Abstract:Abstract:The holin-lysin two-step lysis system widely exists in double stranded DNA bacteriophages for the release of progeny bacteriophage from an infected bacterial cell at the final stage of phage infection． Lambda bacteriophage is a prototype for studying holin． The S gene in Lambda bacteriophage has a dual-start motif and encodes holin S105 and antiholin S107． Here，we reviewed the progress in topological structure of holin from Lambda bacteriophage and its formation of membrane lethal holes．We also discussed the potential of the holin in the control of bacterial infection．

Abstract:Abstract:Tuberculosis ( TB) is one of the world’s deadliest diseases． Approximately eight million individuals develop active tuberculosis annually，and two million die of tuberculosis． The emergence of multi-drug resistance strains，HIV coinfection，and an increasing aging population further worsen this scenario． Mycolic acids (MAs，also mycolate) are integral cell wall components of Mycobacterium tuberculosis，other mycobacterium and most actinomycetes，engaging in the remarkable survival ability of Mycobacterium tuberculosis within infected hosts，virulence and evasion of immunity． The biosynthesis and regulation of mycolic acids are rife with anti-tuberculosis drug targets． First-line tuberculosis drugs such as isoniazid and ethambutol target this pathway． In-depth investigation of this aspect will provide more opportunities to find better measures to combat tuberculosis． To this end，we reviewed the structures，classification，biosynthesis pathway，regulation factors in pathway of mycolic acid，as well as promising drug targets．

Abstract:Abstract:The occurrence of off-flavor problems caused by prokaryotes cyanobacteria and actinomycetes is a worldwidewater and food quality issue． Based on literatures on two earthy-muddy-smelling metabolites，(i.e.,geosmin and 2-methylisoborneol (2-MIB))，we reviewed their chemical characteristics，biosynthetic pathways，genes and enzymes that are involved in biosynthesis． Then we discussed current research questions related to off-flavor and future directions．Finally，we addressed the significance of probable key regulatory mechanism for the production and release of geosmin and 2-MIB，which could provide more scientific strategies to better control off-flavors outbreaks in drinking and aquaculture water．

Abstract:Abstract:［Objective］ To isolate myxobacteria and investigate their diversity in saline-alkaline soils from Akesu in Xinjiang． ［Methods］Conventional culture-dependent methods，e． g． baiting technique，water agar，soil extract agar and mineral agar，were used to isolate myxobacteria from 25 soil samples collected from Akesu areas of Xinjiang． Combining with physicochemical properties (acidity /alkalinity，salt concentration，vegetation and geographical locations) of the soil samples，myxobacterial diversity was studied．［Results］In total 58 strains were isolated，and identified as belonging to 6 different genera，i． e． Myxococcus，Cystobacter，Corallococcus，Sorangium，Nannocystis and Polyangium of Myxococcales．The most frequent genus isolated was Myxococcus which may better adapt in harsh environments． Different myxobacterial diversity was detected in different habitat．［Conclusion］Myxobacteria diversity was low in saline-alkaline soils of Akesu in Xinjiang．

Abstract:Abstract: ［Objective］To determine the pathogenic bacterium infecting giant salamander (Andrias davidianus). ［Methods］Bacterium was isolated from the liver of diseased Chinese giant salamander and identified by the Biolog Microbial Identification System and molecular biology method． Healthy Chinese giant salamander and crucian carp were used for experimental infection with bacterial suspension． ［Results］A bacterial strain JZ01 was isolated and identified from diseased giant salamander． Infection with the bacterial suspension to healthy giant salamander could reproduce the diseased symptoms as occurred naturally and the same bacterium could be recovered from these infected giant salamanders．The isolated bacterium also has certain pathogenicity to crucian carp． Identification by the Biolog Microbial Identification System，and further 16S rDNA sequence and phylogenetic analysis demonstrated that the bacterium isolated from diseased giant salamander was Citrobacter freundii． The susceptibility test to antibiotics demonstrated that the bacterial strain JZ01 was susceptible to aztreonam，cefepime and cefotamine． ［Conclusion］Citrobacter freundii is a pathogen for cultured Chinese giant salamander．

