Abstract:Abstract: Recently，some research reports showed varied endophytic bacteria in the root nodules of some legumes，which attracts great interest in research field of rhizobia． Here，we reviewed the discovery，identification of some endophytic bacteria ( Agrobacterium，non-symbiotic rhizobia，and other bacteria) in root nodules and their influence on symbiosis or plant growth，to understand the microecosystem of root nodule and to extend the field of rhizobia research．

Abstract:Abstract: The recently discovered Clustered Regularly Interspaced Short Palindromic Repeat (CRISPRs) can protect bacteria and archaea with adaptive and heritable defense systems against the invasion of phage- and plasmid- associated mobile genetic elements． Here，we review the structure，diversity，mechanism of interference and self versus non-self discrimination of CRISPR systems． We also discuss the potential applications of this novel interference system．

Abstract:Abstract: Anaerobic ammonium oxidation (anammox) is a biological process by which ammonium is oxidized to dinitrogen gas by using nitrite as the electrons acceptor． Anaerobic ammonium-oxidizing bacteria play an important role in nitrogen removal from wastewater and global N-cycle． The study of metabolism of anammox bacteria will help us understand the anammox mechanism and develop anammox biotechnology． Anammox bacteria are chemoautotrophic bacteria that use CO2 or HCO－3 as carbon source and obtain their energy from the conversion of ammonium and nitrite into dinitrogen gas．Hydrazine has been detected as an intermediate in the anammox pathway，while hydroxylamine and nitric oxide have not been detected yet． The genomic data indicate that anammox bacteria fix carbon dioxide through acetyl-CoA pathway．The proposed anammox pathway is consistent with the available experimental data， thermodynamical calculation and biochemical determination and as well as the Ockhams razor principal．

Abstract:Abstract: ［Objective］ This study aims at investigating the diversity of actinobacteria in Aiding Lake，a hypersaline lake and the lowest land point in China． ［Methods］ The diversity of actinobacteria in the sediment from Aiding Lake was investigated by culture － independent method based on phylogenetic analysis of 16S rRNA gene sequences and selective isolation． Specific primers were used to amplify the actinobacterial 16S rRNA gene，and corresponding clone libraries were constructed for the sediment samples． Different clones selected on the basis of HaeⅢ digestion patterns were sequenced．Nine selective media with different salinities were used to isolate actinobacteria from the sediment samples．[Results]The analysis of 16S rRNA gene sequences showed that 273 clone sequences belonged to subclasses Actinobacteridae(208) ，Acidimicrobidae (13) and Rubrobacteridae (52).The dominant actinobacteria was genus Rothia，which accounted for 37% of total clones． The similarity between 45. 8% of 273 detected sequences and published sequences were less than 97% ， which might represent new taxa． Some sequences，which formed several distinct clades in phylogenetic tree may represent new taxonomical groups of actinobacteria． Fifty-five strains were isolated by different selective media． They belonged to six suborders of the order Actinomycetales，of which Streptomyces and Nocardiopsis were the dominant groups．Six potential new species were obtained．[Conclusion] Aiding Lake harbors abundantactinobacteria，including large number of unknown actinobacterial groups．

Abstract:Abstract: ［Objective］ In order to enable the caerulomyicn biosynthetic study by in vivo gene disruptions，it is crucial to develop a genetic modification system for the producer Actinoalloteichus sp. WH1-2216-6. Methods］The spore germination timing and the concentration of MgSO4 in the medium were investigated for the optimal conjugal transfer of exotic pSET152 DNA into Actinoalloteichus sp． WH1-2216-6． Using the PCR-targeting system，we disrupted a putative caerulomycin 2，3 -dihydroxybenzoate-AMP ligase gene by“in-frame deletion”in E． coli，to afford the cosmid pCSG2104，which was then transferred into Actinoalloteichus sp． WH1-2216-6 by conjugation under optimized conditions．［Results］The putative caerulomycin 2，3-dihydroxybenzoate-AMP ligase in Actinoalloteichus sp． WH1-2216-6 was successfully disrupted by in-frame replacement with the aac3IV gene cassette． The resulting mutant strain was unable to produce caerulomycins．［Conclusion］ The presence of high concentration of MgSO4 in the medium can promote the conjugation efficiency between E． coli and Actinoalloteichus sp． WH1-2216-6 and lead to the successful development of a genetic modification system for Actinoalloteichus sp． WH1-2216-6， enabling the functional characterization of caerulomycin biosynthetic genes in vivo． A positive example was provided for other Actinobacteria recalcitrant to genetic modification．

