Abstract:Abstract: 2-keto-L-gulonate (2-KGA) is the key intermediate of vitamin C，which can be biosynthesized by Ketogulonigenium vulgare．There are five reactions related to 2-KGA metabolism，including: (1) Oxidation of D-sorbitol to L-sorbose; (2) Oxidation of L-sorbose to L-sorbosone;(3) Oxidation of L-sorbosone (Pyranose form) to 2-KGA; (4) Oxidation of L-sorbosone (Furanose form) to vitamin C，and (5) Reduction of 2-KGA to L-idonate．L-sorbose/Lsorbosone dehydrogenase (SSDH) is responsible for the reaction of 1 through 3，L-sorbose dehydrogenase (SDH) is responsible for the reaction of 2 and 3，L-sorbosone dehydrogenase (SNDH) is responsible for the reaction of 3 and 4，aldehyde dehydrogenase (ALDH) is responsible for the reaction of 3，2-KGA reductase (2-KGR) is responsible for the reaction of 5．Enzymes of SDH，SSDH and ALDH belong to Quinoprotein Type Ⅰ that uses PQQ as the only prosthetic group．SNDH belongs to Quinoprotein Type II that is quinohemoprotein assembling heme c and PQQ．They are all soluble in the periplasm and coupled with the respiratory chain．The substrate respiration to generate ATP directly on the outside cellular membrane means this strain can use the substrate quickly in the natural environment for the necessary bioenergy required．

Abstract:Abstract:Methylmercury (CH3Hg+，or MeHg) is the most poisonous form of mercury (Hg) because it can enter into human bodies through the consumption of Hg-contaminated fish and shellfish．A first step toward bioaccumulation of MeHg in aquatic foods is the methylation of inorganic mercury，a process that is predominantly mediated by anaerobic bacteria，such as sulfate reducing bacteria and iron reducing bacteria． Many researches have confirmed that microbial methylation of mercury is an intracellular reaction．Therefore，MeHg production rates are not only related to the presence and productivity of methylating bacteria and also the biouptake of Hg to these anaerobic bacteria．To understand the pathways of Hg biouptake is indispensable to elucidate the mechanisms of microbial methylation．In this review，we systematically evaluated the current state of knowledge regarding the four pathways of mercury biouptake，Mer-based transport system，passive diffusion，facilitated diffusion and active transport．In the future，facilitated diffusion and active transport of inorganic mercury to the cytoplasm of microbial cells should be emphasized．

Abstract:Abstract:［Objective］The aim of this study was to analyze the bacterial diversity of land-water transition zone in littoral wetland of Wuliangsuhai Lake，and to study the effect of eutrophic substrate on the bacterial composition．［Methods］The denaturing gradient gel electrophoresis (DGGE) with PCＲ-amplified 16S rDNA fragments was used to determine bacterial diversity，and the ordination technique of canonical correspondence analysis (CCA) was used to evaluate its effects on bacterial community composition．［Results］DGGE result shows that the microbial quantity decreased gradually from eutrophic lake sediment to desert soil．Diversity index analysis shows that the Shannon-Wiener index (H)，Evenness index (E)，richness index (S) and Simpson index (DS) decreased gradually from water to land (eutrophic lake sediment＞swamp sediment ＞ saline soil ＞ desert soil)．Based on the sequencing results，there are 3 phyla in 4 samples，including Proteobacteria (78.6%)， Acidobacteria (7.1%)，Bacteroidetes (14.3%)， Proteobacteria (52.6%) were dominant species，in which the Epsilon proteobacteria were predominant subgroup．The results of CCA suggest that that NH4 + -N，total nitrogen，organic carbon，total soluble salt，Cl－and K + have most significant influenced the distribution of bands related species．［Conclusion］The bacterial community structure of transition zone in Wuliangsuhai wetland differed significantly，and eutrophic relative factors had a strong impact on the bacterial community structure．This work presented a certain reference for the preliminary understanding of the bacterial composition and diversity and provided a scientific foundation for the research of spatial heterogeneity in this transition zone．

