Abstract:Abstract:Due to the potential for producing soluble，correctly folded protein with high yield，Pichia pastoris is currently one of the most effective hosts for the expression of heterologous proteins． However，limitations of expression efficiency are often reported for many different heterologous proteins． Accumulating evidences suggest that protein folding and processing of heterologous protein is a major bottleneck during secretion process in yeast． Apart from optimization of the fermentation process，the current strategies for strain engineering for improving protein secretion are focused mainly on folding and processing，and systematic engineering by high-throughput screening for potential secretion enhancers． This article reviews the genetic engineering progresses of these aspects．

Abstract:Abstract:The viable but non-culturable ( VBNC) is a microbial state，in which microbial cells are metabolically active but cannot be cultivated by routine methods． In this article，we address the formation mechanism，change and variety，resuscitation，research significance and application prospects of VBNC state in bacteria． Furthermore，we report our research findings on VBNC state of bacteria in the past 10 years，including resuscitation，culturable，phylogenetic relationship and potential functions．

Abstract:Abstract:Bacteroidales has been proposed as a fecal pollution indicator． Microbial Source Tracking ( MST) based on Bacteroidales host-specific gene markers has recently been applied in the fecal pollution identification，which does not require culturing the fecal pollution indicator organisms． This method needs to design specific primers． The primers are designed based on Bacteroidales specific 16S rRNA gene． Once a pair of specific primers was amplified，the fecal pollution can be identified． In this paper，the progress of specific primers of Bacteroidales in human，swine，ruminant feces were reviewed and discussed． The advantages and disadvantages were put forward． Future researchers should be focused on the new biological markers and the combination of different MST methods．

Abstract:Abstract:Canine parvovirus(CPV-2) ，first recognized in 1978 as a new pathogen of dogs，was probably derived from a very closely related virus in cats，feline panleukopaenia virus ( FPLV) or a closely related carnivore parvovirus ( FPLVlike virus) ． CPV-2 is responsible for either myocarditis or fatal gastroenteritis in pups with high morbidity and mortality．Shortly after its emergence，CPV-2 has become endemic in the global dog population． The original CPV-2 continued to evolve，and was subsequently replaced by three different but closely related antigenic variants，designated CPV-2a，CPV- 2b and CPV-2c，which now coexist in dog populations worldwide． The genetic and antigenic variation in CPV-2 also correlated with changes in the host range and tissue tropisms of the virus． Here，we reviewed variation and evolution of CPV-2 in past 30 years and discussed CPV-2 as an important model to study virus evolution．

Abstract:Abstract:［Objective］We studied the microbial diversity in the sediments of different depth in a gravity piston core HSPC500 from Shenhu Area，the northern of South China Sea． ［Methods］ Total DNA was extracted from the sedimental materials; the archaeal and bacterial 16S rRNA gene sequences were amplified． The clone libraries were used to analyze the microbial systematic development． ［Results］Group C3 was the predominant archaeal group in the top layer(0 － 5 cm bsf) sediments，and the Marine Benthic Group(MBG) -B group became predominant with depth，reaching 38. 9% and 62. 5% in the middle ( 350 － 355 cm bsf ) and bottom ( 790 － 795 cm bsf ) sediments． Some belonged to MBG-A，Miscellaneous Crenarchaeotic Group(MCG) ，Thermoprotei，Novel Group Crenarchaeota( NGC) ，Halobacteriales，MBGE，South African Gold Mine Euryarchaeotic Group( SAGMEG) ． Proteobacteria was the dominant bacterial group in the top of the core，but became minor deeper within the sediments． As depth increased，Chloroflexi and candidate division JS1 became the predominant groups and reached up to 28. 1% ，29. 2% and 39% ，24. 7% ． Other sequences respectively belonged to Nitrospirae，Actinobacteria，Acidobacteria，candidate division OP8，Spirochaetes，candidate division TM6，Deferribacteres and Plantomycete． ［Conclusion］ Down-core variation in microbial abundance in sediments of HS-PC500 was consistent with the changes of methane concentration in the same core; the lower microbial abundance might be probably due to lower total organic carbon ( TOC) in sediments; However，microbial diversity was relatively high and community structure varied apparently with depth; the community was dominated by clusters that was dominated in sulfate reduction condition，suggesting that microbial etabolization played very important role in the material cycle of marine sediments．

