Abstract:Abstract:The roles of CRISPR-Cas systems in bacterial host protection against mobile genetic elements (MGEs) are now well known，but there is mounting evidence that these systems modulate other processes，such as the genetic regulation of group behavior and virulence，DNA repair and genome evolution． Here，we reviewed the structure，types and mechanism of interference of CRISPR-Cas system as well as the additional functions of CRISPR-Cas beyond adaptive immunity．Furthermore，we discussed the mechanisms for phages to overcome bacterial CRISPR-Cas system，and the prospective evolution of interactions between phage and host．

Abstract:Abstract:Filamentous microorganisms are important biocatalysts for the fermentation industry．They usually present three types of mycelial morphology in submerged cultivation: dispersed mycelium，clumps and pellet，which have an important relationship with the product quality and yield．This paper summarizes the effect of mycelial morphology on the fermentation results as well as the effect of adding microparticles on mycelial morphology，mycelial structure and fermentation yield during the fermentation process of filamentous microorganisms．

Abstract:Abstract:Eutrophication has raised increasing concerns due to its adverse effects on creatures．It is widely accepted that microbes are capable of removing nitrogen (N) and phosphate (P) via denitrification and P accumulation．So far，several strains can do this work．Therefore，more studies are focused on looking for micro-organisms that have both denitrification and P accumulation ability．Whether exposed to aerobic or anaerobic environment，microbial N and P removal mechanisms differ．Proton Motive Force and Electron Acceptor Theory are involved in the chemical process，whereas denitrifying enzymes polyphosphate kinases are regarded as the leading participators in the enzymatic systems．Studies have shown the influences of N on P accumulation，but further investigation should identify the influences of P on N removal．Here we reviewed the aspects of N and P removal mechanisms in denitrifying phosphorus accumulating organisms (DPAOs) and their potential to remove N and P from water system．Moreover，future works on clarifying denitrifying phosphorus accumulating mechanisms in depth and improving efficiency of removing N and P by DPAOs are provided．

Abstract:Abstract:［Objective］ We analyzed phylogenetic relationships between asexual endophytes isolated from western Chinese Elymus species.［Methods］ Fifteen act sequences were cloned from asexual endophytes of the western Chinese Elymus species．The phylogenetic tree and network topology structure were constructed using act sequences obtained and Genbank.［Results］ The act sequences from asexual endophyte were single-copy gene．The western Chinese asexual endophye (Neotyphodium spp.) has a different origination from the North American sexual endophyte (Epichlo elymi) from Elymus species，but there is a closer relationship between Neotyphodium spp. and Neotyphodium chisosum from North American Achnatherum eminens．Further，endophyte gene-flow exists between sexual Neotyphodium chisosum from North American Achnatherum eminens (Hap 8) and asexual Neotyphodium sp．from western Chinese Elymus species (Hap 3)．

Abstract:Abstract:［Objective］To use efficiently the insoluble K-bearing minerals in forest soil as nutrients for plant growth，we studied the weathering mechanism of muscovite by ectomycorrhizal fungi．［Methods］Cenococcum geophilum，Paxillus involutus，Laccaria bicolor，Pisolithus tinctorius，and Suillus tomentosus were used to dissolve muscovite in three culture mediums． Furthermore，K release from muscovite，biomass，remaining glucose，pH value andorganic acids were measured during the 21-day incubation of C．geophilum in Modified Melin-Norkrans (MMN) culture medium．We also compared K release amounts，organic acids and polysaccharides in external environment with those in micro-environment after 15-day and 21-day bioleaching．The weathering phenomenon on muscovite surface was observed by Scanning Electron Microscope (SEM).［Results］Among five ectomycorrhizal fungi，C．geophilum showed the best K-releasing ability in MMN culture medium．Fungal biomass，remaining glucose and pH value had significantly effects on K release．With the aid of exopolysaccharides，the hypha adhered to the mineral surface forming the micro-environment．In the microenvironment，organic acids were concentrated to promote K-release from muscovite，and obvious weathering marks on muscovite surface were found by SEM image．［Conclusion］Ectomycorrhizal fungi could weather muscovite to provide Ksolubilizing microbial resource for plant growth，and the synergism action of hyphae，organic acids and polysaccharides would accelerate K mobilization.