Abstract:Abstract:［Objective］In order to determine the effect of bkdR deletion on Cry protein production．We analyzed the transcriptional regulation of bkd gene cluster and the phenotype of bkdR mutant．［Methods］Sequence of bkd gene cluster in Bacillus thuringiensis was analyzed by sequence alignment．RT-PCRwas used to reveal the transcriptional units of the bkd gene cluster．bkdR insertion mutant was constructed by homologous recombination．Transcriptional activity was analyzed by promoter fusions with lacZ gene．Comparison of the Cry1Ac protein production was determined by protein quantitation．［Results］The bkd gene cluster was composed of eight genes．The ptb-bkdB formed one transcriptional unit．The transcriptional activity of ptb sharply decreased in sigL and bkdR mutants．Deletion of bkdR decreased the motility of cells，but no effect on growth，sporulation efficiency and Cry protein production．The bkd gene cluster is controlled by Sigma 54 and activated by BkdR． Deletion of bkdR has no effect on Cry protein production，but decreased the motility of the cells．［Conclusion］The bkd gene cluster is controlled by Sigma 54 and activated by BkdR． Deletion of bkdＲ has no effect on Cry protein production，but decreased the motility of the cells．It suggested that deletion of bkdR do not affect the Cry protein production the same as sigL mutant．It means decreasing of Cry protein productioninsigL mutant was not caused by only one EBP mutation，but might be multiple roles．

Abstract:Abstract:［Objective］To discover new elements for cry gene expression，PexsY，which is the promoter of the exosporium basal layer structural gene exsY，was used to express cry1Ac gene in Bacillus thuringiensis．［Methods］We used be tagalactosidase assays by promoter-lacZ fusion to analyze the transcriptional activity of exsY promoter and truncated exsY promoter．The cry1Ac gene was directed by the non-cry gene promoter PexsY and was then expressed in Bacillus thuringiensis HD73．Transmission electron microscope (TEM) was used to observe the formation of crystal inclusion．The Cry1Ac yieldswere evaluated by protein quantification and SDS-PAGE analysis．Bioassays against Ostrinia furnacalis were used for the functional verification．［Results］ Beta-galactosidase assays showed that the exsY promoter had a strong transcriptional activity in the acrystalliferous mutant strain HD73－on the late sporulation phase．Cry1Ac expression products directed by the PexsY could form diamond crystals．SDS-PAGE analysis showed that the cry1Ac gene directed by the cry8E promoter has the highest protein yield among the four promoters while the cry1Ac gene under the direction of PexsYorcry3A promoters showed similar protein yields．The bioassay results showed that the Cry1Ac protein directed by the PexsY promoter was toxic against Ostrinia furnacalis．［Conclusion］The cry1Ac gene under the direction ofthe non-cry gene promoter PexsY was able to express the Cry proteins at the late sporulation phase and could form crystal inclusion in a B．thuringiensis strain．Our finding provides applicationpotential for the genetically modification of engineered Bt strains．

Abstract:Abstract:［Objective］We aimed to study the carbohydrate metabolism and lactic acid biosynthesis of Lactococcus lactis KLDS4.0325．［Methods］Whole genome shot gun strategy was used for genome sequencing of strain L．lactis KLDS 4.0325．Then，using bioinformatics method，we compared a series of protein－coding genes involved in transporting extracellular carbohydrate，sugar metabolism and lactic acid biosynthesis of strain L． lactis KLDS4．0325 with other 9 reference strains．［Results］In L．lactis KLDS4．0325 genome，where possesses more key enzyme coding genes related to the whole pathway of sugar metabolism than reference strains．［Conclusion〗In gene level，therefore，strain L．lactis KLDS4．0325 shows a remarkable characteristic by utilizing various sugar to produce lactic acid，is a lactic acid bacteria with industrial potential of high yield L－lactic acid．

Abstract:Abstract:［Objective］ We identified an endophytic fungus of Vigna unguiculata，as well as the influence of carbon sources on the production of kojic acid by the isolated fungus．［Methods］ This kojic acid producer was identified as Aspergillus flavus F52 according to morphological characteristics and ITS region of rDNA．The metabolite of strain F52 was obtained by recrystallization，and identified as kojic acid based on the spectral data of NMＲ，HＲ-ESI/MS and IR．The fungus was cultivated in medium containing various carbon sources，and the production of kojic acid in the fermentation broth was quantified by high performance liquid chromatography．［Results］ The complex carbon source which was composed of glucose and sucrose was preferential，whereas the presence of lactose was not beneficial to the production of kojic acid．The content of kojic acid in the fermentation broth reached 24. 44 g/L．［Conclusion］Aspergillus flavus F52 might be a potent producer of kojic acid for commercial use．