Abstract:Abstract:［Objective］To develop a novel Escherichia coli cell surface display system by using C-terminally truncated NCgl1221 as the anchoring protein，which greatly enriched or optimized the bacterial displayed systems．［Methods］We amplified the sequence of C-terminally truncated NCgl1221 and β-amylase，and constructed the fusion expression vector．Then we transformed the recombinant plasmids PET-NA and PET-28a into Rosetta (DE3) pLysS．The fusion protein expression was induced by IPTG and identified by SDS-PAGE and Western blot analysis．The IPTG induced strains were immunostained and investigated by fluorescence microscope and flow cytometry to detect the displayed β-amylase．Finally， we analyzed the activity of β-amylase and starch hydrolization in order to determine whether the displayed β-amylase has the activity or not． ［Results］The fusion protein was successfully expressed in E． coli，and the active β-amylase was displayed on the cell surface by fusing it to the C terminus of the anchor． The recombinant strain displaying β-amylase can utilize soluble starch in the medium．［Conclusion］A novel E． coli surface display system by using C-terminally truncated NCgl1221 as the anchor motif was successfully developed． The active enzyme with a molecular size of 56 kDa was displayed on E． coli by this system，which provided the basis for the application of the system in whole-cell biocatalyst or biosorbent．

Abstract:Abstract:［Objective］To clone outer membrane protein A (ompA) gene from Aeromonas hydrophila HBNUAh01 and transiently express this protein in tobacco (Nicotiana tabacum) leaf cells． ［Methods］ A． hydrophila outer membrane protein A (AhompA) gene was amplified by PCR using A． hydrophila HBNUAh01 cells as template，and cloned into the pEASY-Blunt Simple vector for sequencing． After sequence confirmation，AhompA gene was inserted into expression vector pCAMBIA1300 containing a yellow fluorescent protein (YFP) gene，to obtain a recombinant expression plasmid． The recombinant expression plasmid was introduced into Agrobacterium tumefaciens GV3101 competent cells，and the positive clones were transfected to tobacco leaf cells． The expression of yellow fluorescent protein fused with AhompA was observed by Confocal Laser Scanning Microscope，and the mRNA of AhompA was detected by RT-PCR． ［Results］The AhompA gene cloned from A． hydrophila HBNUAh01 was 1032 bp． The fusion protein of AhompA and YFP was successfully expressed in tobacco leaf cells． ［Conclusion］The successful expression of the AhompA gene in tobacco leaf cells has laid a foundation for further investigation of prevention of limnobios diseases caused by A． hydrophila with plant vaccine．

Abstract:Abstract:［Objective］We used a minicircle DNA vector system to express small interfering RNA (siRNA) and studied the inhibition of hepatitis B virus (HBV ) replication and gene expression in vitro． ［Methods］ siRNA targeting HBV Sgene (siHBS) was designed ，synthesized and cloned into a minicircle DNA vector pMC． BESPX-MCS2. After sequencing，we transformed the recombinant pMC-H1-siHBS-U6 into E.coli ZYCY10P3S2T， and induced the degradation of its bacterial backbone by adding L-arabinose into the bacterial growth medium． As expected，a minicircle RNA interference (RNAi) vector pmc-H1-siHBS-U6 was generated only consisting of gene expression cassette． Then pmc-H1-siHBS-U6 was co-transfected into Huh-7 cells with HBV expression vector pHBV1. 3. ELISA and Real-time PCR were performed to evaluate the inhibition effect of the secretion of HBsAg and HBeAg and the levels of HBV DNA and mRNA in Huh-7 cells． ［Results］We Successfully established the minicircle-based RNAi vector pmc-H1-siHBS-U6，which can significantly inhibit the secretion of HBsAg and HBeAg in Huh-7 cells for two to three weeks． Real-time PCR results show that HBV DNA and mRNA levels were also down-regulated about 71% and 80%． ［Conclusion］ The minicircle DNAbased RNAi vector pmc-H1-siHBS-U6 can suppress HBV replication and gene expression specifically，efficiently and steadily． Thus，this study provided us a new siRNA delivery system and a new gene therapy strategy of HBV infection．