Abstract:Abstract: ［Objective ］ To investigate the influence of isolated predominant aerobic bacteria on daidzein biotransformation capacity by co-culture with daidzein biotransforming bacteria．［Methods］ Predominant aerobic bacteria were isolated from diluted feces solutions of different healthy animals，including ICR mice，Luhua chicken，Landrace pigs and Rex rabbits． Daidzein biotransforming bacteria were anaerobically co-cultured with the isolated predominant aerobic bacteria and the cultural broth was extracted and detected by high performance liquid chromatography (HPLC).［Results］ Twenty two predominant aerobic bacteria were isolated from the four different healthy animals mentioned above． Based on the analyses of 16S rRNA gene sequences，morphology study and relative biophysicobiochemical characteristics，all 22 isolates belong to the 5 genera，i.e.Escherichia (10)，Proteus (5),Enterococcus(4), Bacillus (2) and Pseudomonas (1). Co-culture between predominant aerobic bacteria and daidzein biotransforming bacteria was carried out under anaerobic conditions． The results showed that the biotransformation capacity was totally lost when different daidzein biotransforming bacterium was co-cultured with either Bacillus cereus (R1) or Pseudomonas aerginosa (R5) and continuously inoculated for 2 or 3 passages． However，no obvious influence was observed when daidzein biotransforming bacteria were co-cultured with all the other isolated predominant aerobic bacteria except R1 and R5. In addition，when strain R1 and R5 was co-cultured with the intestinal microflora of the ICR mice anaerobically and continuously inoculated for 5 passages，about 90% of the co-cultures totally lost the activity to convert daidzein to equol effectively．［Conclusion］Different predominant aerobic bacteria showed different influence on daidzein biotransformation capacity after being co-cultured with different daidzein biotransforming bacteria． Among all the isolated predominant aerobic bacteria used for co-culture，both Bacillus cereus (R1) and Pseudomonas aerginosa (R5) were detected significant inhibition on biotransformation activity of different daidzein biotransforming bacteria．

Abstract:Abstract: ［Objective］ The fusion protein SL2B with both xylanase and N-acyl-homoserine lactonase activities was expressed in Pichia pastori． Characterization of the purified xylanase and N-acyl-homoserine lactonase fusion protein SL2B was investigated．［Methods］ The fusion gene sl2b was amplified from the N-acyl-homoserine lactonase gene aiiA-B546 and the xylanase gene xynAS27cd via overlap PCR technique． After the recombinant vector pPIC9/sl2b was transformed into P． pastoris，transformants with both xylanase and N-acyl-homoserine lactonase activity were screened． The purified SL2B was obtained with ammonium sulfate precipitation and molecular sieve． Both N-acyl-homoserine lactonase and xylanase activities of SL2B were characterized． ［Results］ The purified SL2B showed that the xylanase had optimal pH and temperature at pH 6. 5 and 60℃，respectively． The enzyme was stable between pH 6. 0 and 8.0，retained over 80% enzyme activity between 50 and 65℃．It resisted various neutral proteases and chemical reagents． With oat spelt xylan as substrate，the Km value of SL2B was 2. 9 mg /L．The N-acyl-homoserine lactonase had optimal pH and temperature at pH 8.0 and 30℃，respectively．The enzyme was stable between pH 4. 0 and 10.0，retained over 80% enzyme activity between 0 and 50℃．It resisted various neutral proteases and chemical reagents． The fusion protein can hydrolyze many Nacyl homoserine lactones substrates． With N-(3-oxo-octanoyl)-L-homoserine lactone as substrate，the Km value of SL2B was 0.050 mmol/L．Conclusion］ High level expression is achieved by fusing N-acyl-homoserine lactonase to the xylanase．