Abstract:Abstract:［Objective］Bacterial strain SE-1 capable of transforming cholesterol was isolated from soil and characterized．The transformation products were identified．Fermentation conditions were optimized for conversion.［Methods］Cholesterol was used as sole carbon source to isolate strain SE-1．Morphology， physiological and biochemical characteristics of strain SE-1 were studied．16S rRNA gene was sequenced and subjected to phylogenetic analysis．Fermentation supernatants were extracted with chloroform，the transformation products were analyzed by silica gel thin layer chromatography and Sephadex LH20．Their structures were identified by 1H-NMR and 13C-NMR．Fermentation medium including carbon and nitrogen，methods of adding substrates and fermentation conditions for Strain SE-1 were optimized．［Results］Strain SE-1 was a Gram-negative bacterium，exhibiting the highest homologs to Burkholderia cepacia based on the physiological analysis．The sequence analysis of 16S rＲNA gene of SE-1 strain and comparison with related Burkholderia show that SE-1 strain was very close to B．cepacia (Genbank No．U96927)．The similarity was 99%．The result of silica gel thin layer chromatography shows that strain SE-1 transformed cholesterol to two products，7β-hydroxycholesterol and the minor product was 7-oxocholesterol．The optimum culture conditions were: molasses 5%，(NH4) 2 SO4 0.3%，4% of inoculation，pH 7.5 and 36℃．Under the optimum culture condition，the conversion rate reached 34. 4% when concentration of cholesterol-Tween 80 was 1 g/L．Cholesterol 7β-hydroxylation conversion rate under optimal conditions was improved by 20.8%．［Conclusion］Strain SE-1 isolated from soil is capable of converting cholesterol at lab-scale．

Abstract:Abstract:［Objective］ Based on the analysis of omics data of Volvariella volvacea，a gene encoding glutathione Stransferase (GSTs) named vv-gto1 was obtained．To reveal the role of GSTs in the growth and development in edible fungi，the structure，the sequence characters and the expression profile of a GST gene vv-gto1 of Volvariella volvacea were analyzed．［Methods］ZOOM software was used to map sequencing read ( reads) from genome and transcriptome against the splicing sequence of genome，to confirm the complete length and the accuracy of the gene sequence，and to visualize gene structure．The MEGA 5.1 was used to do the multiple sequence alignment and phylogenetic tree analysis． Ｒeal time fluorescent quantitative PCR was used to determine the expression levels of vv-gto1 at different growth periods of Volvariella volvacea． ［Ｒesults］The full sequence of vv-gto1 covered 2083bp，containing 11 exons and 10 introns，and encoded a protein with 356 amino acids．5'UTＲ was 305bp which contains one intron region，and 3'UTＲ was 86bp． Two intron retentions could be recognized during RNA processing，and the transcripts formed by the intron retention could not translate the correct conservative functional domains．The full-length of vv-gto1 had more than 50 accurate positioning genome sequencing reads，suggesting that genome sequencing and assembly results are accurate and reliable． The phylogenetic tree showed that GTO1 of Volvariella volvacea belonged to the subclass I of the Omega class of glutathione S-transferase superfamily，and had the closest relationship with GTO1 and GTO2 in Phanerochaete chrysosporium． The analysis of digital gene expression profiling，fluorescence quantitative PCＲ and proteomics showed that vv-gto1 had the highest expression level in the heterokaryotic hyphae．［Conclusions］This is the first time to obtain a gene encoding glutathione S-transferase from Volvariella volvacea which belongs to Omega class．Our study showed that the gene may play an important role during the special biological functions of heterokaryotic hyphae． This study also suggested that Volvariella volvacea heterokaryotic hyphae in H1521 had stronger resistance ability than other samples． In addition，vv-gto1 could form different alternative splicesome to regulate gene transcription and translation，and ultimately affect the function of the protein．

Abstract:Abstract:［Objective］To obtain a new homoserine dehydrogenase with better properties from Corynebacterium pekinense by the spatial structure transfromation．［Methods］Double mutants L200F/D215A，L200F/D215E，L200F/D215G and L200F/D215K were constructed by site-directed mutagenesis and expressed in E．coli BL21．L200F /D215K was characterized for its highest catalytic efficiency and compared with that of L200F．［Results］The Vmax of L200F/D215K was 36.92 U/mg，1.24 times as that of L200F．The optimum reaction temperature of L200F/D215K was 37℃，2℃ higher than that of L200F．The optimum pH of L200F/D215K was 7.5，the same as that of L200F．The half-life time of L200F/D215K under optimum temperature was 4.16 h and was 1.12 times as that of L200F．Both L200F/D215K and L200F had good resistance to organic solvents and metal ions．［Conclusion］Through the spatial structure transformation，the enzymatic activity was increased，and the enzymology properties was optimized．