Abstract:Abstract:［Objective］Streptomyces hygroscopicus ATCC29253 has attracted much interests due to its capacity of producing various secondary metabolites with strong bioactivities，including immunosuppressant rapamycin，nigericin，hexaenes，elaiophylin and hygrocins． ［Objective］To investigate biosynthetic pathway of these metabolites and construct high-yield strains by genetic engineering，establishment of a highly efficient genetic manipulation system is critically required in this strain． ［Methods］We tested the effects of conjugation media and donor strains on conjugal transfer from Escherichia coli to S． hygroscopicus ATCC29253 and other Streptomycetes． ［Results］ We found that both casamino acid and MgCl2 supplemented in conjugation media improved conjugation frequency in S． hygroscopicus ATCC29253. A random experiment led to the disclosure of an optimal combination of casamino acid and MgCl2 by which the conjugation frequency in S． hygroscopicus reached 1. 5 × 10 － 4 ． Meanwhile，we also found significant changes in conjugation frequencies of S．lividans，S． albus and S． avermitilis when casamino acid was supplemented in conjugation media． ［Conclusion］Casamino acid has significant influence on conjugation frequency in not only S． hygroscopicus ATCC29253 but also other Streptomyces such as S． lividans，S． albus and S． avermitilis．

Abstract:Abstract:［Objective］To determine the best conditions for Bacillus globisporus Q12 and Rhizobium sp．Q32 to produce organic acids and extracellular polysaccharides，respectively，and further elucidate the weathering mechanism of the two potassium-bearing mineral-solubilizing bacteria．［Methods］Different contents (0－1.2 g/L) of (NH4)2SO4 were added to media to analyze the ability of the strains to produce organic acids and extracellular polysaccharides，and assess the ability of Q12，Q32 and their mixture to dissolve potassium feldspar． Scanning electron microscope (SEM) was also used to observe the distribution of the bacterial cells on the surfaces of the feldspar and the mineral weathering． ［Results］ Results show that Bacillus globisporus Q12 produced more organic acids，when the contents of (NH4) 2SO4 were 0.6 g/L;Rhizobium sp． Q32 produced more extracellular polysaccharides，when there was no (NH4) 2SO4 in the media; and the mixture of two strains produced more organic acids and extracellular polysaccharides，when the contents of (NH4)2SO4 were 0.3 g/L． Mineral dissolution experiment showed that Bacillus globisporus Q12，Rhizobium sp． Q32 and the mixture (Q12 + Q32) significantly dissolved the feldspar and released the elements from the mineral，of which the mixture of Q12 and Q32 had the best weathering ability than strain Q12 or Q32; SEM also indicated that the mixture of Q12 and Q32 had more ability to weather feldspar than each tested strain． ［Conclusion］ The contents of (NH4)2SO4 in the media could affect the growth and metabolites of the strains Q12 and Q32 and the mineral bioweathering，the mixture of strains Q12 and Q32 had the more potential of feldspar weathering through the combined action of organic acids and extracellular polysaccharides produced by strains Q12 and Q32．

Abstract:Abstract:［Objective］The aim of this study is to clone and express the nucleotidylytransferase encoding gene-amiE from the biosynthetic gene cluster of amicetin，a disaccharide nucleoside antibiotic，and to characterize AmiE in vitro．［Methods］ The amiE，encoding a ucleotidylytransferase of 257 amino acid，was PCR amplified and cloned into pET28a，resulting in the plasmid pCSG4001，which was transformed into E.coli BL21(DE3) for expressing N-(His)6-tag AmiE． The recombinant AmiE was purified by affinity chromatography via AKTA Purifier 10 system． The AmiEcatalyzedreactions were performed using TTP (or UTP) and glucose-1-phosphate as substrates． The enzyme assays were analyzed by HPLC; the substrate flexibility of AmiE was probed with three unnatural sugars-1-phosphate，including galactose-1-phosphate，galactosamine -1-phosphate and mannos-1-phosphate． ［Results］ The N-(His)6-tag AmiE was expressed in E． coli in soluble form and was successfully purified via Ni +2 mediated affinity chromatography; in vitro biochemical experiments showed that AmiE could convert glucose-1-phosphate into TDP-glucose (or UDP-glucose) in the presence of TTP ( or UTP)． However，galactose-1-phosphate，galactosamine-1-phosphate and mannos-1-phosphate were not substrates of AmiE． ［Conclusion］The amiE was successfully cloned and expressed in E． coli，and the purified AmiE was biochemically confirmed to be a nucleotylyltransferase in amicetin biosynthesis pathway．

