic MRS medium.

Abstract:[Objective] To isolate and characterize a glyphosate-resistant strain from extremely polluted environment. [Methods] A glyphosate-resistant strain was isolated from extremely polluted soil taking glyphosate as the selection pressure. Its glyphosate resistance, growth optimal pH and antibiotic sensitivity were detected. Its morphology, cultural characteristics, physiological and biochemical properties, chemotaxonomy and 16S rDNA sequences were studied. Based on these results, the strain was identified according to the ninth edition of Bergey’s manual of determinative bacteriology. [Results] The isolate was named SL06500. It could grow in M9 minimal medium containing up to 500 mmol/L glyphosate. The cell growth optimal pH of SL06500 was 4.0. It was resistant to ampicillin, kanamycin, tetracycline and chloromycetin. The 16S rDNA of SL06500 was amplified by PCR and sequenced. Compared with the published nucleotide sequence of 16S rDNA in NCBI(National Center for Biotechnology Information), SL06500 showed high identity with Achromobacter and Alcaligenes. Based on morphological, physiological and biochemical characteristics, the strain was identified as Alcaligenes xylosoxidans subsp.xylosoxidans SL06500 according to the ninth edition of Bergey’s manual of determinative bacteriology. [Conclusion] Strain SL06500 is worthy to be studied because of its high glyphosate resistance.

Abstract:[Objective] In this study we labeled the ylyA gene of Bacillus subtilis with green fluorescent protein (GFP) to investigate the subcellular localization of YlyA protein. [Methods] Chromosomal DNA was prepared from different strains of Bacillus subtilis, ylyA amplified by PCR and the products sequenced. Full-length ylyA was then amplified and inserted into the GFP plasmid vector pSG1729, to give pNG426 containing a gfpmut1-ylyA fusion. Finally, Bacillus subtilis 168 was transformed with pNG426, resulting in insertion of the gfpmut1-ylyA fusion into the chromosome at the amyE locus. Double crossover integrants (subsequently named BS363) were identified by their inability to hydrolyse starch and verified by colony-PCR. Following induction of gfpmut1-ylyA expression with 0.5% xylose, localization of the fusion protein was determined by epifluorescence microscopy. [Results] The correct sequence and translation start site of ylyA was identified from sequence analysis of the several amplified PCR products permitting construction of a gfpmut1-ylyA fusion. Microscopic observation of strain BS363 showed that the GFP labeled YlyA was distributed around the cell periphery, closely juxtaposed with the cytoplasmic membrane. [Conclusion] GFP labeled YlyA produced by BS363 cultured on the nutrient agar solid medium distributed around the cell periphery.This suggests it may play a role in membrane biology.

Abstract:[Objective] To observe the morphological recovery of rabies virus strain SRV 9 after rejuvenation in suckling mice and to study its morphogenesis in BHK-21 cells. [Methods] The long freeze-preserved rabies virus strain SRV 9 was rejuvenated through intracerebral inoculation of sucking mice twice, followed by propagation in BHK-21 cells. After cell culture the virus was purified through sucrose gradient density ultracentrifugation. [Results] Electromicroscopy of the purified virus showed that effective recovery of viral shape was obtained after the rejuvenation with majority of viral particles having a typical bullet-like shape and intact spikes on viral membrane. The proportion of DI particles (with short triangle and irregular shapes) in rejuvenatd virus super-natant was significantly decreased compared to un-rejuvenated virus. Viral morphogenesis in cells showed that typical virus parti-cles could form in intracytoplasm 24 hours p.i. and the number of matured viral particles in cytoplasm increased significantly as culture was prolonged from 24 hours to 96 hours p.i.. Furthermore, the rejuvenated virus was observed budding from vacuole membrane in different patterns. [Conclusion] (1) The proportion of DI particles can be significantly decreased by rejuvenated through intracerebral inoculation of sucking mice.(2) The optimal harvest opportunity of SRV9 is after being 1~2 undiluted pas-saged. (3) This research provided more information about morphogenesis of rabies virus.

Abstract:The alternative sigma factor sB modulates the stress response of several Gram-positive bacteria. Not only does sB play a prominent role in sporulation in the Gram-positive model organism Bacillus subtilis, but it also contributes both directly and indirectly to bacterial virulence in the food-borne human pathogens Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. As sB has been shown to regulate expression of both virulence and virulence-associated genes, it indicates that sB is a key player in pathogen ecophysiology.

Abstract:Lipid A, the hydrophobic group of lipopolysaccharide, covers the surface of most Gram-negative bacteria. Lipopolysaccharide, known as endotoxin, can cause fatal disease like sepsis syndrome. Resent studies have shown that it is only the lipid A part of lipopolysaccharide that has the function of endotoxin. After entering the human body, lipid A on the surface of bacteria can stimulate the Toll-like-receptor 4 on the surface of host cells, cause a series of reaction, and produce different cytokines. Here we have discussed the structure, biosynthesis and pathogenesis of lipid A. The application of lipid A in vaccine development was proposed.