Abstract:Abstract: Polyketides have played an important role in antibiotic drug discovery with most antibacterial drugs being derived from a natural product or natural product lead. Furthermore, the biosynthetic gene clusters for numerous bioactive polyketides have been intensively studied over the past 15 years. This paper focuses on the polyketide drugs approved by US-FDA and takes a general view in the antibiotics produced by polyketide synthase in streptomyces.

Abstract:Abstract: In eukaryotes, Ca2+ is an important second messenger and regulates diverse cellular activities ranging from muscle contraction to fertilization. In bacteria, growing evidence suggests that Ca2+ also plays important regulatory roles in various physiological processes. Here we review current understanding of calcium regulation in bacteria from the following aspects: 1） the concept of bacterial [Ca2+]i and its determination; 2）cellular processes affected by [Ca2+]i changes; 3）transportation of Ca2+ across bacterial membrane; 4）eukaryotic calcium binding proteins in bacteria and their functions.

Abstract:Abstract: [Objective] Puerarin was a Chinese traditional medicine. We isolated a fungal from soil which could convert puerarin to 3′-hydroxypuerarin. [Methods] Strains were cultivated on potato dextrose agar (PDA) medium. The biotransformation product was extracted with an organic solvent and detected on high performance liquid chromatography (HPLC). [Results] A total of 186 fungal strains were isolated from soil, and strain NT-01was determined to transform puerarin into 3′-hydroxypuerarin. The product was identified as 3′-hydroxypuerarin by MS and 1H-NMR and 13C-NMR. The morphology characteristic of strain NT-01 was essentially consistent with Gliocladium deliguescens. Phylogenic analysis on ITS sequences showed that strain NT-01 was clustered together with G.deliguescens and G.viride with high boostrap supporting, so that strain NT-01 was identified as Gliocladium sp. [Conclusion] Gliocladium strain NT-01 can convert puerarin to 3′-hydroxypuerarin.

Abstract:Abstract: [Objective] To analyze the structural characteristics of tdh gene and its adjacent loci of Vibrio parahaemolyticus isolates from seafoods. [Methods] Long distance PCR and genome walking were used to amplify the DNA sequences flanking the tdh gene, and the sequences were analyzed by blastn against the NCBI database. [Results] The genetic structure of tdh-adjacent loci (VPA1312-VPA1327) from isolate ZS34 was similar to that of the reference strain RIMD2210633, with the nucleotide identity of 98.3%. The tdh gene of isolates FJ14 and WZ64 was located in the loci different from the reference strain and ZS34, and showed high nucleotide similarity to tdh3 gene. In the genome of isolate FJ14, tdh was 15kb away from the trh-ure cluster, with IS-like elements and transposase genes inserted therein. Isolate WZ64 lacked trh gene, but also harbored IS-like elements upstream of tdh. [Conclusion] The tdh-adjacent loci of V. parahaemolyticus seafood isolates exhibit high diversity, an additional evidence of lateral gene transfer in this particular species.

Abstract:Abstract: [Objective] We studied the physiological function of α-ketoglutarate dehydrogenase complex (KGDH) on the metabolism of Torulopsis glabrata. [Methods] With manipulation of KGDH in Torulopsis glabrata, we screened a mutant strain T. glabrata kgd1::kan, in which the kgd1 gene encoding the E1 subunit of KGDH was deleted. [Results] Disruption of KGDH resulted in: (a) the enhancement of glyoxalate pathway as a complementarity for carbon metabolism in TCA cycle; (b) compared with that of the control, the ratio of NADH/NAD+ and ATP/ADP decreased by 33.7% and 31.8%, respectively. But the specific activities of pyruvate dehydrogenase, isocitrate dehydrogenase and malate dehydrogenase increased by 58.1%, 33.3% and 32.5%, respectively; (c) the intracellular concentration of pyruvate was reduced by 49.9%, while the intracellular concentration of succinate, malate and α-ketoglutarate was higher 172.7%, 66.1% and 41.1% than the corresponding values of the control; (d) The content of pyruvate-family amino acid was 29.3% lower while the level of glutamate-family amino acid and aspartate-family amino acid were 34.7% and 26.8% higher than that of control. [Conclusions] Those results present here demonstrated that α-ketoglutarate dehydrogenase complex plays essential role on the metabolism of yeast.