Abstract:Abstract:［Objective］We studied the formation of carbonate minerals by bacteria to understand the mechanism of microbial mineralization． ［Methods］Cultures of carbonate precipitation using Lagoa Vermelha medium with 6:1 molar ratio of Mg /Ca within 35 days were made under the mediation of Clostridium sp． (MH18 strain) isolated from soil． At the same time，aseptic experiments without the inoculation were done as the control． Mineral species were determined by Xray diffraction，and the morphologies of precipitated carbonates were observed using optical microscopy and scanning electron microscopy． ［Results］In the LV medium，MH18 strain mediated the formation of carbonate mineral，in which high-magnesium calcite was dominant． In the initial stage，the minerals had shapes with dumbbell-like morphology，and finally transformed to spheres． Only a small amount of precipitation appeared in the control，but X-ray diffraction patterns showed that these precipitations were amorphous substance． ［Conclusions］MH18 strain could induce crystallization of carbonate.

Abstract:Abstract: ［Objective］ To explore the mechanism of cell death in Microcystis aeruginosa PCC 7806．［Method］According to the water environment of the later period of algal bloom，M． aeruginosa PCC 7806 was treat with dark and O2 limitation． We observed the morphological changes using transmission electron microscope (TEM) and detected the reactive oxygen species ( ROS) activity and Caspase3 activity in M． aeruginosa PCC7806 subjected to dark and O2 limitation． DNA status was also examined with the methods of Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and agarose gel electrophoresis． ［Result］Massive algae cell died after 48 h treatment under dark and O2 limitation． During cell death process，we observed some changes of cell organelles including ribosomes and thylakoids disorganization，cytoplasmic vacuolation，nucleoplasm diffusion and plasmolysis in M． aeruginosa PCC7806 subjected to darkness and O2 limitation． Meanwhile，we found that increased ROS reactivity and capase 3 activity were related to the cell death process of M． aeruginosa． DNA breakage and fragmentation were proved by TUNEL staining and agarose gel electrophoresis during cell death process． ［Conclusion］All results showed that cell death with characteristics similar to eukaryotic programmed cell death could be induced in M． aeruginosa PCC 7806 after treatment with darkness and O2 limitation． Therefore，we suggested that the mechanism of cell death are conserved during evolution according to the characteristics of cell death shared between eukaryotes and Microcystis．

Abstract:Abstract:［Objective］Glacier is a unique ecological system． This study focused on the isolation and characterization of a cold-active bateriophage from Mingyong glacier area in northwest Yunnan． ［Methods］ Bacterial strains isolated from glacial melt water were used as host cells to isolate and purify bacteriophages by double-layer plate method． The morphology of the isolated phages and their host strains were observed by electron microscope． Restriction fragment length polymorphism (RFLP) analysis of genomic DNA，constituent proteins and physiological analysis of the bacteriophages were further carried out to characterize the phages． ［Results］A lytic cold-active bacteriophage，designated as MYSP03，was isolated from Mingyong glacier． Its host strain MYB03 was identified as a member of genus Flavobacterium，based on the 16S rRNA sequence analysis． The bacteriophage MYSP03 has a isometric head (about 72 nm in diameter) and a long tail (about 240 nm in length and 10 nm in width)，but no envelope was detected． Physiological analysis results showed that MYSP03 had infection activity at 4℃，and clear and transparent plaques were formed on double-layer plates between 4 and 20℃． Its optimum infection temperature was 10℃ and optimal pH 9. 4，respectively． It is insensitive to chloroform．Furthermore，the genome of MYSP03 consists of double-stranded DNA and is approximately 66 kb．