Abstract:Abstract:［Objective］ To study the inhibitory effect of wasp gut fungus against the radicle growth of Amaranthus retroflexus for the development of herbicides from microorganisms.［Methods］Eleven strains were isolated from wasp gut．Among them the fermentation broth of strain MF06 showed potent herbicidal activity against A．retroflexus．MF06 was identified by morphological observation and molecular biology identification．Fermentation product was isolated and purified by silica column chromatography，TLC and Sephadex LH-20 column chromatography．Metabolite 1 was obtained from fermentation product，and the inhibitory effect of metabolite 1 against the radicle growth of A．retroflexus was studied．The structure was determined by mass spectrum and nuclear magnetic resonance analyses.［Results］By the morphological observation and ITS sequence analysis，MF06 was identified as Fusarium oxysporum． The ethyl acetate extract of MF06 had strong activity against A．retroflexus with inhibition rate of more than 68% under the concentration of 100 μg/mL． It is better than other polarities crude extracts．Metabolite 1 was separated from ethyl acetate extract，and it was determined as a mixture of fusaric acid and 9，10-dehydrofusaric acid．The mixture is co-crystallizing in a 1:1 molar stoichiometry．It inhibited radical growth of A． retroflexus with IC50 value of (0.51±0.18)μg /mL，comparable to that of 2-(2，4-dichlorophenoxy) acetic acid (0.30±0.14 μg/mL) used as a positive control．［Conclusion］ Strain MF06 could be potentially developed as a microbial herbicide．

Abstract:Abstract:［Objective］We studied the effects of trophic modes related to glucose and light (photoautotrophy，mixotrophy and heterotrophy) on growth，cellular components and carbon metabolic pathway of Chlorella vulgaris．［Methods］The parameters about growth of algal cells were investigated by using spectroscopy and chromatography techniques.［Results］When trophic mode changed from photoautotrophy to mixotrophy and to heterotrophy successively，the concentrations of soluble sugar，lipid and saturated C16/C18 fatty acids in C．vulgaris increased，whereas the concentrations of unsaturated C16，C18 fatty acids，proteins，photosynthetic pigments and 18 relative amino acids decreased． Light and glucose affect the growth，metabolism and the biochemical components biosynthesis of C．vulgaris．Addition of glucose can promote algal biomass accumulation，stimulate the synthesis of carbonaceous components，but inhibit nitrogenous components．Under illumination cultivation，concentration and consumption level of glucose decided the main trophic modes of C． vulgaris．［Conclusion］Mixotrophic and heterotrophic cultivation could promote the growth of algal cells．

Abstract:Abstract:［Objective］A polar marine actinobacterium (XE-1) was selected and used to produce a protease with special characteristics.［Methods］The XE-1 was identified as Streptomyces based on morphological，biochemical and molecular characterizations (16S rRNA gene sequence analysis)．The protease was purified by 3 purification steps，including ethanol precipitation，ion exchange and gel chromatography．Its apparent molecular mass was estimated by SDS-PAGE.［Results］A solvent，detergent and oxidizing agent stable alkaline serine protease (with a low weight molecular，14 kDa by SDS-PAGE)，secreted by strain XE-1，was purified and characterized．The protease was stable in the pH range between 5 and 10，with optimal pH 8.2 and optimal temperature 55℃ ．Km and Vmax towards casein activity were 1.9 mg/mL and 973 U/mL，respectively．The protease was more active and stable in various hydrophilic organic solvents (such as dimethylformamid and toluene)．Moreover，it was also active and stable in bleaching agents (such as hydrogen peroxide); and stable in denaturant agents (such as urea and guanidine hydrochloride) at the concentration from 0.2 mol/L to 4 mol/L，which were the new characteristics． ［Conclusion］These biochemical characteristics suggest this enzyme has the potential value in numerous industrial applications.