Abstract:Abstract: ［Objective］ Denitrifying bacteria play an important role in the biological nitrogen removal process，especially the aerobic denitrifying bacteria． However，there are few studies on aerobic denitrifying bacteria． The present study aimed at the isolation of aerobic denitrifying bacteria with high ammonium and nitrite nitrogen removing ability from environmental samples，and its phylogeny and denitrifying characteristics.［Methods］ Based on the aerobic denitrifying activity，ammonium and nitrite nitrogen removing ability， the strains were isolated from sludge，water and sediment in a eutrophicated pond． A strain with the highest activities was identified according to its morphological，physiological and biochemical properties and phylogenetic analysis of its 16S rRNA sequence． By using NO－3-N，NH+4-N and NO－2-N as the sole nitrogen source respectively，its denitrifying characteristics，and the effects of culture conditions such as initial pH of medium，temperature，carbon source，shaking speed on the ability of removing ammonium and nitrite nitrogen，were investigated under aerobic condition．［Results］Among the isolated strains，strain C-4 showed the highest ability of removing ammonium and nitrite nitrogen． Strain C-4 was identified as Acinetobacter sp．Under the conditions of sodium citrate as carbon source，temperature 30℃ ，shaking speed 120 r/min，cell age of 18 h，pH 8.5 for 200 mg/L NH4+-N medium and pH 7.5 for 100 mg/L NO2 －-N medium，the net removal efficiency of nitrogen were 65.8% and 47.8% after 15 h and 12 h，respectively．［Conclusion］ An aerobic denitrifying strain Acinetobacter sp．C-4 ( HQ896038) was isolated from water pond，and it exhibited high net removal efficiency of nitrogen in relative media． The net removal efficiency of nitrogen of strain C-4 was 73. 04% in dealing with a eutrophicated pond water．

Abstract:Abstract: ［Objective］ The pine wood nematode，Bursaphlenchus xylophilus，morphologically similar to B． mucronatus，is the pathogen of pine wilt disease． This study was focused on the endophytic bacteria present in these nematodes．［Methods］ Detailed observations were made on sections of all parts of the two types of nematodes by transmission electron microscope． The nematodes were surface-sterilized by soaking in 1% mercuric chloride and antibiotic mixture，and then ground and cultured on nutrient agar plate． The physiological and biochemical characteristics combined with molecular characterization of bacteria were analyzed and identified．［Results］Endophytic bacteria were found in intestines of the two nematodes by transmission electron microscope observations． On the basis of surface sterilization，total three bacteria strains were obtained from B． xylophilus and B． mucronatus． These bacteria belong to Stenotrophomonas and Ewingella．［Conclusion］ It confirms the presence of endophytic bacteria in Bursaphelenchus xylophilus and B． mucronatus and these bacteria may play a physical and ecological roles in nematodes．