Abstract:Abstract:［Objective］ To investigate functions of flgDxoc and flgExoc genes regulated by diffusible signal factor (DSF) in Xanthomonas oryzae pv． oryzicola(Xoc)Rs105．Methods］The flgDxoc and flgExoc genes were amplified by PCR．We constructed ΔflgDxoc and ΔflgExoc，the deletion mutants from Rs105 by using double crossover method，and determined cell morphology，motility，pathogenicity in host rice and hypersensitive response (HR) in nonhost tobacco． We tested the differential expression of flgDxoc and flgExoc gene by reverse transcriptional polymerase chain reaction(RT-PCR) between the wide type and ΔrpfFxoc ( the deletion mutant of rpfFxoc gene，which could not produce DSF)．Results］We cloned flgDxoc and flgExoc from genomic DNA of Rs105． PCR and Southern blot analysis demonstrated that the flgDxoc and flgExoc genes were knocked out successfully． Both mutants were non-flagellated and significantly attenuated motility on the 0. 3% semi-solid medium． The pathogenicity on rice were obviously attenuated in ΔflgDxoc and ΔflgExoc compared to the wild type． All the changes in mutant could be restored through the complementation． However，there was no significant difference in bacterial growth in MMX medium and induction of HR between mutant (ΔflgDxoc or ΔflgExoc ) and the wild type． In addition，the results of RT-PCR demonstrated that the transcription level of flgDxoc and flgExoc were downregulated in ΔrpfFxoc．Conclusion］ This study showed that expressions of flgDxoc and flgExoc were positively regulated by DSF，and necessary for flagellar hook assembly and flagellar structure in Xoc． Meanwhile，FlgD and FlgE contributed to pathogen's virulence，motility and chemotaxis，but no differences at growth rate in MMX medium and HR in nonhost．In addition，our results provided molecular evidences that the contribution of DSF-type quorum sensing to pathogen's virulence might be，at least partially，dependent on acterial flagellar in Xoc．

Abstract:Abstract:［Objective］ Nucleotide guanosine-3'，5'-( bis) pyrophosphate ( ppGpp ) synthesized by ( ppGpp ) synthesase RelA or bifunctional ppGpp synthase / degradase RelA / SpoT，mediates bacterial stringent response to various stressful conditions． Here we characterized the slr1325 ( syn-rsh ) gene encoding a RelA / SpoT homolog ( Syn-RSH ) of the cyanobacterium Synechocystis sp． PCC6803． ［Methods］We performed phenotypic complement test using Escherichia coli strain with( p) ppGpp-synthesis defect to determine Syn-RSH function( s) ，and employed chromatographic analysis of32 Plabeled cellular mononucleotides to detect the accumulation of ppGpp in Escherichia coli strains expressing Syn-RSH and in Synechocystis sp． PCC6803． ［Results］ Syn-RSH expression in E． coli relA / spoT double mutant was able to restore the cell growth arrest; Chromatographic analysis of32 P-labeled cellular mononucleotides revealed that Syn-RSH expression resulted in the synthesis of ppGpp in E． coli strain with relA and spoT mutant mutation． Additionally，Synechocystis cells accumulated a low level of ppGpp under laboratory growth conditions． ［Conclusion］Syn-RSH possesses ppGpp synthase /degradase activities，and ppGpp is required for Synechocystis cell viability under normal growth conditions．

Abstract:Abstract:［Objective］We developed an efficient method of gene knockout in Verticillium dahliae，an important soil-borne fungal pathogen that causes cotton vascular wilt diseases． ［Methods］By using fusion PCR，we constructed gene knockout vectors． By using Agrobacterium tumefaciens-mediated transformation and applying a herpes simplex virus thymidine kinase (HSVtk) gene in T-DNA as a conditional lethal gene to counter-select against ectopic transformants，we developed an efficient method to select gene knockout transformants． ［Results］Gene knockout frequency for ADE4 and ChsV was 87% and 44% ，respectively． ［Conclusion］We developed an efficient tool for gene knockout in Verticillium dahliae，which would help clarify the infection mechanism of this fungal pathogen．