Abstract:Abstract:［Objective］Ferric reductases play a central role in iron acquisition and mobilization in C．albicans．This study focuses on stress response strategies exhibited by several ferric reductase genes through function and expression analyses．［Methods］ Northern blot analysis was used to examine ferric reductase genes expression levels in different iron deficiency．We constructed ferric reductase-null mutants by a PCR-based homologous recombination，and examined the effects of gene deletion on cell-surface ferric reductase activity and growth ability under different conditions． Sub-cellular localization of Frp1-GFP fusion was imaged and analyzed by confocal laser scanning fluorescence microscopy． ［Ｒesults］FRE10 was highly expressed at acidic pH，compared to that at alkaline pH，whereas the expression of FＲE2 was just the opposite． Deletion of FＲE10 resulted in a significant decreased surface reductase activity at acidic pH，with 75. 5% downregulation compared to wild-type levels．The fre2Δ/Δ mutant showed significantly attenuated growth ability and cellsurface ferric reductase activity at alkaline pH．Sub-cellular localization revealed that the green fluorescence was accumulated in the vacuoles．［Conclusion］The expression of both FRE10 and FRE2 is induced in a pH-dependent manner．FRE2 encodes a major cell surface ferric reductase under alkaline pH condition．Frp1 localizes to the vacuole，and might support mobilization and transport of vacuolar ferric iron stores．

Abstract:Abstract:［Objective］To study the adaptation of A．hospitalis W1 to oligotrophic and acidic hot spring environments at the whole genome level．［Methods］We annotated the gene functions and constructed metabolic pathways of strain W1 by using different databases，such as NCBI non-redundant database (NRDB)，UniProt，Sulfolobus protein database and Kyoto Encyclopedia of Genes and Genomes (KEGG)．The metabolic pathways were polished according to the results of comparative genomics．［Results］ Strain W1 grew autotrophically by fixing CO2 as carbon source through 3-hydroxypropionate/4-hydroxybutyrate or dicarboxylate-4-hydroxybutyrate cycle，and gained energy for growth by oxidation of reduced inorganic sulfur compounds (RISCs)．Strain W1 differenced from A．ambivalens because its genome did not possess sulfur-metabolizing genes encoding sulfite: acceptor oxidoreductase，adenosine phosphosulfate reductase，sulfate adenylyl transferase and phosphoadenosine phosphosulfate reductase．Glucose was metabolized by strain W1 through nonphosphorylated Entner-Doudoroff pathway and tricarboxylic acid cycle．In addition，the sugar and amino acids transporters，as well as related hydrolysis enzymes were identified in the genome．These results suggest that strain W1 could also grow facultative autotrophically．Strain W1 cannot use H2 as electron donor due to lack of hydrogenase encoding genes．［Conclusion］The versatile metabolic patterns afforded A．hospitalis W1 the ability to adapt to oligotrophic and acidic hot spring environments．Furthermore，the unique metabolic features of strain W1 will help to better understand the metabolic diversities of Acidianus.

Abstract:Abstract: ［Objective］ To study the inhibitory effect of biochanin A on efflux system of Methicillin-resistant Staphylococcus aureus (MRSA)． ［Methods］ Inhibitory effects of biochanin A on efflux system of Strain MＲSA41577 were evaluated using double dilution method，two plate method and fluorescence spectrophotometry．Real time PCＲ and SDS-PAGE were applied to detect the expression of MRSA41577 norA and to analyze the changes of MRSA41577 efflux protein before and after dosing biochanin A in association with liquid chromatography mass spectrometry to determinate protein variation．［Results］Biochanin A alone had no inhibitory effect on MRSA41577，but it showed synergy effect with ciprofloxacin in inhibition MRSA41577 in which 40μg/mL biochanin A decreased the minimum inhibitory concentration (MIC) value of ciprofloxacin from 64 μg/mL to 8 μg/mL．Biochanin A significantly increased the accumulation of ciprofloxacin in MRSA41577 in a time-dependent manner．At 15 min，biochanin A increased ciprofloxacin in MRSA41577 by 83%，which is similar to that of reserpine (positive control)．Further mechanism studies indicated that biochanin A could reduce the expression of norA in ciprofloxacin-treated MＲSA41577．After incubated with biochanin A and ciprofloxacin for 16 h，the relative expression of norA of MＲSA41577 was reduced by 65%．SDS-PAGE analysis showed that the total protein profiles of MRSA41577 were significantly changed after treatment with biochanin A for 16h，in which both norA protein and efflux system ABC transporter ATP-binding protein were significantly decreased．［Conclusion］Biochanin A could inhibit Methicillin- resistant Staphylococcus aureus efflux system through reducing pathogen’s expression of norA and norA protein.