Abstract:Abstract: [Objective] To obtain hydroxyectoine-producing strain with tolerance to osmotic shock and improve hydroxyectoine productivity by adopting “bacteria milking” process. [Methods] We isolated a strain from salt lake, and then carried out the identifications of morphology, physiological and biochemical characteristics of the strain. We also investigated the effects of the medium and its NaCl concentration on the hydroxyectoine synthesis of this strain. Under optimal condition, hydroxyectoine was produced by adopting“bacteria milking”process. [Results] A hydroxyectoine-producing strain was obtained and identified as Cobetia marina CICC10367 (C. marina CICC10367). It could enhance hydroxyeceoine synthesis when the medium adopting monosodium glutamate as the sole source of carbon and nitrogen at 90 g/L NaCl. The highest synthesized hydroxyectoine concentration was 694.5 mg/L in the above described medium. After the “bacteria milking” process of six osmotic shock, the total concentration of hydroxyectoine was 4179.0 mg/L, the productivity was 597.0 mg/L/d, and the conversion rate of hydroxyectoine on substrate was 80.2 mg/g. [Conclusion] C. marina CICC10367 was able to synthesize hydroxyectoine under NaCl induction and tolerant to osmotic stress. The hydroxyectoine productivity and conversion rate of hydroxyectoine on substrate were both significantly improved by adopting “bacteria milking” process.

Abstract:Abstract: [Objective] In order to study the structure and function of β2 microglobulin (β2m). [Methods] We sub-cloned the mature peptide of β2m into the p2X plasmid and transformed them to the the Esherichia coli (E. coli) TB1. The recombinant bacteria was induced to be expressed and the expressed fusion protein was detected by SDS-PAGE and western blot. After purifying and cleaving with Factor Xa, we separated the monomer protein β2m from MBP. Finally, we determined the secondary structure of the β2m protein by circular dichroism (CD) spectrum. [Results] The results indicated that MBP-β2m was 52.1 kDa, and the monomer protein β2m was 10.6 kDa. The a-helix, b-sheet, turn, and random coil of the fusion protein showed 0, 45, 8 and 45 amino acids, respectively, detected by CD estimation. [Conclusion] The results concluded that the β2m protein had correct secondary structure and could be used to have further research of peptide binding in vitro.

Abstract:Abstract: [Objective] By exploring Ssp DnaE intein-catalyzed protein trans-splicing we aimed to investigate the ligation of expression product of ATP-binding cassette transporter A1(ABCA1) gene in E. coli. [Methods] The ABCA1 cDNA was broken into two halves of N-part and C-part before Cys978 codon which meets the splicing required conserved residue, and then fused to 123 and 36 amino acid-containing N terminal and C terminal coding sequences of Ssp DnaE intein naturally occurring trans-splicing intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pET-28a(+). After transformation into E coli BL21(DE3) cells followed by induction the expression of recombinant proteins and the ligation of ABCA1 were observed. [Results] Through IPTG induction for expression of recombinant protein it displayed an obvious protein band as predicted size of ABCA1 on SDS-PAGE gel. Western blotting using His-Tag specific antibody confirmed that this protein band is trans-spliced ABCA1. [Conclusion] The data demonstrated that Ssp DnaE intein can efficiently catalyze the ligation of ABCA1 providing an evidence for our ongoing study on ABCA1 gene transfer by a dual AAV vector system to circumvent AAV volume limitation in gene therapy of Tangier disease which resulted from ABCA1 gene mutations.