Abstract:Abstract: ［Objective］To investigate xylose metabolism in the Saccharomyces cerevisiae stains overexpressing the xylulokinase gene XKS1 at different levels by replacing the promoter in the chromosome.［Methods］ Based on S．cerevisiae CEN．PK 113-5D，we constructed xylose-metabolizing strains where the promoter of xylulokinase gene XKS1 was replaced by TEF1 promoter，PGK1 promoter and HXK2 promoter on the chromosome． We quantitated the transcriptional level of XKS1 gene ( accumulated mRNA) and measured the activity of xylulokinase in each stains． Furthermore，we also determined the intracellular level of ATP and evaluated the xylose-fermenting abilities of the engineered strains．［Results］ The engineered strains exhibited higher expression of xylulokinase than the parental strain at both transcription and enzyme activity levels． The highest xylulokinase activity was observed in the strain whose XKS1 was controlled by PGK1p，and was decreasingly followed by the strains whose XKS1 was controlled by TEF1p，HXK2p and native promoter．The expression level of xylulokinase negatively correlated with intracellular level of ATP and positively correlated with ability of ethanol production from xylose． The highest ethanol yield was 0. 35 g / g consumed sugars while the lowest xylitol yield，which was 0. 18 g / g consumed xylose，was observed． ［Conclusion］ By promoter replacement，xylulokinase was overexpressed at different levels． In this work，higher expressional level of xylulokinase improved the conversion of xylose to ethanol．

Abstract:Abstract:［Objective］To screen mutants with high yield of taxol，and construct cDNA subtractive library of obtained mutant and primary strain HD1-3 ． ［Methods］The spores of taxol-producing fungus HD1-3 were treated by diethyl sulphate (DES)，ultraviolet radiation and diethyl sulphate (UV + DES) ． cDNA subtractive library of taxol producing fungi from the mRNA of obtained mutant with high yield of taxol tester and HD1-3 driver was constructed by using suppression subtracted hybridization ( SSH) ．［Results］ The optimal conditions for mutagenesis of strain HD1-3 were as follows: the spore suspension was treated with 8% DES for 15 min，followed by UV irradiation (30w，30cm distance) for 45sec under magnetic stirring，a mutant UD14-11 which was able to produce taxol with high yield and could be stably passed on genetics was found． Its ability to produce taxol was improved from 232. 73 ± 4. 61 μg /L (strain HD1-3) to 312. 81 ± 7. 51 μg /L (strain UD14-11)．The tilter of the constructed cDNA library was 1. 2×107 cfu /mL，the recombinant rate reached to 75. 3% and the length of the inserted fragments was mostly 300 bp-1. 0 kb． ［Conclusion］ A mutant UD14-11 with high yield was obtained，and cDNA subtractive library of the mutant UD14-11 and strain HD1-3 was constructed． The study laid solid foundation for isolation of taxol biosynthesis related genes and construction of engineering strains with high yield of taxol by genetic techniques.

Abstract:Abstract:［Objective］To explore new resource from inactive actinomycete strains，we screened resistant mutant strains by ribosome engineering，and analyzed the products derived from the selected mutant strains．［Methods］ Three Gorges reservoir area-derived actinomycete strains including BD20、FJ3、WZ20 and FJ5 were used as initial strains，which showed no-antibacterial activities． The streptomycin-resistant (strR) mutants and rifampicin-resistant( rifR ) mutants were screened by single colony isolation on streptomycin-containing plates and rifampicin-containing plates according to the method for obtaining drug-resistant mutants in ribosome engineering． The four initial strains and their strR -mutants and rifR -mutants were fermented in a liquid medium with the same composition． Mutants with anti-Staphylococcus aureus activity were obtained by paper chromatography．The components of fermentation broth were analyzed by high performance liquid chromatography (HPLC) and high performance liquid chromatography-mass spectrometry (LC-MS)．Furthermore，FJ3 strain was identified by 16S rDNA and morphology．［Results］ The minimal inhibitory concentration (MIC) of streptomycin and rifampicin for FJ3 was: 0.5μg/mL and 110μg/mL，respectively．Twenty-four strR-utant strains and 20 rifR -mutant strains of FJ3 mutant strains were selected for bioassay． The result of the antibacterial activity screening demonstrated that six strains inhibited bacteria．Two strains (FJ3-2 and FJ3-6) were screened from the streptomycinresistance mutants of nactive strain FJ3. The result of bioassay showed that the fermentation broth of FJ3-2 and FJ3-6 exhibited obvious anti-Staphylococcus aureus activity． The assay of paper chromatography showed that the active substance may be nucleic acid class antibiotic via using solvent system Doskochilova． Moreover，the results of HPLC and LC-MS exhibited that this substance may be thiolutin． ［Conclusion］Ribosome engineering for changing the secondary metabolic function of the inactive wild-type actinomycete strains was a feasible method for the acquirement of active mutant strains， which will be beneficial to exploit the new medical actinomycete strains．