Abstract:Abstract:［Objective］ The aim of our study is to express Coprinus cinereus peroxidase (CIP) in Pichia Pastori efficiently.［Methods］ We synthesized CIP gene with P．pastori codon bias by our Gene Synthesis and site-specific mutagenesis platform，using DNAWorks 3.1 program to design and optimize primers． Then，we sequenced the PCR products，inserted the correct gene into expression vector pPICZαA and transformed the linearized pPICZαA-Cip DNA into P．pastori GS115．We integrated CIP gene into the genome of P．pastori，using the α-mating factor from Sacchoramyces cerevisiae as signal peptide to direct the secretion of the recombinant protein．To obtain transformants with high CIP activity，we checked transformants by nested PCR and stained 82 positive ones on YPD agar plate with 1000 mg/L Zeocin．Then，we got 6 transforments with high resistance to Zeocin and expressed them in small scale; the one exhibiting the highest activity was chosen as engineered strain and named CIP /GS115．［Results］We purified CIP from culture medium after induction with ethanol，the maximum activity reached 487.5 U/mL on the 4th day．The purified CIP exhibited maximal activity at pH 5.0 and 25℃ with ABTS as substrate．The enzyme had 61.5% of the maximal activity at 45℃ and was stable below 40℃．However，the stability was drastically reduced above 45℃ ． The recombinant CIP remained stable between pH 4.5 and 6.5．We studied the substrate specificity on different substrates with the purified enzyme，and the optimal substrates were in the order of ABTS＞2，6- Dimethoxyphenol ＞ guaiacol ＞ 2，4- Dichlorophenol ＞ phenol．［Conclusion］The highly secretory expression of CIP and high special activity lay the good foundation for it’s industrial applications in waste water treatment，decolouration of dyestuffs．

Abstract:Abstract:［Objective］To clarify the composition of endophytes community in American ginseng，to know the differences of the endophytes community structure of different producing area and colonization part，and to grasp the resource situation of endophytes in American ginseng of Jilin Province． ［Methods］ Endophytes were separated with tissue explant and identified with 16S rDNA and ITS sequence analysis，by combining the cultivation forms． Moreover，ecological index analysis was conducted for the endophytes separated.［Results］Endophytes in American ginseng root obtained included 13 genera and 26 species，endophytes of the stem obtained included 10 genera and 18 species，whereas endophytes of the leaves obtained included 9 genera and 16 species． Bacillus and Pseudomonas were advantageous genera in the endophytes in American ginseng，and the species contained occupied 17.6% and 14.7% of the total endophytes respectively．According to the diversity index(H')，Hunchun＞Jingyu＞Fusong＞Ji’an＞Changbai; according to the evenness index(J)，Hunchun＞Ji’an＞Jingyu＞Fusong＞Changbai; according to the dominance index (D)，Jingyu＞Hunchun＞Fusong＞Ji’an＞Changbai.［Conclusions］ Endophytes in American ginseng were characterized by abundant genetic diversity. The structure of the endophytes community in American ginseng was complicated，there were differences of the endophytes community structure of different producing area and colonization part，the community diversity of producing area with high altitude was decreased.

Abstract:Abstract:［Objective］Our aim was to know response of spring bacteria and metabolic characteristics of sensitive bacteria to felt earthquake． ［Methods］ Water samples were collected from January 31 to December 31，2012，during which period 5 felt earthquakes occurred and the epicenter was 100 kilometers away from the No．10 Spring in Urumqi．We monitored the spring bacterial activities and function diversity changes from No．10 Spring in Urumqi during the pre- and post-earthquake stages by using plate culture counting methods and BIOLOG GEN III bacteria plate．［Results］ The spring bacterial numbers presented stochastic dynamic changes through the year．The culturable bacteria numbers and average well color development (AWCD) of carbon source utilization of bacterial community were higher after the earthquake．Besides，there were some correlations with magnitude and epicenter distance of earthquake．The main carbon source utilization types of sensitive bacteria group for felt earthquake were sugar alcohol at the No.10 Spring． ［Conclusion］The results indicated that the BIOLOG GEN III plate can be used for spring bacterial metabolism diversity research．Culturable bacteria numbers and carbon source utilization of bacterial communities showed some reflecting earthquake law.

Abstract:Abstract:［Objective］Nitrite reductase encoded by nirK is a key enzyme to denitrification，and is found in ammoniaoxidizing archaea (AOA)．Based on the diversity of nirK，it was good to study the functions of nitrite reductase to AOA on denitrification.［Methods］ We constructed nirK gene clone libraries based on the nirK gene PCR products of water，sediment and soil，screened the positive clones by restriction fragment length polymorphism (RFLP)，and sequenced the representative fragments from positive clones．［Results］RFLP analysis of the clone libraries shows that there were 10 OTUs in fresh water and sediment，8 in vegetable soil and its nearby water．Phylogenetic analysis indicated that the amino acid sequences of these nirK were most closely related to the Candidatus Nitrosopumilus koreensis AＲ1 and Nitrosopumilus maritimus SCM1 with similarities ranging from 53% to 68%．Diversity index of clone libraries shows there were many different types of nirK genes in all samples．Diversity and evenness index of nirK gene of water samples was higher than soil samples whreas vegetable field samples were the richest.［Conclusion］Thaumarchaeote nirK gene had high diversity in soil and freshwater environments which were very different from ocean sample． The nirK gene encoding nitrite reductase might be important for thaumarchaeote denitrification．