Abstract:Abstract: [Objective] To investigate the biochemical characteristics of the chitosanase from Fusarium solani, and its application in chitooligosaccharides production, and express the chitosanase gene (csn) in an Saccharomyces cerevisiae industrial strain. [Methods] The chitosanase cDNA (EU263917) was amplified by reverse transcription-mediated PCR (RT-PCR). A His-chitosanase fusion protein was expressed in E. coli DE3. This protein was purified and characterized. Thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) were used to analyze the hydrolysates of this enzyme when it acted on 85% deacetylated chitosan. In addition, the csn cDNA was also fused to the inulinase (INU1A) signal sequence from Kluyveromyces marxianus. The fusion sequence was transferred into S. cerevisiae industrial strain N-27. [Results] The purified chitosanase had a specific activity of 2.5 U/mg. The enzyme showed the maximum activity at 50oC, which was different from the first reported chitosanase that from F. solani f. sp. Phaseoli. TLC and HPLC results indicated that most of the hydrolysis products were chitooligosaccharides with a polymerization below 10, and no monomers were detected. Furthermore, chitosanase activity was detected in the culture medium of the recombinant S. cerevisiae cultures, and the volume activity reached 50.2 mU/mL. This result indicated that the recombinant protein was secretively expressed in S. cerevisiae. [Conclusion] The chitosanase from F. solani 0114 is an endochitosanase. Because of its special characteristics, this enzyme is very useful in chitooligosaccharides production, and the construction of recombinant S. cerevisiae made a step towards the large-scale production of this enzyme.

Abstract:Abstract: [Objective]The enzyme elastase is an important virulent factor of the opportunistic pathogen Aeromonas hydrophila. To know better about the molecular characterization of this enzyme, we purified the elatase and investigated its property and activity. [Methods] We cloned a structural gene ahyB encoding for the extracellular elastase of A. hydrophila J-1 and expressed it in E. coli BL21 by using pET-32a as vector. We purified the recombinant enzyme using His Bind Resin Purification Kit and recovered the elastolytic activity of the purified protein by incubation in a buffer containing 6 mol/L guanidine HCl and subsequent removal of denaturant by dilution. In addition, we also purified the native enzyme from the culture supernatant of A. hydrophila J-1 by 30 to 60% ammonium sulfate fractionation, anion exchange chromatography and sephaceryl chromatography. We compared the properties of the two elastase preparations. [Results] Native enzyme showed a pH optimum at 8.5, but recombinant enzyme at 10.0. Compared with the native enzyme, the recombinant enzyme was more stable for heat. Both the elastase preparations showed some identical properties concerning inhibitors, which were both mainly inhibited by EDTA, the cation chelator, and OPA, a Zn2+/Fe2+protease inhibitor. However, the recombinant elastase had higher tolerance than native enzyme against the inhibitors. And the metal cation Zn2+ and Fe2+ strongly inhibited the enzyme activity. [Conclusion] The recombinant product showed the similar enzymatic characteristics as the native enzyme from A. hydrophila J-1 and the two elastases belonged to the zinc-and-iron-dependant metalloendopeptidase. This is the first report on the recombinant expression and subsequent molecular chracterization analysis of A. hydrophila elastase.

Abstract:Abstract: [Objective] To clone the chitin synthase gene PstChsII from Puccinia striiformis and to analyze its expression pattern. [Methods] We isolated the cDNA and genomic DNA of PstChsII via RT-PCR and PCR, analyzed the sequences with different bioinformatic tools, characterized the gene expression pattern via real-time PCR. [Results] The coding region of PstChsII (Genbank accession no: GQ329851), interrupted by 15 introns, corresponded to a 2727 bp open reading frame encoding 908 amino acids. PstChsII showed a highest 94% similarity to PgtChsII from Puccinia graminis. PstChsII had 7 transmembrance regions and several conserved chitin synthase domains and motifs such as “QXRRW”,“GXGPL”and “DXD”. PstChsII belonged to the class II sub-family and had a closer phylogenetic relationship to its homologs from basidiomycetes than those from ascomycetes. PstChsII expression level increased by 10-fold at the urediospore germination stage. [Conclusion] PstChsII might be involved in the cell wall synthesis during the germ tube elongation process. The cloning and expression analysis of PstChsII served as a good foundation for further analyzing the role of this gene in the pathogenesis process.