Abstract:Abstract:［Objective］Bacillus firmus was a common probiotics bacteria in nature and widely used in the shrimp aquaculture．In order to construct expression vectors for secretive proteins，we identified several major secretive proteins of Bacillus firmus by mass spectrometry and analyzed their gene sequences to find signal peptide sequences．［Methods］The secreted proteins of Bacillus firmus were extracted and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)．The 3 highly expressed protein bands in the SDS-PAGE were identified by mass spectrometry Matrix Assisted Laser Desorption Ionization (MALDI)-Time of Flight (TOF)/Time of Flight (TOF) and the sequences were downloaded from National Center for Biotechnology Information (NCBI) database．Then Polymerase Chain Reaction (PCR) primers were designed according to the downloaded sequences and specific DNA bands were amplified sequenced and bioinformatics analyzed．［Results］The proteins were identified as the putative chitinase，enterotoxin A and protein BCG9842 of Bacillus firmus．Three signal peptides were conformed by using the online software SignaIP 3. 0，namely bf-43，bf-37 and bf-16. The cellular localization of the secreted sequences were analyzed by PSORT． And we found that bf-43 located in the outer membrane of cells，bf-37 and bf-16 located in the extracellular cell．［Conclusion］3 major secreted proteins of Bacillus firmus have been identified．3 possible signal peptides were obtained and will be useful for the construction of expression vectors for secretive proteins．

Abstract:Abstract:［Objective］We investigated the structure model and function of xylitol dehydrogenase from Aspergillus oryzae．［Methods］ Xylitol dehydrogenase (XDH) gene from Aspergillus oryzae was cloned and sequenced．We constructed four tertiary structure models of XDH by homology modeling with Swiss-MODEL and Modeller and obtained the best quality model by evaluation of PROCHECK and Prosa2003. The dockings of NAD+，Zn2+ and xylitol with XDH were performed by Molsoft program． ［Results］ Structure analysis suggested that XDH was a member of medium-chain dehydrogenase/reductase (MDR) family．This was supported by the presence of the zinc-containing alcohol dehydrogenase signature and a typical alcohol dehydrogenase Rossmann fold pattern composed by NAD + binding domain present in MDR superfamily．The molecular docking indicated that amino acid residues Asp206，Arg211，Ser255，Ser301 and Arg303 in XDH binding domain had hydrogen bonding with NAD + ，His72 and Glu73 in catalytic domain had hydrogen bonding with Zn2 + ，Ile46，Ile349，Lys350 and Thr351 in catalytic domain had hydrogen bonding with xylitol． ［Conclusion］ These key amino acid residues might play a vital role in the XDH catalytic reaction and can instruct the further directed modification of XDH．

Abstract:Abstract:［Objective］We studied the ability of Lactobacillus plantarum 121 and Lactobacillus pentosus ML32 to bind benzo(a) pyrene．［Methods］ The percentage of benzo (a) pyrene bound by the lactobacilli strains was quantitated by HPLC after bacterial cells and benzo (a) pyrene were co-incubated in MRS media at 37℃ for 4 h．［Results］The percentage of benzo (a) pyrene-binding was 65.9% for 121 and 64. 9% for ML32．Physical factors affecting binding ability included incubation time，temperature，bacterial cell viability，pH and concentrations of Ca2 + and Mg2 + ． Different chemical and enzymatic treatments to cells affected the binding of the two strains to benzo(a) pyrene． The simulation of gastrointestinal environments showed that the binding ability of the two strains depended largely upon pH and bile salt concentrations，but less upon treating time． Trypsin had only influence on the ability of 121 to bind benzo(a) pyrene．The presence of benzene in washing cell impaired the ability of the two strains to bind benzo (a) pyrene．［Conclusions］Strains 121 and ML32 had potential to bind benzo(a) pyrene．

Abstract:Abstract:［Objective］ In order to investigate antibody responses by musosal DNA vaccines encoding the codon-optimized F protein of human respiratory syncytial virus (RSV) delivered with attenuated Salmonella typhimurium aroA strain SL7207 (SL7207) via intranasal or intragastric routes．［Methods］After the codon-optimized F gene was synthesized，we constructed eukaryotic expression plasmid pcDNA3.1/Fsyn and transformed it into SL7207 to get recombinant SL7207 of SL7207/pcDNA3.1/Fsyn．Then，SL7207/pcDNA3.1/Fsyn was exploited to evaluate its immunogenicity in BALB/c mice administered intranasally or intragastrically． The resultant serum and mucosal antibody responses were analyzed by indirect ELISA． ［Results］Compared with intragastric immunization group，intranasal immunization group achieved higher level of RSV specific serum IgG and secretion IgA (SIgA)(P＜0.05)．Moreover，F protein expressed by the codon-optimized F gene of Fsyn was of elevated immunogenicity，compared with wild type F (Fwt)(P＜0.05)．［Conclusion］ Intranasal immunization and codon optimization approaches can improve antibody responses of RSV DNA vaccine delivered by attenuated Salmonella typhimurium aroA strain SL7207．