Abstract:Abstract:［Objective］To investigate the mechanism of gp96 raised during hepatitis B virus (HBV) infection and the pathological mechanism． ［Methods］ The mechanism of NF-κB activating gp96 expression was determined by bioinformatics analysis，luciferase reporter assay，real-time PCＲ and Western blot．The effect of over-expression and knockdown gp96 expression by transfection or ＲNA interference on hepatocyte proliferation，apoptosis and cell cycle was examined by CCK-8 and flow cytometry．The role of gp96 for HCC development was determined by epithelial-mesenchymal transition (EMT) and colony formation assay．［Results］NF-kB significantly increased the gp96 expression by binding to the NF-kB binding site．Over-expression and knockdown studies both show that gp96 promoted hepatocyte proliferation，inhibited apoptosis，and induced G0/G1 to S phase cell cycle progression．Moreover，gp96 induced epithelialmesenchymal transition and increased colony formation ability of hepatocytes．［Conclusion］Our results therefore provide insights in chronic HBV infection-induced gp96 expression，and indicate that elevated gp96 may contribute to HCC development during chronic inflammation．

Abstract:Abstract:［Objective］In this study，we constructed two recombinant Escherichia coli strains to produce phospholipase C (PLC) from Acinetobacter calcoaceticus．The recombinant enzymes were purified to homogeneity and characterized．［Methods］ We cloned the PLC encoding gene plc1，plc2 from genome DNA of A. calcoaceticus ATCC17902．The amplified fragments were inserted into pET28a (+) to obtain expression plasmids．E．coli BL21 (DE3) harboring the above plasmids were cultivated and induced with isopropyl-β-D-thiogalactopyranoside to express PLCs．The recombinant PLCs were purified by affinity chromatography and their catalytic properties were characterized．［Results］Two PLCs from A．calcoaceticus were cloned and functional expressed in E．coli．The recombinant enzymes have activities of 31160 ± 418 U/mg for PLC1 and 13640 ± 354 U/mg for PLC2，when using p-nitrophenyl phosphorycholine as substrate．The purified PLC1 and PLC2 exhibited optimum temperature at 65o C and 50o C，respectively．Their optimal pH were 8 and 7. 5，respectively．PLC2 was stable under 40oC and pH at 8，whereas the residual activity of PLC1 was less than 25% in the same condition．Mg2 + and Ca2 + stimulated two enzymes activity，whereas Zn2 + stimulated PLC1 and inhibited PLC2． PLC1 and PLC2 hydrolyzed phosphatidylinositol．［Conclusion］It is the first time to express and characterize the PLC gene from A． calcoaceticus ATCC17902．These research results provide reference for the study of food-safety microbiological PLC．

Abstract:Abstract:［Objective］To compare the abundance of 16S rRNA gene of intestinal Fusobacterium and butyrate-producing bacteria in patients with colorectal adenomas patients and colorectal cancer and to reveal the correlation between the target bacteria and the development of colorectal cancer．［Methods］Feces were collected from colorectal cancer patients (n=19)，colorectal adenomas patients (n= 12) and healthy subjects (n=19) ．Bacteria genome DNA from the fecal samples was used to quantitate the Fusobacterium，two butyrate-producing bacteria Eubacterium rectal，Faecalibacterium prausnitzii and total bacteria by real-time polymerase chain reaction． Then the variation of the target bacteria among different groups were assayed using Mann-Whitney U test．［Results］ The abundance of Fusobacterium was significantly higher in colorectal cancer patients than that in healthy subjects (P=0.000) and colorectal adenomas patients (P=0.013)，and it was significantly higher in colorectal cancer patients than that in colorectal adenomas patients (P=0.002)．F．prausnitzii was significantly lower in colorectal adenomas patients compared to healthy subjects (P=0.033)．The total bacteria count was significantly lower in the colorectal adenomas samples than that in the healthy samples (P=0.002)．There was no significantly difference of E．rectal between the three groups．［Conclusions］ The shifts in the colonic bacterial population may potentially contribute to the development of colorectal cancer．