Abstract:Abstract: [Objective] The aim of our study was to isolate and identify merA gene from a bacterial strain with high resistance to mercury. [Methods] A bacterial strain with resistance to mercury was isolated from the river sediment of Liangshui river in Beijing, The strain was identified according to the sequence analysis of 16S rRNA gene and its physiological and biochemical properties. A pair of PCR primers was designed according to the merA gene sequences of some bacteria published in the GeneBank to amplify the complete merA gene using the genomic DNA of KHg2 as a template. The PCR-amplified DNA fragment was expressed in Escherichia coli BL21(DE3). Mercury resistance of the expression strain was tested. [Results] A bacterial strain which could grew on LB medium plate containing 70 mg/L HgCl2 was isolated , it was named as KHg2. The strain KHg2 shares 96% sequence identity with the type strain DSM12223T of Bacillus silvestris. The morphological characteristics and the results of physiological and biochemical properties of the strain were agreement with that of Bacillus silvestris. One PCR fragment of 1680bp was obtained from the strain, which shares 99% sequence identity with merA gene of Pseudomonas putida . The PCR-amplified DNA fragment was cloned into a pET-30a (+) vector to obtain a expression plasmid pZY2 . The expression plasmid was transformed into E.coil host strain to obtain a expression strain E.coli BL21(DE3)﹒pZY2, a protein of 33 kDa was expressed by the expression strain after induced with isopropyl-b-D-thiogalactoside (IPTG). The result of heavy metal resistance test showed that the expression strain could grew on LB medium containing 20 mg/L HgCl2 , meanwhile a negative control , E. coli BL21(DE3), harbouring pET-30a (+) , could not grew on LB medium containing 20 mg/L HgCl2 . [Conclusion] The isolated strain KHg2 was closely related to Bacillus silvestris. A merA gene was cloned from the strain KHg2 and was expressed successfully in E. coli. The expression strain E.coli BL21(DE3)﹒pZY2 has resistance to heavy metal mercury.

Abstract:Abstract: [Objective] The aim of this study is to isolate novel and efficient hydrocarbon-degrading bacteria (HDBs) from pelagic ocean. [Methods] The surface water sample from Atlantic Ocean was enriched in NH medium with crude oil: diesel oil (in a ratio of 1:1) as the sole carbon source. HDBs were isolated and their degradation ability was tested in MMC medium, and further subjected to genotypic and phenotypic analysis. Polymerase chain reaction with degenerate primers was performed to detect the genes encoding integral-membrane non-heme iron monooxygenase (AlkB) and cytochrome P450 CYP153 family. Meanwhile, the production of biosurfactant was examined by surface tension measurement, then extracted and purified for component characterization. [Results] A hydrocarbon-degrading bacterium named S14-10 was obtained, which can utilize C10?C36 as the sole carbon source. Analysis of 16S rRNA sequence showed that it belonged to Gordonia genus showing the highest similarity (99.86%) with type strain of Gordonia terraeT, while secA1 sequence showed a low identity only 93.69%. Furthermore, two genes alkB and CYP153 P450 were obtained with PCR, which had highest similarities 88.76％and 99.61％ to that of Gordonia sp. TF6, respectively. In addition, strain S14-10 was found to produce glycolipid biosurfactant, which lowered the surface tension of water to 31.6 mN/m. [Conclusion] Strain S14-10 is possibly a novel species of Gordonia, and the first hydrocarbon-degrading bacterium isolated from open sea. It has potential in bioremediation of oil contaminated environment.

Abstract:Abstract: [Objective] To investigate the accumulation mechanism of volatile fatty acids (VFAs) the shift of bacterial community and the contribution of syntroph acetogenic bacteria during the anaerobic fermentation of sewage sludge under different pH values. [Methods] The VFAs were determined at different pH conditions during the sludge anaerobic fermentation process. The Terminal Restriction Fragment Length Polymorph (T-RFLP) and Fluorescence In Situ Hybridization (FISH) were used to explore the variation of bacterial community and the quantity of syntroph acetogenic bacteria, respectively. [Results] When the pH was controlled at 10.0, yields of VFAs and acetate were 652.6 mg COD/g-VS and 322 mg COD/g-VS,respectively (COD, Chemical Oxygen Demand; Volatile Solid, VS). They are much higher than that under neutral and acid pH conditions. The results by T-RFLP showed that Granulicatella was the dominant bacterial population at pH 12.0, while Peptostreptococcus dominated at pH 10.0. When the pH was adjusted from 7.0 to 3.0, the dominant bacterial populations were Clostridium and Bacillus, respectively. Based on the FISH result, the amount of syntrophic acetogenic bacteria at neutral condition was higher than that under the acid and alkali conditions. When the pH value was 10.0, the relative abundance was below 0.01% in total microorganism. [Conclusions] pH not only altered the yields of VFAs and acetate, but also changed the bacterial community structure. Under the alkali condition, the VFAs accumulation was mainly from the hydrolysis fermenting bacteria. At the neutral and acid conditions, the syntroph acetogenic bacteria made more contribution for the acetate accumulation.