Abstract:Abstract:［Objective］We investigated the distribution of autotransporter adhesin gene B11 and other putative virulence genes of APEC IMT5155，which contributed to study the pathogenic mechanism of Avian pathogenic Escherichia coli (APEC)．［Methods］PCR and Dot blot were used to detect the distribution of putative virulence genes in E．coli strains isolated from different country (101 Chinese strains and 121 German strains)，which were from different sources (human，avian and pig)．The association of E． coli phylogenetic group and distribution of putative virulence genes was also analyzed．［Results］The putative virulence genes were most prevalent among APEC isolates，36. 4% (32/88) of APEC isolates harboring A1，53.4% (47/88) for A8，63.6% (56/88) for A10，37.5% (33/88) for B11，59.1% (52/88) for F3．The isolates positive for putative virulence genes were linked to E． coli phylogenetic group B2． Among Neonatal meningitis E．coli isolates，60%,80% and 80% were positive for putative virulence genes D1，E9 and F11，respectively． However，all the Neonatal meningitis E． coli strains were negative for B11．Conclusion］ Autotransporter adhesin gene B11 and other putative virulence genes were associated to APEC． However，putative virulence genes D1，E9 and F11 were associated to Neonatal meningitis E． coli，which indicated that APEC might be a virulence gene reservoir for Neonatal meningitis E.oli．

Abstract:Abstract:［Objective］ We searched optimal signal peptide for heterologous and exogenous secretion of xylanase in Bacillus subtilis． ［Methods］We constructed a screening vector for signal peptides from B．subtilis． The Alkali resistance xylanase gene(xynA) from Bacillus pumilus was chosen as reporter gene and cloned into E． coli and B． subtilis shuttle vector pGJ148 which has maltose-inducible promoter Pglv and spectinomycin resistant gene． 24 Sec-type signal peptides (SPs) was amplified from B． Subtilis 1A747 and cloned into the screening vector for the expression of xynA in B．Subtilis WB700． The xylanase activity of the culture supernatant were detected after 24h incubation．Results］ The screening of these signal peptides revealed differences in xylanase activity of the culture supernatants， The recombinant strain containing YnfF signal peptide showed the highest xylanase acitivity (37.2IU/mL)．［Conclusion］ Experiment proved screening of signal peptides is ffective way for optimization of the export of heterologous protein in B．subtilis．

Abstract:Abstract:［Objective］We established a cell line stably expressing Mycobacterium tuberculosis secretory protein EspB in order to provide evidences for studying EspB in modulating the functions of macrophage．［Methods］ The recombinant plasmid pEGFP-C1-EspB was first constructed，then RAW264. 7 cell was transfected with pEGFP-C1-EspB and pEGFPC1 by liposome respectively． After screening with a high level of G418，the macrophage cell lines that stably expressed EGFP-EspB fusion protein or EGFP were established． The gene and protein expression levels were further analyzed by RTPCR，fluorescence microscopy and western blot．［Results］ The EGFP-EspB fusion gene was integrated into the chromosome and the protein was stably expressed in the selected macrophage cell line．The macrophage cell lines that stably expressed EGFP-EspB fusion protein or EGFP were established．［Conclusion］ These results gave us a tool for the future study in the effects of EspB protein in modulating the functions of macrophage and its interaction with other molecules of macrophage．

Abstract:Abstract:［Objective］We constructed a recombinant eukaryotic expression vector harboring green fluorescent protein (GFP) and the hygromycin resistance gene hph，and observed its expression in Clonostachys rosea．［Methods］We used PCR， enzyme digestion， phosphorylation， ligation and transformation to construct the plasmid． Using protoplast preparation and transformation technologies，we expressed the plasmid in the fungi C．rosea．［Results］We created the eukaryotic expression vector，transformed it into C．rosea and observed green fluorescence with fluorescence microscope．［Conclusion］The successful construction of the pANGH3 recombinant plasmid and its expression in C． rosea establishes a new model for studying fungal infection mechanisms．