Abstract:Abstract:［Objective］We investigated the correlation between the in vitro immune profiling of 5 lactobacilli strains and their in vivo protective effect in a mouse β-lactoglobulin (BLG) allergy model for selecting the candidate strains with potential anti-allergy activity． ［Methods］In vitro immunomodulation was assessed by measuring interleukin (IL)-4 and interferon γ (IFN-γ) release by primary lymphocytes stimulated with 5 active/heat-killed lactobacilli． A mice model of β-lactoglobulin allergy was then used to evaluate the alleviating allergy capacity of the same set of strains． The rats were randomly divided into blank group，BLG allergy group and different lactobacilli strains group． The total IgE and BLGspecific IgE contents in the serum of rats were measured with ELISA． Splenic lymphocytes were isolated and cultured in vitro，the levels of Thl/Th2 type cytokine were detected by ELISA． ［Results］ Protection of BLG-induced allergy was strain-specific． The strains displaying an in vitro capacity to induce higher levels of the Thl type cytokine (IFN-γ) and lower levels of the Th2 type cytokine (IL-4)，significantly decreased the levels of total IgE and BLG-IgE in allergic rat serum (P＜0.05)．In contrast，strains leading to a low IFN-γ/IL-4 cytokine ratio could not significantly attenuate allergic symptoms． ［Conclusion］We could predict the in vivo protective capacity of the studied lactobacilli strains based on the cytokine profile established in vitro． Oral consumption of specific strain may be effective in preventing and alleviating BLG allergic symptoms by the improvement of the Th1 /Th2 cell balance toward Th1 dominance，and the inhibition of IgE production.

Abstract:Abstract:［Objective］To identify and characterize an acetonitrile degrading strain BX2，thus to assess its potentials in the treatment of acetonitrile containing wastewater.［Methods］ By means of phenotype and physio-biochemical characterization as well as phylogenetic analysis，we identified strain BX2． The optimum culture conditions of the strain were studied with single factor test，and the degradation of acetonitrile under the optimal growth conditions was determined． Additionally， NaCl tolerance was investigated.［Results］ The phenotype and physio-biochemical characteristics of strain BX2 were similar to those of Rhodococcus sp. The phylogenetic analysis based on 16S rRNA， gyrB and secA1 gene suggested strain BX2 was the closest relative of Rhodococcus rhodochrous with 99.37%，99.29% and 97.87% sequence similarity respectively．The optimal conditions for cell growth were 35℃，initial pH 7.5，and 1% inoculum． Under these conditions，the degradation rate of acetonitrile was 95.87% (800mg/L) within 16 h.Strain BX2 was able to grow in defined medium containing NaCl up to 6%.［Conclusion］Strain BX2 was identified as Rhodococcus rhodochrous and named Rhodococcus rhodochrous BX2． It showed great environmental adaptation and high capability of degrading acetonitrile．

Abstract:Abstract:［Objective］We identified genes that regulate the expression of aphB，the gene encoding a key virulence regulator in Vibrio cholerae O1 El Tor C6706－．［Methods］We constructed a transposon library in V． cholerae C6706－strain containing a PaphB-luxCDABE and PaphB-lacZ transcriptional reporter plasmids． Using a chemiluminescence imager system，we rapidly detected aphB promoter expression level at a large scale． We then sequenced the transposon insertion sites by arbitrary PCR and sequencing analysis． ［Results］We obtained two candidate mutants T1 and T2 which displayed reduced aphB expression from approximately 40，000 transposon insertion mutants． Sequencing analysis shows that Tn inserted in vc1585 reading frame in the T1 mutant and Tn inserted in the end of coding sequence of vc1602 in the T2 mutant． ［Conclusion］By using a genetic screen，we identified two potential genes that may involve in regulation of the expression of the key virulence regulator AphB． This study sheds light on our further investigation to fully understand V．cholerae virulence gene regulatory cascades.

Abstract:Abstract:［Objective］ To develop a PCR method for detecting the newly reported genotype (variant 2c，V2c) of Mycoplasma pneumoniae strains． ［Methods］Specific primer was designed for detecting the V2c type based on the variant region of V2c strain p1 gene． A nested multiple PCR method for V2c strain detection was set up and confirmed by related gene sequencing． In total 214 clinical strains isolated from Beijing between 2008 and 2011 were analyzed by this typing method． ［Results］Nest multiple PCR typing method is effective to detect the V2c strain． Of the 214 M． pneumoniae strains 90. 2% (193/214) were type 1，0.9% (2/214) were variant 2a，and 8. 9%(19/214) were V2c． No type 2 was detected． ［Conclusion］This typing method is effective to distinguish the V2c strains from other variant M． pneumoniae strains，and important for the epidemiological study of Mycoplasma pneumonia infection．