Abstract:Abstract: [Objective] To construct an infectious clone for studying functions of duck hepatitis virus(DHV) type1 genome by reverse genetic technique. [Methods] Three fidelity DNA fragments covering the full genome of DHV type1 CL strain were amplified by RT-PCR, and inserted into pBR322 vector, resulting in the full-length cDNA clone BR-CL. The in vitro-transcribed RNA from BR-CL was transfected into duck embryo renal cells and the rescued virus was identified using RT-PCR, indirect immunofluorescence assay and colloidal gold immunoelectron microscopy after six generations. After inoculating the rescued virus into SPF chick embryos, embryo death and pathological changes were observed. [Results] The results of RT-PCR, indirect immunofluorescence assay and immunoelectron microscope showed that infectious virus was rescued. After inoculating into SPF chick embryos, the rescued virus was able to kill embryos with pathogenic changes. [Conclusion] This is the first report on generation of infectious cDNA clone of DHV, which provides a valuable platform for further research on functions of DHV genome.

Abstract:Abstract: [Objective] To analyze the phylotypes of symbiotic archaea in the gut of Reticulitermes chinensis Snyder using non-cultivating method. [Methods] We amplified 16S rRNA genes of the symbiotic archaea from the DNA extract of the whole gut with the universal archaeal 16S rDNA primer. A clone library of archaeal 16S rDNA was established and selected clones were sequenced and phylogeneticly analyzed. [Results] Five 16S rDNA sequences with the similarity of 93.2%～99.2% to each other were obtained from Reticulitermes chinensis Snyder. Phylogenetic analysis showed that the five clones were clustered with Methanobrevibacter clones or isolates obtained from Reticulitermes speratus and Reticulitermes flavipes. [Conclusion] The results showed that symbiotic archaea in the gut of Reticulitermes chinensis were affiliated with Methanobrevibacter.

Abstract:Abstract:[Objective] To investigate the effects and mechanism on immune regulation activity in mice of two Tibetan Kefir exoploysaccharides(EPS) with different molecular weight of 0.1×105~3×105(fraction 1) and 1.8×103(fraction 2). [Method] The immune regulation activity experiment was carried out in vitro based on the Functional Assessment Procedure and Test Methods of Health Food, which was issued by Ministry of Health of China. First, we treated mice subjects with EPS at doses of 40mg/kg, 80mg/kg, 120mg/kg through ig. Then we detected the index of immune organs, the ability of antibody production (tested by HC50), activity of NK cell, delayed type hypersensitivity (DTH) and phagocytosis of macrophage in mice. Finally, we examined the expression of Erk protein in Macrophages by Western Blot assay. [Results] Fraction 1 could promote HC50, activity of NK cell and DTH in mice which low dose showed better. Fraction 2 could promote DTH, phagocytosis of macrophage which high dose showed better. The expression of Erk and COX-2 had the same trend with Phagocytic index. [Conclusion] We verified the two fractions of Tibetan Kefir EPS could enhance immune functions in mice. Fraction 1 regulated immune function through NK cell and B cell while fraction 2 through macrophage cell and T cell. The effects to macrophage of Tibetan Kefir EPS in mice may realize through extra cellular signal-regulated kinase Erk pathway.

Abstract:摘要: 本文将介绍2009年度国家自然科学基金委员会微生物学学科各类项目受理与资助概况，并对面上项目的受理与资助情况按照依托单位和研究领域进行分析。