Abstract:Abstract: ［Objective］Phytophthora melonis is the casual agent of wax gourd and cucumber Phytophthora blight which becomes a constraint for sustainable production of the related crops．Metalaxyl is one of the principal fungicides for controlling the disease now． The objectives of the present study were: (1) to investigate the baseline sensitivity and field resistance of P．melonis to metalaxyl in South China;(2) to test the occurrence of metalaxyl-resistant mutants from metalaxyl-sensitive wild type strains exposed to the fungicide; and (3) to monitor the development of metalaxyl resistance in P． melonis population．［Methods］ Over 400 samples of wax gourd and cucumber Phytophthora blight were collected from Guangxi Zhuang Autonomous Region and Guangdong province during 2007-2010，and 193 strains of P．melonis were isolated and purified．The sensitivity of the isolated strains to metalaxyl was tested using mycelial growth rate method in vitro and floating-leaf-disk method in vivo，respectively． The metalaxyl-sensitive strains were induced on PDA plates containing 10 μg/mL metalaxyl．［Results］The sensitive，moderately resistant and resistant strains were recorded as 29.0%，18.1% and 52.8%，respectively，among 193 tested strains．The frequency and level of resistance of P．melonis from Guangdong were higher than that from Guangxi．The strains from cucumber was generally more resistant to metalaxyl than those from wax gourd． The metalaxyl-resistant strains were frequently detected as predominant populations in most of the sampling sites and the highest resistance index (4226.9) was confirmed．Metalaxyl-resistant (Mrt) mutants could be isolated from approximately 60% of the sensitive wild-type strains．The resistance level of the Mrt mutants was 189－407 times higher than that of their sensitive parental strains．The EC50 values of 9 sensitive strains from a sampling site without a record of phenylamide fungicide application ranged from 0.0429 to 0.5461 μg/mL．Their mean EC50 value (0.3200±0.1617μg/mL) was considered as the baseline sensitivity of P． melonis to metalaxyl in South China．［Conclusion］Metalaxyl-resistant strains universally occur in South China，especially in the vegetable-growing areas with a longer history of metalaxyl application． The establishment of the baseline sensitivity of P． melonis to metalaxyl will provide a sciencebased guide for evaluating and further monitoring resistance of the pathogen to the fungicide．

Abstract:Abstract:［Objective］To study the degradation of pyridine and quinoline by two Pseudomonas．［Methods］Based on the analysis of 16S rRNA gene sequence homology and the intergenic spacer region sequence，the two isolates were identified．The degradation capability of pyridine and quinoline was etermined according to spectrophotometry and Electrospray Ionisation /Mass Spectrometry (ESI/MS)．The degrading plasmids were detected by plasmid curing and the possible degrading genes were also cloned． ［Results］ The two isolates were identified as Pseudomonas and nominated XJUHX-1 and XJUHX-12．The two Pseudomonas were tolerant with pyridine and quinoline and two and four possible metabolites were detected in the culture medium containing quinoline and pyridine，respectively． The degrading capability of curing plasmids was lower than the crude isolates．The gene segments coding for the NADH (acceptor) reductase component OxoR for quinoline degradation and nitrogenase reductase (NifH) of denitrification for pyridine degradation were amplified from the genome of XJUHX-1 and XJUHX-12，both were cloned and expressed in E．coli BL 21 producing recombinant proteins with molecular mass of 43 kDa and 16 kDa． ［Conclusion］ The two isolates could degrade pyridine and quinoline respectively．

Abstract:Abstract: ［Objective］To study the biological characteristics and pathogenicity of a recombinant rabies virus Flury LEP (low egg passage) that has two glycoprotein genes (Ggene)．［Methods］By using reverse genetics techniques，we constructed a recombinant virus Flury LEP that has an additional G gene between P and M gene (rLEP-PGM)．Then we studied the biological characteristics of the recombinant virus and its pathogenicity on mice．［Results］The in vitro growth characteristic of rLEP-PGM were similar to the LEP strain． Western blot analysis of glycoprotein expression showed that the glycoprotein expression level of rLEP-PGM was 1.5 times higher than LEP． The LD50 of rLEP-PGM and LEP was 3 FFU and 1 FFU by intracerebral injection． However，the LD50 of intramuscular injection was 4×104 Lg FFU and 3.2×105 Lg FFU，respectively．［Conclusion］Insertion of an additional G gene between P and M gene can significantly raise the expression level of glycoprotein and enhance the ability to invade central nervous system from peripheral sites．