Abstract:Abstract:［Objective］To evaluate the effect of planting genetically modified cotton on soil denitrifer.［Methods］The impact of transgenic Bt + CpTI cotton (SGK321) and its receptor cotton (SY321) on rhizosphere denitrifier abundance and diversity were investigated by using quantitative PCR (qPCR) and terminal restriction fragment length polymorphism (T-FLP)．We collected rhizosphere soil before cotton planting (Pre) and along with the cotton growth stage (budding，flowering，belling and boll opening).［Results］The abundance of denitrifier in both cottons changed significantly across the growth stage，but the variation tendency was different．In the rhizosphere of transgenic cotton，the denitrifier abundance increased from 3.12×106 copies/g dry soil (Pre) to 2.81×107 copies/g dry soil (belling). The denitrifier abundance in non-transgenic cotton was significantly affected by the growth stage: increased at budding，decreased at flowering，and then increased at belling．Canonical correspondence analysis and partial canonical correspondence analysis show that the denitrifier diversity was more correlated with pH，concentration of NO3 － and budding and flowering．Additionally，cotton genotype was an important factor of influencing the diversity of denitrifier.［Conclusions］ This indicates the abundance and diversity were influenced by both the cotton growth stage and the cotton genotype by adjusting the soil pH and concentration of NO3－．Planting of transgenic Bt + CpTI cotton leads an increase in the soil pH，which results in an increase in abundance and diversity of denitrifier．

Abstract:Abstract:［Objective］This study was aimed at sequence analysis and function annotation of plasmid pKKma carrying a mariner transposon.［Methods］ Primers were designed based on the partial known sequence and used for directly sequencing plasmid pKKma．Transposon mutagenesis libraries were constructed to analyze the mutagenesis efficiency of plasmid pKKma.［Results］pKKma comprises 6879 bp with 7 open reading frames (ORFs). Among them，ORF6 encodes a mariner transposase of 348 amino acids (aa)，a C9 variant of Himar1 type transposase．Two inverted terminal repeats (ITRs) are identified and of 27 bp each．ORF7 encodes gentamycin resistance gene aacC1，locating between two ITRs．Transposable sequence alignment with other mariner transposons shows that the coverage is 2.0%－47.7% and the homology is 3.2% to 99.7%. The result indicates pKKma is significantly different from the other vectors with mariner transposon. The transposition efficiency is also analyzed．It’s (3.1×10－4)－(4.8×10－4) for S. marcescens and (1.3×10－3)－(1.7×10－3) for C.freundii，respectively.［Conclusion］pKKma carries a new mariner transposon and could be used to study the role of genes by constructing transposon libraries in bacteria.

Abstract:Abstract:［Objective］ Effects of pellet morphology，diameter，density，and interior structure on L-lactic acid fermentation by Rhizopus oryzae were characterized for different inoculum sizes and concentrations of peptone and CaCO3.［Methods］ Different initial spore concentrations were inoculated in the preculture medium with different peptone and CaCO3 concentrations，and cultivated at 30℃ for 36 h. Representative pellets were chosen for interior structure analysis and L-lactic acid production.［Results］ Inoculum size was the most important factor determining pellet formation and diameter． Peptone concentration had the greatest effect on pellet density. L-lactic acid production depended heavily on pellet density but not on pellet diameter． Low-density pellets formed easily under conditions of low peptone concentration and often had a relatively hollow structure．This structure greatly decreased production．The production of L-lactic acid increased until the density reached a certain level (50－60 kg/m3)，which the compact part distributed homogeneously in the thick outer layer of the pellet，and loose in the central layer．Homogeneously structured，denser pellets limited mass transfer．CaCO3 concentration only had a slight influence on pellet diameter and density.［Conclusion］ This work provides the insight into pellet structure and its relationship with productivity.