Abstract:Abstract: ［Objective］ Targeting the important enzyme in human glucose metabolic pathway，we established a high throughput screening model for human pancreatic α-amylase inhibitors． ［Methods］ Pichia pastoris expression system was used to clone and express the human pancreatic α-amylase; we established the α-amylase inhibitor screening model using the catalytic properties of enzyme; this model was applied in screening of actinomycete' metabolites; the taxonomic status of positive strains were analyzed by constructing 16 S rRNA phylogenetic tree． ［Results］ We cloned and expressed the intact gene of human pancreatic α-amylase successfully; the high-throughput screening model of α-amylase inhibitors was established; nearly 2000 actinomycete' metabolites were screened，14 α-amylase inhibitor producing strains were obtained finally， and showed taxonomically rich diversity． ［Conclusion］ The α-amylase inhibitor high-throughput screening model had high practical value for developing new hypoglycemic drugs．

Abstract:Abstract: ［Objective］In order to find the best extraction method for proteins of Alexandrium tamarense for twodimensional electrophoresis (2-DE) analysis．［Methods］Three methods for extracting proteins from A．tamarense were compared，including trihydroxymethyl aminomethane (Tris-HCl) buffer extraction，trichloroacetic acid (TCA)/acetone precipitation and lysis buffer extraction． Alga was cultivated in normal f /2 media (control) and supplemented with algicidal substances． Proteins obtained using the best extraction method were separated with 2-DE． Protein-expression differences were identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS)．［Results］ Among the three protein extraction methods，lysis buffer extraction shows the best detection of the number and quality of protein spots with a clear background． Then，the lysis buffer extraction method was successfully applied to profiling protein expression in algicidal substances stress conditions and 14 differential expression proteins were identified using MALDI-TOF/MS．［Conclusion］Lysis buffer extraction was the most effective protein extraction method for Alexandrium tamarense．

Abstract:Abstract: Objective］The aim of this study was to establish a new EvaGreen real-time IAC-PCR for the rapid detection of Salmonella．［Methods］ We used Salmonella genomic comparison analysis to mine Salmonella-specific targets，and Primer Premier 5.0 to design primers which were evaluated by specificity and sensitivity tests．［Results］We obtained a Salmonella-specific gene that encodes putative type Ⅲ secretion protein (ssaQ)，and specific primers (SsaQ6L/SsaQ6R) were designed based on this gene．Then we established IAC-PCR and EvaGreen real-time IAC-PCR assays，which showed 100% inclusivity and 100% exclusivity on all strains tested． Their detection limits of purified Salmonella genomic DNA were 14. 9 copies /PCR and 2. 76 copies /PCR respectively． Artificial contamination assays showed that Salmonella could be detected after 10 hours and 8 hours enrichment when the original bacterial concentration was 4. 2 cfu /10 mL．Conclusion］A new EvaGreen real-time IAC-PCR with high specificity and sensitivity was successfully developed for the rapid detection of Salmonella．

Abstract:Abstract: ［Objective］An antagonistic bacterial strain YB-3 against Rhizoctonia solani was isolated from soils．［Methods］ Antagonistic strains were isolated by a reporter strain method． YB-3 was identified based on morphology observation，physiological and biochemical characterizations，Biolog，G + C content and 16S rDNA sequence analysis．The antagonistic spectrum and the properties of the inhibitor produced by Bacillus amyloliquefaciens YB-3 against plant pathogenic fungi and bacteria were investigated by means of plate two-way cultivation and disc diffusion method．［Results］ The strain YB-3 against Rhizoctonia solani was identified as Bacillus amyloliquefaciens． The antagonistic results showed that it had distinctively inhibitive effects on 14 pathogenic fungi and 7 bacteria． In addition，it also had inhibitive effects on strains from genus Bacillus to which YB-3 belongs． Antagonistic properties of B．amyloliquefaciens YB-3 was thermostable，acid resistant，and protease sensitive． ［Conclusion］ Bacillus amyloliquefaciens YB-3 was isolated and characterized which had distinctively inhibitive effects on Rhizoctonia solani and had broad-spectrum，highly efficient to plant pathogens．